PRECURSOR BISPECIFIC ANTIBODY CONSTRUCTS AND METHODS OF USE

THEREOF

SEQUENCE FISTING STATEMENT

[001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 25, 2019, is named P-5797l3-PC-SQL-SEPl9_ST25.txt and is 173 KB in size.

FIELD OF THE INVENTION

[002] Disclosed herein are precursor bispecific antibody constructs and methods of use thereof for these precursor constructs. Methods of use include for treatment of cancer, wherein the precursor constructs comprise prodrugs having tumor restricted activation.

BACKGROUND

[003] The functionality of monoclonal antibodies (non-conjugated or naked antibody) currently approved by drug regulatory agencies worldwide for clinical use in oncology setting are known to use one or a combination of the following mechanisms: 1) blocking cell growth signaling, 2) blocking the blood supply to cancer cells, 3) directly mediating cell apoptosis, 4) eliciting immunological effector functions such as antibody dependent cellular cytoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC), and 5) promoting adaptive immunity towards tumors.

[004] Monoclonal antibody therapies have demonstrated survival benefits in the clinic. However, the overall response rates in cancer patients are low, and the survival benefits are marginal (several months) compared to chemotherapy. Although the underlying reasons for the lack of robust clinical anti-cancer activities are not fully understood, research has suggested that cancer cells often quickly develop compensating signaling pathways to escape cell death. Also, cancer stem cells (CSC), which are considered as potent cancer initiating cells, are less active at cell proliferation therefore they tend to sustain the lack of growth signal better.

[005] In an attempt to improve anti-tumor activity of monoclonal antibodies, multi- specific antibodies are being developed. In contrast to classical monoclonal antibodies, which are the standard first-line therapy in several tumor entities, these multi- specific antibodies may bring together a tumor cell and the means to destroy the tumor cell, thereby increasing the efficiency of treatment. These multi- specific antibodies provide for new treatment options for cancer patients.

[006] Another anti-cancer therapeutic approach is to utilize T-cells. T-cells provide defense against cancer throughout life by patrolling the body in search for newly arisen cancer cells and eliminating them effectively and promptly. Therapeutic approaches utilizing T-cells have proved successful in cancer treatment of at least metastatic melanoma, metastatic kidney cancer, asymptomatic metastatic hormone refractory prostate cancer, and advanced melanoma.

[007] By engaging multiple receptors, for example, providing a bispecific antibody that can bind an antigen receptor on a tumor cell and bind an antigen receptor on a cell able to provide toxic activity to kill the tumor cell (e.g., a T-cell), the means to both specifically target and to destroy a tumor cell may be combined in a single therapeutic entity. Thus, bispecific antibodies offer the potential to improve upon single-agent - single target antibody, checkpoint blockades.

[008] Bispecific antibodies targeting a tumor antigen and a T-cell cell surface antigen have been developed. However, while bispecific antibodies have demonstrated potent tumor cell killing potential, severe side effects, including systemic immune activation, immunogenicity (anti-drug antibody response) and general poor manufacturability of these molecules remain and to a large extent, have prevented this class of drugs from broad applications.

[009] Recently bispecific antibody technology platform referred to as bi-specific T cell engagers, or BiTEs, attracted a lot of attention because of its outstanding potency demonstrated in preclinical and clinical tests (Bargou et al., Science (2008) 321:974-7). In particular, patients with non- Hodgkin's lymphoma showed tumor regression, and in some cases complete remission during a clinical trial of blinatumomab administration. However, blinatumomab caused severe side effects including central nervous system side effects manifested by the loss of language ability and disorientation. Symptoms were transient and reversible once administration of the drug was stopped. It was hypothesized that the direct binding of the CD3 (on T cells) by the drug causing T- cell partial activation and cell redistribution. Some of the redistributed T-cells adhere to the brain miscro-vasculature, partially activate the endothelial cells and lead to the enhanced permeability of the micro-vasculature in the brain. It was observed in the clinic trial that the incidence of side effects was lower in patients with high B cell to T cell ratios than those with low ratios. It has also been reported that using different CD3 binding antibody fragments can alleviate or avoid T cell redistribution in Monkeys. These observations strongly suggest that antibody binding to CD3 together with the binding epitope on CD3 and the resultant partial activation of the T-cell may be the direct cause of the severe CNS side effects.

[0010] A pitfall of antibody therapeutics used in cancer treatment is the“off-target” binding of the antibody to non-cancer tumor-associated-antigen-expressing cells, especially if such binding leads to cytotoxicity. Thus,“off-target” binding by multi- specific and bispecific antibodies presents a potential challenge to controlling their "off-target" activity against normal tissues that also express antigen, even at extremely low levels. These "off-target" effects are a serious limitation to multi- specific and bispecific antibody therapeutics.

[0011] Another drawback of many bispecific or multi- specific antibodies is their short half-life. In addition, bispecific antibodies having the structure of an scFv-scFv fusion protein, have a tendency to aggregate. Therefore, production of these bispecific antibodies requires highly complicated antibody engineering skills and it is laborious to make them stable and manufacturable.

[0012] Various technologies and bispecific antibodies are being developed. However, there remains a need to provide a bispecific antibody with qualities that reduce toxic side effects and preserve the antibodies effectiveness until it has both (1) engaged the target associated with a tumor cell/ tumor-associated cell and (2) localized to a tumor microenvironment, thereby reducing any non-specific toxic side effects. Further, it is essential that such bispecific antibodies do not reduce significantly the immunogenicity to a tumor or tumor-associated target. The precursor bispecific antibody constructs described herein addresses this need by attaching a regulatable half-life enhancing component to the antibody and a blocking component that inhibits the antibody from engaging a toxicity providing cell prior to binding to a tumor or tumor-associated target.

SUMMARY

[0013] In one aspect, disclosed herein is a precursor bispecific antibody construct comprising

(a) a first binding domain binding to a cell surface tumor associated antigen (TAA) binding domain;

(b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain comprising a cleavable half-life prolonging domain comprising a protease cleavage domain and a human serum albumin (HS A) polypeptide.

[0014] In a related aspect, it is disclosed that a second binding domain comprises a variable heavy chain (VH2) region and a variable light chain (VL2) region; wherein said first binding domain is located N-terminally to said VL2 or VH2 region of the second binding domain; wherein when said first binding domain is located N-terminally to said VL2 region, said regulatory domain is located N-terminally to said VH2 region, and when said first binding domain is located N-terminally to said VH2 region, said regulatory domain is located N-terminally to said VL2 region. In another related aspect, the human serum albumin polypeptide is located N-terminally or C-terminally to said protease cleavage domain. I

[0015] In a related aspect, the precursor bispecific antibody construct comprises two polypeptides, polypeptide A and polypeptide B, wherein polypeptide A comprises components having an order N-terminal to C-terminal: the human serum albumin, the protease cleavage domain, the CD3 second binding domain VH2; or the human serum albumin, the protease cleavage domain, the CD3 second binding domain VL2; and wherein polypeptide B comprises components having an order N- terminal to C-terminal: the TAA first binding domain, the CD3 second binding domain VL2 region; or the TAA first binding domain, the CD3 second binding domain VH2 region. In another related aspect, the cleavable half-life prolonging domain further comprises a CAP component that reduces the ability of the second binding domain to bind the extracellular epitope of human CD3e. In a further related aspect, the CAP component comprises an amino acid sequence comprising the second binding domain extracellular epitope of human CD3e. In another related aspect, the amino acid sequence of the CAP component is set forth in SEQ ID NO: 4 or a homolog thereof.

[0016] In a related aspect, the order of the cleavable half-life prolonging domain components comprises, N-terminal to C-terminal, the CAP component, the human serum albumin, the protease cleavage domain; or the CAP component, the protease cleavage domain, the human serum albumin.

[0017] In a related aspect, the precursor bispecific antibody construct comprises two polypeptides, wherein polypeptide A comprises components having an order N-terminal to C-terminal the CAP component, the human serum albumin, protease cleavage domain, CD3 second binding domain VH2 region; or the CAP component, the human serum albumin, protease cleavage domain, CD3 second binding domain VL2 region; or the CAP component, protease cleavage domain, the human serum albumin, CD3 second binding domain VH2 region; or the CAP component, protease cleavage domain, the human serum albumin, CD3 second binding domain VL2 region; and wherein polypeptide B comprises components having an order N-terminal to C-terminal cell surface TAA first binding domain, CD3 second binding domain VL2 region; or cell surface TAA first binding domain, CD3 second binding domain VH2 region.

[0018] In a related aspect, the first binding domain (cell surface TAA binding domain) comprises a single chain variable fragment (scFv). In another related aspect, the second binding domain (CD3e binding domain) comprises an antigen binding fragment (Fab). In another related aspect, the tumor associated antigen (TAA) an EGFR, FcyRI, FcyRIIa FcyRIIb FcyRIIIa FcyRIIIb, CD28, CD 137, CTFA-4, FAS, fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, glucocorticoid-induced TNFR-related (GITR) protein, lymphotoxin-beta receptor (FTpR), toll-like receptors (TER), tumor necrosis factor-related apoptosis-inducing ligand-receptor 1 (TRAIF receptor 1) and TRAIF receptor 2, pro state- specific membrane antigen (PSMA) protein, prostate stem cell antigen (PSCA) protein; tumor-associated protein carbonic anhydrase IX (CAIX), epidermal growth factor receptor 1 (EGFR1), EGFRvIII, human epidermal growth factor receptor 2 (Her2/neu; Erb2), ErbB3 also known as HER3, Folate receptor, ephrin receptors, PDGFRa, ErbB- 2, CD20, CD22, CD30, CD33, CD40, CD37, CD38, CD70, CD74, CD56, CD40), CD80, CD86, CD2, p53, cMet also known as tyrosine-protein kinase Met or hepatocyte growth factor receptor (HGFR), MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE -A 12, BAGE, DAM-6, -10, GAGE-l, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, NY-ESO- 1, BRCA1, BRCA2, MART-l, MC1R, GplOO, PSA, PSM, Tyrosinase, Wilms' tumor antigen (WT1), TRP-l, TRP-2, ART-4, CAMEL, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, P-cadherin, Myostatin (GDF8), Cripto (TDGF1), MUC5AC, PRAME, P15, RU1, RU2, SART-l, SART-3, WT1, AFP, b-catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-l, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPEmbcr-abl, ETV6/AML, LDLR/FUT, Pml/RARa, TEL/AML1, CD28, CD137, CanAg, Mesothelin, DR5, PD-l, PD1L, IGF-1R, CXCR4, Neuropilin 1, Glypicans, EphA2, CD138, B7-H3, gpA33, GPC3, SSTR2, ROR1, 5T4, or a VEGF-R2. In a related aspect, the cell surface TAA comprises EGFR, and the first binding region comprises the amino acid sequence set forth in any of SEQ ID NOs: 9-10, or a combination thereof.

[0019] In a related aspect, the VL2 region comprises CDR-L1 (selected from SEQ ID NOs: 74-76), CDR-L2 (SEQ ID NO: 77), and CDR-L3 (selected from SEQ ID NOs: 78-79), and said VH2 region comprises CDR-H1 (SEQ ID NO: 71), CDR-H2 (SEQ ID NO: 72), and CDR-H3 (SEQ ID NO: 73). In another related aspect, the VL2 region comprises an amino acid sequence selected from the group set forth in any of SEQ ID NO: 41-70, or an amino acid sequence having at least 80% homology thereto. In another related aspect, the VH2 region comprises the amino acid sequence set forth in any of SEQ ID NO: 13-29, or an amino acid sequence having at least 80% homology thereto.

[0020] In a related aspect, the protease cleavage domain comprises the sequence set forth in SEQ ID NO: 6. In another related aspect, the at least one of said first or second binding domains comprises a humanized binding domain.

[0021] In addition, in one aspect, disclosed herein is a pharmaceutical composition comprising the precursor bispecific antibody construct of claim 1, and a pharmaceutically acceptable carrier.

[0022] In addition, in one aspect, disclosed herein are isolated nucleic acid sequences, which encode polypeptides A and B that together form a precursor bispecific antibody construct comprising (a) a first binding domain binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain comprising a cleavable half-life prolonging domain comprising a protease cleavage domain and a human serum albumin (HSA) polypeptide.

[0023] In addition, in one aspect, disclosed herein is an expression vector comprising the isolated nucleic acid sequences of claim 20, wherein said expression vector comprises a single expression vector comprising both nucleic acid sequences or comprises two expression vector each comprising a nucleic acid sequence wherein one sequence encodes polypeptide A and one sequence encodes polypeptide B. [0024] Further, in one aspect, disclosed herein is an isolated host cell comprising the isolated nucleic acid sequences of claim 20, or the expression vector of claim 21, wherein said host cell is a single cell comprising both nucleic acid sequences, or wherein said host cell comprises two host cells each comprises a nucleic acid sequence encoding a polypeptide A or B, respectively.

[0025] In one aspect, the application discloses a method of producing a precursor bispecific antibody construct comprising (a) a first binding domain binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain comprising a cleavable half-life prolonging domain comprising a protease cleavage domain and a human serum albumin (HSA) polypeptide, said method comprising steps (i) culturing a host cell comprising a nucleic acid sequence encoding precursor bispecific antibody construct polypeptides A and B, (ii) expressing said polypeptides A and B, (iii) isolating said expressed precursor bispecific antibody construct polypeptides A and B, and (iv) dimerizing said polypeptides A and B. In a related aspect, the expression comprises expression from a same host cell or wherein said host cell comprises two host cells each expressing a different polypeptide, polypeptide A and polypeptide B, respectively.

[0026] In one aspect, disclosed herein is a method of treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject, or any combination thereof, comprising a step of administering a pharmaceutical composition comprising a precursor bispecific antibody construct comprising (a) a first binding domain binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain comprising a cleavable half-life prolonging domain comprising a protease cleavage domain and a human serum albumin (HSA) polypeptide; to said subject, wherein said method treats, prevents, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in said subject, or reduces the minimal residual disease, increases remission, increases remission duration, reduces tumor relapse rate, prevents metastasis of said tumor or said cancer, or reduces the rate of metastasis of said tumor or said cancer, or any combination thereof, compared with a subject not administered said pharmaceutical composition. In a related aspect, disclosed herein is a method of treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject, or any combination thereof, comprising a step of administering a pharmaceutical composition comprising a nucleotide sequence encoding a precursor bispecific antibody construct, said construct comprising

(a) a first binding domain binding to a cell surface tumor associated antigen (TAA binding domain);

(b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and

(c) a regulatory domain comprising a cleavable half-life prolonging domain comprising a protease cleavage domain and a human serum albumin (HSA) polypeptide;

to said subject, wherein said method treats, prevents, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in said subject, or reduces the minimal residual disease, increases remission, increases remission duration, reduces tumor relapse rate, prevents metastasis of said tumor or said cancer, or reduces the rate of metastasis of said tumor or said cancer, or any combination thereof, compared with a subject not administered said pharmaceutical composition.

[0027] In a related aspect, the size or the growth rate or a combination thereof, of said cancer or tumor is reduced, or wherein the survival of said subject is increased or a combination thereof. In another related aspect, the subject is a human subject. In another related aspect, the cancer or tumor comprises a solid tumor or non-solid tumor, or wherein said cancer or tumor comprises a metastasis of a cancer or tumor. In a further related aspect, the non-solid cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cell myeloma), an acute lymphoblastic leukemia, an acute myelogenous leukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, a non- Hodgkin lymphoma, or plasma cell leukemia; or wherein said solid tumor comprises a sarcoma or a carcinoma, a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endotheliosarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor, an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilm's tumor, a cervical cancer or tumor, a uterine cancer or tumor, a testicular cancer or tumor, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodenroglioma, a schwannoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma. [0028] In another related aspect, a precursor construct described herein may be used in a method of treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject, as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The subject matter regarded as the precursor bispecific antibody constructs disclosed herein, is particularly pointed out and distinctly claimed in the concluding portion of the specification. The precursor bispecific antibody constructs, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0030] Figures 1A - 1C present schematic embodiments of precursor bispecific antibody constructs, wherein the Fab portion recognizes a CD3 surface antigen, wherein the components and regions of the different construct domains are identified. In the embodiments shown here, the precursor bispecific antibody construct is formed by two polypeptides, wherein one of the polypeptides includes the anti-tumor associated antigen (TAA) first binding domain (Red; scFv) and components of the anti-CD3 Fab second binding domain (Brown; Fab), and the other polypeptide includes a regulator domain comprising a CAP region (Green triangle) or a HS A region (Grey oval) or both, linked to a polypeptide of the second binding domain by a protease cleavable linker (blue rectangle), and the other components of the anti-CD3 Fab second binding domain (blue - constant regions CH1 and CL). As shown here, the anti-TAA is a single chain variable fragment (ScFv). As shown here in Figure 1A, the N-terminal to C-terminal order of the variable regions of the ScFv is a Variable Light chain region (VL) followed by a linker (Ll) followed by a Variable Heavy chain region (VH). In the embodiments presented in Figure IB, the N-terminal to C-terminal order of the variable regions of the ScFv is a Variable Heavy chain region (VH) followed by a linker (Ll) followed by a Variable Light chain region (V _L ). The linkers between components and between domains are identified by an“L” followed by a numeral, e.g., Ll, L2, L3, L4, L5, L6, and L7. V _L ! is a variable light-chain region of binding site one (1), VL2 is a variable light-chain region of binding site two (2), V _H l is a variable heavy-chain region of binding site one (1), and VH2 is a variable heavy-chain region of binding site two (2). The oval shape indicated as HSA is the human serum albumin component. The shape indicated as CP is the cleavage peptide. The triangle shape is the CAP component. Following the same shape and color scheme, Figure 1C provides schematics of embodiments of constructs made and analyzed, in the Examples 1-15, including an active Micro- Environment Activated T cell Engager (MATE) construct, a Precursor Tumor Micro-Environment Activated T cell Engager (PTMATE) construct, a PTMATE lacking a CAP region (PTMATE (D- CAP), also termed HSA-MATE-C wherein C indicates a cleavable linker), and a PTMATE lacking a half-life extension domain (PTMATE (A-HSA), also termed CAP-MATE -C wherein C indicates a cleavable linker). Not shown are similar constructs with non-cleavable linkers (NC) in place of the protease cleavable linker, which were also produced and used in the Examples.

[0031] Figures 2A-2F present various embodiments of precursor bispecific antibody constructs described herein. Figures 2A and 2B present schematic embodiments of a precursor bispecific antibody construct comprising an anti-tumor associated antigen binding domain, in which the N- terminal to C-terminal order of variable regions is either VL-L!-VH (Figure 2A) or VH-L!-VL (Figure 2B). In these embodiments, the anti-TAA is an anti-EGFR ScFv binding domain. The precursor bispecific antibody construct further includes an anti-CD3e Fab domain, and a regulatory domain comprising a protease cleavable linker, a human serum albumin (HSA) polypeptide sequence, and a CD3 CAP, wherein is some embodiments the amino acids of the CAP are amino acids 1-27 of the mature CD3e polypeptide (SEQ ID NO: 4), wherein the order of components in the regulatory domain is N-terminus to C-terminus is CAP, HSA, protease cleavable linker. Ll, L2, etc represent possible linkers between the different domains or domain components. Linkers may or may not be present. Figure 2C presents a schematic of a precursor bispecific antibody construct comprising an anti-tumor associated antigen (TAA) scFv domain, an anti-CD3e Fab domain, and a regulatory domain comprising (N-terminal to C-terminal) a CD3e CAP, which may comprise amino acids 1-27 of CD3e (SEQ ID NO: 4), a protease cleavable linker, and a human serum albumin (HSA) polypeptide sequence. The linkers between components and between domains are identified by an“L” followed by a numeral, e.g., Ll, L2, L3, L4, L5, L6, and L7. Figure 2D presents a schematic of a precursor bispecific antibody construct comprising an anti-tumor associated antigen (TAA) scFv domain, an anti-CD3e Fab domain, and a regulatory domain comprising (N-terminal to C-terminal) a human serum albumin (HSA) polypeptide sequence and a protease cleavable peptide (CP) sequence. The linkers between components and between domains are identified by an “L” followed by a numeral, e.g., Ll, L2, L3, L5, L6, and L7. Figure 2E presents a schematic of a precursor bispecific antibody construct comprising an anti-tumor associated antigen (TAA) scFv domain, an anti-CD3e Fab domain, and a regulatory domain comprising (N-terminal to C-terminal) a CD3e CAP that may comprise amino acids 1-27 of CD3e (SEQ ID NO: 4), and a protease cleavable linker sequence. The linkers between components and between domains are identified by an“L” followed by a numeral, e.g., Ll, L2, L3, L4, L6, and L7. Figure 2F presents a schematic of an active cleaved precursor bispecific antibody construct comprising an anti-tumor associated antigen (TAA) scFv domain, an anti-CD3e Fab domain. The linkers between components and between domains are identified by an“L” followed by a numeral, e.g., Ll, L2, L3, L6, and L7. [0032] Figures 3A-3B shows a flow diagrams of protease specific activation inside tumor tissue of a precursor bispecific antibody construct, wherein T-cell engagement and activation is limited to tumor sites. Figure 3A shows the origins of anti-Tumor associated antigen (TAA) scFv domain sequences and anti-human CD3e Fab sequences. The precursor bispecific antibody construct remains intact with an extended in vivo half-life when in circulation or present in normal tissue. Upon binding to a TAA target antigen and wherein that target antigen is present on the surface of a tumor (tumor microenvironment), protease specific activation may occur, leading to cleavage of the regulatory domain and exposure of the anti-CD3e binding site. The activated bispecific antibody construct antibody has a reduced limited half-life of hours compared with days to weeks for the precursor bispecific antibody construct. Figure 3B shows the influence of a cancer (tumor) microenvironment on a precursor bispecific antibody construct. The precursor bispecific antibody construct presented in Figure 3A comprises a protease cleavable domain C-terminal to an HSA half-life extendable polypeptide and C-terminal to a CAP component that may specifically be bound by the second binding domain. Entry into a cancer microenvironment, that are known to be rich in cancer-cell secreted proteases, results in protease cleavage and removable of the HSA and CAP, wherein in some embodiments the CAP comprises an extracellular CD3e epitope. The resultant activated antibody (activated bispecific antibody construct) may now bind and activate a T-cell. Though not depicted in the diagram due to drawing constraints, the precursor bispecific antibody construct may in some embodiments bind to the tumor associated antigen on the target cell, prior to protease cleavage. Further, were the precursor construct to bind to a TAA not in a tumor microenvironment, protease cleavage would not occur and neither would T-cell activation.

[0033] Figures 4A-4J present embodiments of amino acid sequences of, and nucleotide sequences encoding, polypeptides of precursor bispecific antibody constructs disclosed herein. Amino acid sequences are presented N-terminal to C-terminal, and nucleic acid sequences are presented 5' to 3'.

[0034] Figure 4A presents one embodiment of an amino acid sequence of a Polypeptide A of a precursor construct, having the order and components as follows CD3e(AAl-27)-G4Sx3 linker- hALB -G-PLGLAG (MMP2/9)-hlF3.5-GlFd-CPPC (SEQ ID NO: 106). The amino acid sequences of the component parts of Polypeptide A shown in Figure 4A include: CAP (red; SEQ ID NO: 4), Linker (L4 e.g., Figure 1) (grey; SEQ ID NO: 98), HSA-human serum albumin (yellow; SEQ ID NO: 116), Linker (L5 e.g., Figure 1) (grey; SEQ ID NO: 99), Cleavage Peptide (CP e.g., Figure 1) (green; SEQ ID NO: 6), Linker (L6 e.g., Figure 1) (grey; SEQ ID NO: 99), anti-CD3 epsilon variable heavy chain and constant heavy chain region 1 (turquoise; SEQ ID NO: 124), followed by two marked cysteine residues (marked red and green), which may participate in disulfide double bond. [0035] Figure 4B presents one embodiment of an optimized nucleic acid sequence (DNA) encoding a Polypeptide A of a precursor construct, having the order and components as follows CD3e(AAl-27)-G4Sx3 linker-hALB -G-PLGLAG (MMP2/9)-hlF3.5-GlFd-CPPC (SEQ ID NO: 107). The nucleic acid sequences encoding the component parts of Polypeptide B include: CAP (red. SEQ ID NO: 117), Linker (L4 e.g., Figure 1) (grey; SEQ ID NO: 118), EISA-human serum albumin (yellow; SEQ ID NO: 119), Linker (L5 e.g., Figure 1) (grey, SEQ ID NO: 120), cleavage peptide (CP e.g., Figure 1) (green; SEQ ID NO: 121), Linker (L6 e.g., Figure 1) (grey; SEQ ID NO: 122), anti-CD3 epsilon variable heavy chain and constant heavy chain region 1 (turquoise; SEQ ID NO: 123), followed by two marked cysteine codons (marked red and green).

[0036] Figure 4C presents one embodiment of an amino acid sequence of a Polypeptide A of a precursor construct, having the order and components as follows CD3e(AAl-27)-G4Sx3 linker- hALB-G-GGSGGS (non-cleavable)- (cloning) -hlF3.5 -GlFd-CPPC (SEQ ID NO: 108). The amino acid sequences of the component parts are the same as for Figure 4A except for the CP region , which here presents a non-cleavable linker peptide (mustard; SEQ ID NO: 99).

[0037] Figure 4D presents one embodiment of an optimized nucleic acid sequence (DNA) encoding a Polypeptide A of a precursor construct, having the order and components as follows CD3e(AAl-27)-G4Sx3 linker-hALB -G-GGS GGS (non-cleavable)- (cloning) -hlF3.5 -GlFd- CPPC (SEQ ID NO: 109). The nucleic acid sequences encoding the component parts are the same as for Figure 4B except for the sequence encoding the CP region, which here encodes a non- cleavable linker peptide (mustard; SEQ ID NO: 125).

[0038] Figure 4E presents one embodiment of an amino acids sequence of a Polypeptide A of an activated bispecific antibody construct, having the order and components as follows hlF3.5-GlFd- CPPC-G4S- (SEQ ID NO: 110). The anti-CD3epsilon variable heavy chain and constant heavy chain region 1 is identical with that shown in Figure 4A (turquoise; SEQ ID NO: 124), which is followed by two marked cysteine residues (marked red and green), which may participate in disulfide double bond.

[0039] Figure 4F presents one embodiment of an optimized nucleic acids sequence (DNA) encoding a Polypeptide A of an activated bispecific antibody construct, having the order and components as follows hlF3.5-GlFd-CPPC-G4S- (SEQ ID NO: 111). The nucleic acid sequence encoding the anti-CD3 epsilon variable heavy chain and constant heavy chain region 1 (turquoise; SEQ ID NO: 123) is the same as shown in Figure 4B, which is followed by nucleic acid sequence of two marked cysteine codons (marked red and green).

[0040] Figure 4G presents one embodiment of an amino acid sequence of a Polypeptide B of a precursor construct, having the order and components as follows anti-EGFR (VH-linker-VL)- GlyGly-hlF3.l- LC-CPPC-S*(SEQ ID NO: 112), see for example Figure 2B. The amino acid sequences of the component parts of Polypeptide B shown in Figure 4G include: anti-EGFR Variable Heavy chain (green; SEQ ID NO: 10), Linker (grey; SEQ ID NO: 100): anti-EGFR Variable Light chain (olive green; SEQ ID NO: 12), linker (no highlighting; SEQ ID NO: 93), anti- CD3 epsilon Variable light chain and constant light chain (pink; SEQ ID NO: 41), which is followed by two marked cysteine residues (marked red and green), which may participate in disulfide double bond.

[0041] Figure 4H presents one embodiment of an optimized nucleic acid sequence (DNA) of a Polypeptide B encoding a precursor construct, having the order and components as follows anti- EGFR (VH-linker- VL)-GlyGly-h 1F3.1 - LC-CPPC-S * (SEQ ID NO: 113). The nucleic acid sequences encoding the component parts of Polypeptide B include: anti-EGFR Variable Heavy chain (green; SEQ ID NO: 126), Linker (grey; SEQ ID NO: 127), anti-EGFR Variable Light chain (olive green; SEQ ID NO: 128), linker (no highlight; SEQ ID NO: 129), anti-CD3 epsilon Variable light chain and constant light chain (pink; SEQ ID NO: 130), which is followed by two marked cysteine codons (marked red and green).

[0042] Figure 41 presents one embodiment of an amino acid sequence of a Polypeptide B of a precursor construct, having the order and components as follows: anti-EGFR (VL-linker-VH)- GlyGly-h 1 F3.1 - LC-CPPC-S* (SEQ ID NO: 114) see for example Figure 2A. The amino acid sequence of the component parts is the same as in Figure 4G, but the order of anti-EGFR variable light and heavy chain sequences has been switched so that the Polypeptide B would have components in the order N-terminal to C-terminal: anti-EGFR Variable light chain region-linker- Variable heavy chain region.

[0043] Figure 4J presents one embodiment of an optimized nucleic acid sequence (DNA) encoding a Polypeptide B of a precursor construct, having the order and components as follows: anti-EGFR (VL-linkcr-VH)-GlyGly-h 1 F3.1 -/,LC-CPPC-S ^::; (SEQ ID NO: 115). The nucleic acid sequences encoding the component parts are the same as in Figure 4H, but the order of anti-EGFR variable light and heavy chain sequences has been switched to encode a Polypeptide B that would have components in the order N-terminal to C-terminal: anti-EGFR Variable light chain region- linker- Variable heavy chain region.

[0044] By combining the different amino acid sequence chains of Figures 4A, 4E, 4G, and 41, different embodiments of precursor bispecific antibody constructs (precursor bispecific antibody construct) may be envisioned, as could active bispecific antibody constructs (activated bispecific antibody constructs) (See, Example 1). For example, in some embodiments, a precursor bispecific antibody construct with a cleavable peptide would be formed by the heterodimer created by the polypeptide of Figure 4A and Figure 41. In other embodiments, a precursor bispecific antibody construct with a cleavable peptide would be formed by the heterodimer created by the polypeptide of Figure 4A and Figure 4G.

[0045] Figures 5A-5F show SDS-PAGE and SEC HPLC analyses of the bispecific antibody constructs. Figure 5A shows an SDS-PAGE analysis of the bispecific antibody construct. Figure 5B shows a SEC-HPLC analysis of the bispecific antibody construct. Figure 5C shows an SDS- PAGE analysis of the non-cleaved bispecific antibody construct. Figure 5D shows a SEC-HPLC analysis of the non-cleaved bispecific antibody construct. Figure 5E shows an SDS-PAGE analysis of the cleaved bispecific antibody construct. Figure 5F shows a SEC-HPLC analysis of the cleaved bispecific antibody construct.

[0046] Figures 6A-6D show ELISA binding assays of bispecific antibody constructs. Figure 6A shows an ELISA binding assay to human epidermal growth factor (hEGFR). Figure 6B shows an ELISA binding assay to rhesus epidermal growth factor (rEGFR). Figure 6C shows an ELISA binding assay to human CD3e (hCD3e). Figure 6D shows an ELISA binding assay to cynomolgus (cyno CD3e). Three antibody constructs were used: an active bispecific antibody construct (circles, fucsia lines), a non-cleaved precursor bispecific antibody (squares, yellow lines), a cleaved precursor bispecific antibody (triangles, green lines).

[0047] Figures 7-28 - The following terms have been used: Micro-Environment Activated T cell Engager construct (MATE); Precursor Tumor Micro-Environment Activated T cell Engager construct (PTMATE); Precursor Tumor Micro-Environment Activated T cell Engager construct with cleavable linker comprising a MMP2/9 cleavage site (PTMATE-C); Precursor Tumor Micro- Environment Activated T cell Engager construct with non-cleavable linker (PTMATE-NC); and Precursor Tumor Micro-Environment Activated T cell Engager construct with multiple cleavage linker having both MMP and Matriptase/uPA/Legumain cleavage sequences in tandem (PTMATE - MC). The target antigen of the first binding site scFv is indicated following the construct name, for example PTMATE-EGFR-MC or PTMATE-C EGFR, wherein the scFv target TAA is EGFR. Additional target TAAs include 5T4 and ROR1. The protease cleavable or non-cleavable linker is between the regulatory arm and a polypeptide of the Fab second binding site, for example see the first and second constructs in Figure 1C showing MATE and PTMATE order of domains.

[0048] Figures 7A and 7B show ELISA binding assays of bispecific antibody constructs. Figure 7A shows an ELISA binding assay to recombinant human epidermal growth factor (human EGFR), wherein precursor structures with and without components of the regulatory domain were analyzed, including constructs with cleavable (C) or non-cleavable (NC) linkers. Figure 7B shows an ELISA binding assay to human CD3e (human CD3e). Antibody precursors constructs analyzed were as follows: Micro-Environment Activated T cell Engager construct (MATE) pink circles; Precursor Tumor Micro-Environment Activated T cell Engager construct (PTMATE)-C orange squares; CAP-MATE -C olive triangles; HSA-MATE inverter green triangles, and PTMATE-NC blue diamonds).“VL-VH” indicates the order of the variable chains within the scFv of the first binding domain, wherein“VL” is the variable light chain and“VH” is the variable heavy chain.

[0049] Figures 8A-8C show ELISA binding assays of bispecific antibody constructs in the presence and absence of protease, wherein the PTMATE construct comprises either a cleavable or non-cleavable linker connecting the regulatory domain with the Fab domain. Figure 8A shows an ELISA binding assay to recombinant human epidermal growth factor (human EGFR), wherein precursor structures with and without cleavable (C) or non-cleavable (NC) linkers were used. Figure 8B shows an ELISA binding assay to human CD3e (human CD3e), wherein PTMATE show decreased binding affinity compared with MATE constructs. Figure 8C shows the effect of incubation with protease for each of the constructs: Antibody precursors constructs analyzed were as follows: MATE pink circles; MATE + protease orange squares; PTMATE-C green triangles; PTMATE-C + protease blue inverted triangles; PTMATE-NC turquoise diamonds; and PTMATE- NC + protease purple squares.

[0050] Figures 9A and 9B show binding analysis of PTMATE variants to the cell surface of tissue culture cells. Figure 9A shows binding analysis of PTMATE variants binding to hEGFR expressing 293F cell (hEGFR binding). Figure 9B shows binding analysis of PTMATE variants to Jurkat T- cells (CD3e binding). Prior to incubating variant constructs with cells, PTMATE binding variants were incubated with proteases. Antibody precursors constructs analyzed were as follows: MATE, PTMATE-C, and PTMATE-NC, as described above wherein“C” refers to constructs comprising a cleavable MMP2/9 linker between the Fab second binding domain and the regulatory arm, while “NC” refers to construct comprising non-cleavable linkers between the Fab second binding domain and the regulatory arm; and PTMATE-MC, wherein“MC” refers to multiple cleavage sites, wherein the linker has both MMP2/9 and Matriptase/uPA/Legumain cleavage sequences in tandem between the Fab second binding domain and the regulatory arm.

[0051] Figures 10A and 10B show dose-dependent T-cell mediated cytotoxicity of breast cancer cells (Figure 10A) and colorectal cancer cells (Figure 10B). Antibody precursors constructs analyzed were as follows: MATE, PTMATE-MC, and PTMATE-NC, as described above. The EC50 for variant constructs is provided underneath the binding curves.

[0052] Figures 11A-11I show T-cell activation and cytokine release using in vitro cell-based assays of PTMATE -EGFR variant constructs. Cytokine release was measure for CD69 (Figures 11A-11C), IFNy (Figures 11D-11F), and TNFa (Figures 11G-11I). MATE-EGFR, PTMATE-C EGFR, and PTMATE-NC EGFR constructs were assayed as follows: MATE-EGFR Figure 11A (CD69), Figure 11D (IFNy), and Figure 11G (TNFa); PTMATE-C EGFR Figure 11B (CD69), Figure HE (IFNy), and Figure 11H (TNFa); and PTMATE-NC EGFR Figure 11C (CD69), Figure 11F (IFNy), and Figure 111 (TNFa). Color scale indicates the concentration of variant constructs analyzed: 10 nM, 1 nM, 100 pM, 10 pM, 1 pM.

[0053] Figures 12A-12C show pharmacokinetics of PTMATE-EGFR constructs in mice. Mice were administered constructs by intravenous (IV) injection, at concentrations of either 0.5 mg/kg or 2 mg/kg. The half-life of the construct was then measured over time (days). Constructs tested were MATE-EGFR-(VF-VH) (Figure 12A), PTMATE-C EGFR-(VF-VH) (Figure 12B), and PTMATE-NC EGFR-(VL-VH) (Figure 12C).

[0054] Figures 13A and 13B provide a schematic of the pilot xenograft studies of PTMATE-EGFR in N O D/SC I D/I L2 Ry ^{nul 1} ( NS G) mice, wherein PTMATE variants were daily tail-injected (Figure 13A; Example 8), and a graft showing the resultant fold-change of tumor size following administration of constructs (MATE, PTMATE-C, or PTMATE-NC) or control cells over time (Figure 13B). Tumor sizes were measured twice weekly in two dimensions using a caliper, and the volume expressed in mm ³ using the formula: V = 0.5 (a) x (b) 2 where a and b are the long and short diameters of the tumor, respectively. The volume of the tumor is calculated at each time point, and then divided by the volume of tumor at day one. This way one normalizes the measurements, coming from tumor volume variability.

[0055] Figure 14 shows the resultant size distribution patterns in an SDS-PAGE gel of PTMATE - MC EGFR and PTMATE-C EGFR constructs following cleavage by Matripase or MMP9.

[0056] Figures 15A and 15B show ELISA binding curves of variant PTMATE constructs to human CD3e (hCD3e) and EGFR antigens in the presence or absence of different proteases. Figure 15A shows ELISA binding assays to hCD3e in the presence or absence of MMP9 (+MMP9 or - MMP9 respectively) or the presence or absence of Matriptase (+Matriptase or -Matriptase, respectively), wherein the PTMATE constructs tested include MATE (control), PTMATE-C, and PTMATE-MC. Figure 15B shows ELISA binding assays to EGFR in the presence or absence of MMP9 (+MMP9 or -MMP9, respectively) or the presence or absence of Matriptase (+Matriptase or -Matriptase, respectively), wherein the PTMATE constructs tested include MATE (control), PTMATE-C, and PTMATE-MC.

[0057] Figures 16A-16F show the resultant size distribution patterns in an SDS-PAGE gel of MATE-5T4 (VL-VH) (Figure 16A), PTMATE-C 5T4 (VL-VH) (Figure 16C), and PTMATE-NC 5T4 (VL-VH) (Figure 16E) constructs under reducing and non-reducing conditions; and the size- exclusion chromatograph (SEC) scans of each of MATE-5T4 (VL-VH) (Figure 16B), PTMATE- C 5T4 (VL-VH) (Figure 16D), and PTMATE-NC 5T4 (VL-VH) (Figure 16F).

[0058] Figures 17A-17D show the resultant size distribution patterns in an SDS-PAGE gel and size exclusion chromatography (SEC) scans, respectively of MATE-5T4 (VH-VL) (Figure 17A), PTMATE-C 5T4 (VH-VL) (Figure 17B), constructs under reducing and non-reducing conditions; and the size-exclusion chromatograph (SEC) scans of each of MATE-5T4(VH-VL) (Figure 17C), PTMATE-C 5T4 (VH-VL) (Figure 17D).

[0059] Figure 18 shows protease cleavage of 5T4 variant constructs in the presence and absence of MMP9 (+MMP9 and -MMP9, respectively). The resultant size distribution pattern is shown in an SDS-PAGE gel.

[0060] Figure 19 show ELISA binding curves of variant PTMATE 5T4 constructs to the human 5T4 antigen. Constructs analyzed include MATE 5T4 (VLVH) - pink circles, PTMATE 5T4-C (VLVH) orange squares, PTMATE 5T4-NC (VLVH) olive triangles, MATE 5T4-C (VH-VL) inverted green triangles, and PTMATE 5T4-C (VH-VL) blue diamonds. The EC50 for each construct is also provided.

[0061] Figures 20A and 20B show FACS binding curves of variant PTMATE 5T4 constructs to CHO-K1 cells expressing or not expressing the human 5T4 antigen. Figure 20A shows FACS binding of variant constructs to CHO-Kl-h5T4 cells, while Figure 20B shows FACS binding of variant constructs to control CHO-K1 cells that do not express h5T4 (empty cassette). Constructs analyzed include MATE 5T4 (VLVH) - pink circles, PTMATE 5T4-C (VLVH) orange squares, PTMATE 5T4-NC (VLVH) olive triangles, MATE 5T4-C (VH-VL) inverted green triangles, and PTMATE 5T4-C (VH-VL) blue diamonds.

[0062] Figure 21 shows ELISA binding curves of variant PTMATE 5T4 constructs to the human CD3e (hCD3e) antigen in the presence and absence of MMP9 protease (+MMP9 and -MMP9, respectively). Constructs analyzed include MATE 5T4 (VLVH) - pink circles, PTMATE 5T4-C (VLVH) -MMP9 green squares, PTMATE 5T4-C (VLVH) +MMP9 green triangles, PTMATE 5T4-NC (VLVH) -MMP9 beige inverted triangles, PTMATE 5T4-NC (VLVH) +MMP9 beige diamond, MATE 5T4-C (VH-VL) purple large circles, PTMATE 5T4-C (VH-VL)-MMP9 blue squares, and PTMATE 5T4-C (VH-VL)+MMP9 large purple triangles.

[0063] Figure 22 shows FACS binding curves of variant PTMATE 5T4 constructs to Jurkat T- lymphoma cells, that naturally express 5T4. MATE 5T4 (VLVH) - pink circles, PTMATE 5T4-C (VLVH) “PT-C”-MMP9 green squares, PTMATE 5T4-C (VLVH) “PT-C” +MMP9 green triangles, PTMATE 5T4-NC (VLVH)“PT-NC” -MMP9 beige inverted triangles, PTMATE 5T4- NC (VLVH)“PT-NC” +MMP9 olive diamond, MATE 5T4-C (VH-VL) purple large circles, PTMATE 5T4-C (VH-VL)“PT-C”-MMP9 blue squares, and PTMATE 5T4-C (VH-VL)“PT-C” +MMP9 large purple triangles.

[0064] Figures 23A-23F show the resultant size distribution patterns in an SDS-PAGE gel of the final purification products following precursor synthesis of: MATE-ROR1 (VL-VH) (Figure 23A), PTMATE-C ROR1 (VL-VH) (Figure 23C), and PTMATE-NC ROR1 (VL-VH) (Figure 23E) constructs under reducing and non-reducing conditions; and the size-exclusion chromatograph (SEC) scans of the purification products of each of MATE-ROR1 (VL-VH) (Figure 23B), PTMATE-C ROR1 (VL-VH) (Figure 23D), and PTMATE-NC ROR1 (VL-VH) (Figure 23F).

[0065] Figure 24 show ELISA binding curves of variant PTMATE ROR1 constructs to the human ROR1 antigen. Constructs analyzed include MATE ROR1- pink circles, PTMATE-C ROR1 orange squares, and PTMATE-NC ROR1 olive triangles. The EC50 for each construct is also provided.

[0066] Figure 25 show ELISA binding curves of variant PTMATE ROR1 constructs to the human ROR1 antigen. Constructs analyzed include MATE (VLVH) ROR1- pink circles, PTMATE-C (VLVH) ROR1 orange squares, PTMATE-NC (VLVH) ROR1 olive triangles, and PTMATE-MC (VHVL) ROR1 green inverted triangles.

[0067] Figure 26 shows the resultant size distribution patterns in an SDS-PAGE gel of ROR1 variant constructs in the presence or absence of MMP9 protease (+ or -, respectively). Constructs analyzed include MATE (VLVH) ROR1, PTMATE-C (VL-VH) ROR1, and PTMATE-NC (VLVH) ROR1.

[0068] Figure 27 shows FACS binding curves of variant PTMATE ROR1 constructs to Jurkat T- lymphoma cells, that naturally express ROR1, wherein certain constructs were first incubated in the presence of MMP9 protease. Constructs analyzed included MATE ROR1 - pink circles, MATE ROR1 +MMP9 - orange squares, PTMATE ROR1-C olive triangles, PTMATE ROR1-C +MMP9 - green inverted triangles, PTMATE ROR1-NC blue diamonds, and PTMATE ROR1-NC +MMP9 purple large circles.

[0069] Figure 28 show ELISA binding curves of variant PTMATE ROR1 constructs to the human CD3e antigen. Constructs analyzed include MATE ROR1 - pink circles, MATE ROR1 +MMP9 - orange squares, PTMATE ROR1-C olive triangles, PTMATE ROR1-C +MMP9 - green inverted triangles, PTMATE ROR1-NC blue diamonds, and PTMATE ROR1-NC +MMP9 purple large circles.

[0070] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. DETAILED DESCRIPTION

[0071] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of precursor bispecific antibody constructs. However, it will be understood by those skilled in the art that the precursor constructs presented herein, the production, and use thereof may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the disclosure.

[0072] Described herein are precursor bispecific antibody constructs comprising cleavable masking and half-life prolonging domains, wherein these cleavable regulatory domains provide reduced binding to T-cells of the precursor bispecific constructs when outside the cancer microenvironment and extended half-life. Half-life extension may be limited to the time a precursor bispecific construct is outside the cancer microenvironment or it may extend to the time a precursor bispecific construct resides within a cancer microenvironment. Also described herein are precursor bispecific antibody constructs comprising cleavable masking regulatory domains, wherein these cleavable regulatory domains provide reduced binding to T-cells of the precursor bispecific constructs when outside the cancer microenvironment.

[0073] Reduction in T-cell binding may lead to a reduction in T-cell activation. In some embodiments, the precursor bispecific antibody constructs described herein are regulatable precursor constructs. The regulatable precursor bispecific antibody constructs describe herein may have an extended half-life, or reduced T-cell binding, or reduced T-cell activation, or any combination thereof.

[0074] In some embodiments, the precursor bispecific antibody constructs described herein provide for a regulatable T-cell activation, wherein the precursor construct provides that T-cell activation is restricted to a tumor microenvironment. In some embodiments, the precursor bispecific antibody construct described herein have an increased half-life and provide that T-cell activation is restricted to a tumor microenvironment, compared with non-precursor bispecific antibodies. In some embodiments, the precursor bispecific antibody constructs described herein have reduced T-cell activation in non-tumor microenvironments, compared with non-precursor bispecific antibodies.

[0075] In some embodiments, the precursor bispecific antibody constructs described herein have an extended half-life in non-tumor microenvironments, compared with non-precursor bispecific antibodies. In some embodiments, the precursor bispecific antibody constructs described herein have reduced T-cell binding and/or activation in non-tumor microenvironments and an extended half-life in a non-tumor microenvironment, compared with non-precursor bispecific antibodies. [0076] In some embodiments, described herein are pharmaceutical compositions comprising a precursor bispecific antibody construct that provides a regulatable T-cell activation in non-tumor microenvironments. In some embodiments, described herein are pharmaceutical compositions comprising a precursor bispecific antibody construct having an increased half-life and providing that T-cell activation is restricted to a tumor microenvironment. In some embodiments, described herein are pharmaceutical compositions comprising a precursor bispecific antibody construct comprising an extended half-life in non-tumor microenvironments. In some embodiments, described herein are pharmaceutical compositions comprising a precursor bispecific antibody construct comprising an extended half-life in non-tumor microenvironments., wherein the half-life is reduced in a tumor microenvironment compared with the half-life in a non-tumor microenvironment.

[0077] In some embodiment, described herein are methods of use of a precursor bispecific antibody construct, as disclosed herein, for use treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or tumor in a subject, or any combination thereof.

Precursor Bispecific Antibody Constructs

[0078] In some embodiments, a precursor bispecific antibody constructs described herein comprises (a) a first binding domain, binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain, binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain. In some embodiments, a precursor bispecific antibody constructs described herein comprises (a) a first binding domain, binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain, binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain, wherein said regulatory domain comprises a cleavable half-life prolonging domain comprising a protease cleavage domain and a human serum albumin (HSA) polypeptide. In some embodiments, a precursor bispecific antibody constructs described herein comprises (a) a first binding domain, binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain, binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain, wherein said regulatory domain comprises a cleavable half-life prolonging domain comprising a protease cleavage domain, a human serum albumin (HSA) polypeptide, and a CAP component. In some embodiments, a precursor bispecific antibody constructs described herein comprises (a) a first binding domain, binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain, binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain, wherein said regulatory domain comprises a cleavable domain comprising a protease cleavage component and a CAP component.

[0079] In some embodiments, a precursor bispecific antibody constructs described herein comprises (a) an scFv fragment comprising a first binding domain, binding to a cell surface tumor associated antigen (TAA binding domain); (b) an Fab fragment comprising a second binding domain, binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain. In some embodiments, a precursor bispecific antibody constructs described herein comprises (a) an scFv fragment comprising a first binding domain, binding to a cell surface tumor associated antigen (TAA binding domain); (b) an Fab fragment comprising a second binding domain, binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain, wherein said regulatory domain comprises a cleavable half-life prolonging domain comprising a protease cleavage domain and a human serum albumin (HSA) polypeptide. In some embodiments, a precursor bispecific antibody constructs described herein comprises (a) an scFv fragment comprising a first binding domain, binding to a cell surface tumor associated antigen (TAA binding domain); (b) an Fab fragment comprising a second binding domain, binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain, wherein said regulatory domain comprises a cleavable half-life prolonging domain comprising a protease cleavage domain, a human serum albumin (HSA) polypeptide, and a CAP component.

[0080] A skilled artisan would appreciate that in some embodiments, a precursor antibody construct encompasses a precursor or derivative form of a pharmaceutically active antibody. In some embodiments, a medicinal preparation comprises a precursor antibody construct. In some embodiments, a formulation comprises a precursor antibody construct. In some embodiments, a precursor antibody construct has reduced adverse effects compared to the activated antibody. In some embodiments, a precursor antibody construct has reduced adverse effects compared to the activated antibody, wherein the precursor antibody may be enzymatically activated or converted into the active form of the antibody. In some embodiments, precursor bispecific antibody construct antibodies described herein are precursor bispecific antibody constructs.

[0081] In certain embodiments, a precursor antibody construct has a prolonged half-life compared to the activated antibody. In certain embodiments, a precursor antibody construct has a prolonged half-life compared to the activated antibody, wherein the precursor antibody may be enzymatically activated or converted into the active form of the antibody and the active form has a decreased half- life compared with the precursor antibody construct.

[0082] In some embodiments, a precursor antibody construct has reduced ability to bind a T-cell. In certain embodiments, a precursor antibody construct has a reduced ability to activate T-cells compared to the activated antibody. In some embodiments, a precursor antibody construct has reduced ability to bind a T-cell. In certain embodiments, a precursor antibody construct has a reduced ability to activate T-cells compared to the activated antibody, wherein the precursor antibody may be enzymatically activated or converted into the active form of the antibody.

[0083] In certain embodiments, a precursor antibody construct has both a prolonged half-life and a reduced ability to activate T-cells compared to the activated antibody. In certain embodiments, a precursor antibody construct has both a prolonged half-life and a reduced ability to bind T-cells compared to the activated antibody. In certain embodiments, a precursor antibody construct has both a prolonged half-life and a reduced ability to activate T-cells compared to the activated antibody, wherein the precursor antibody may be enzymatically activated or converted into the active form of the antibody. In certain embodiments, a precursor antibody construct has both a prolonged half-life and a reduced ability to bind T-cells compared to the activated antibody, wherein the precursor antibody may be enzymatically activated or converted into the active form of the antibody.

[0084] In some embodiments, a precursor antibody construct is synthesized in vitro. In some embodiments, a precursor antibody construct is not converted to an active form of the antibody, when the precursor is present in vivo (e.g., in circulation) in a non-tumor microenvironment.

[0085] A skilled artisan would appreciate that as used throughout, in some embodiments the terms “precursor bispecific antibody construct”,“precursor bispecific antibody construct”,“precursor antibody”,“precursor construct”,“precursor antibody construct”,“precursor bispecific antibody”, “bispecific antibody”,“antibody”,“bispecific antibody construct”, and“bispecific construct” may be used interchangeably having all the same qualities and meanings. In some embodiments, a precursor bispecific antibody construct is identified as a“PTMATE”, wherein stands for“Precursor Tumor Micro-Environment Activated T cell Engager”. In some embodiments, an activated PTMATE is identified as a“MATE”, a“Micro-Environment Activated T cell Engager”.

[0086] Bispecific antibodies can be designed to bind both a tumor cell antigen and a T-cell antigen for their goals to bind and kill tumor cells more selectively over normal cells, and ultimately to increase the efficacy and safety over a monospecific reagent, wherein a bispecific antibody comprises a binding domain binding a cell surface tumor associated antigen (TAA) and a binding domain binding an extracellular epitope of a T-cell. However, such bispecific antibodies fail to regulate the order of binding and therefore, may bind a T-cell prior to or in the absence of binding a cell surface TAA, wherein cytotoxicity provided by the activated T-cell may actually cause harmful side effects by non-specifically causing non-tumor cell death. In some embodiments, a cell surface TAA comprises a human antigen.

[0087] In some embodiments, a precursor antibody construct comprises a regulatory domain, in addition to antigen binding domains. In some embodiments, a precursor antibody construct comprises an enzymatically cleavable regulatory domain, in addition to antigen binding domains. In some embodiments, a precursor antibody construct comprises a regulatory domain in addition to antigen binding domains, wherein a portion of said regulatory domain is enzymatically cleavable.

[0088] In some embodiments, a precursor bispecific antibody described herein comprises enhanced selectivity at targeting tumor cells over normal cells prior to cytotoxic activation of T-cells. In some embodiments, a precursor bispecific antibody construct comprises a first binding domain binding to a cell surface tumor antigen associated antigen (TAA), a second binding domain binding to an extracellular epitope of a T-cell, for example an extracellular epitope of CD3e, and a third regulatory domain.

[0089] Immunological binding generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example by way of illustration and not limitation, as a result of electrostatic, ionic, hydrophilic and/or hydrophobic attractions or repulsion, steric forces, hydrogen bonding, van der Waals forces, and other interactions. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K _d ) of the interaction, wherein a smaller K _d represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (k _on ) can be determined by calculation of the concentrations and the actual rates of association and the "off rate constant" (k _off ) and can be determined by the actual rates of dissociation. The ratio of k _off /k _on is thus equal to the dissociation constant K _D . See, generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

[0090] A skilled artisan would appreciate that a "binding domain" or related expressions such as a domain that“binds” or has "reactivity with/to" a specific target encompasses the ability of the domain to discriminate between the respective antigens and to specifically associate with a target antigen. A "binding domain" or "binding region" according to the present disclosure may be, for example, any protein, polypeptide, oligopeptide, or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, or a component thereof, e.g., an extracellular component thereof). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest. For example, and as further described herein, a binding domain may be antibody light chain and heavy chain variable region regions, or the light and heavy chain variable region regions can be joined together in a single chain and in either orientation (e.g., VL- VH or VH-VL). A variety of assays are known for identifying binding domains of the present disclosure that specifically bind with a particular target, including Western blot, ELISA, flow cytometry, or surface plasmon resonance analysis (e.g., using BIACORE.TM. analysis).

[0091] In some embodiments, binding domain or a portion thereof "specifically binds" to a target molecule if it binds to or associates with a target molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of l/M) of, for example, greater than or equal to about HEM ¹ . In certain embodiments, a binding domain or a portion thereof binds to a target with a K _a greater than or equal to about 10 ⁶ M ¹ , 10 ⁷ M ¹ , 10 ⁸ M ¹ , 10 ⁹ M ¹ , 10 ¹⁰ M ¹ , 10 ¹¹ M ¹ , 10 ¹² M ¹ , or 10 ¹³ M ¹ , "High affinity" binding domains may encompass those binding domains with a K _a of at least 10 ⁷ M ¹ , at least 10 ⁸ M ¹ , at least 10 ⁹ M ¹ , at least 10 ¹⁰ M ¹ , at least 10 ¹¹ M ¹ , at least 10 ¹² M ¹ , at least 10 ¹³ M ¹ , or greater. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 ⁵ M to 10 ¹³ M, or less). Affinities of binding domain polypeptides and portions thereof, as described herein can be readily determined using conventional techniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173; 5,468,614, or the equivalent, which are incorporated herein in full).

[0092] Illustrative binding domains are described herein. In certain embodiments, the target molecule may be a cell surface expressed protein, such as a receptor or a tumor antigen. In some embodiments, the target molecule is a tumor associated antigen (TAA). Illustrative binding domains include immunoglobulin antigen-binding domains such as scFv, scTCR, extracellular domains of receptors, ligands for cell surface molecules/receptors, or receptor binding domains thereof, and tumor binding proteins. In certain embodiments, the antigen binding domains can be an scFv, a VH, a VL, a domain antibody variant (dAb), a camelid antibody (VHH), a fibronectin 3 domain variant, an ankyrin repeat variant and other antigen- specific binding domain derived from other protein scaffolds (Owen, B. (2017) Nat Biotechnol Jul l2:35(7):602-603).

[0093] Thus, in certain embodiments, a binding domain comprises an antibody-derived binding domain but can be a non-antibody derived binding domain. An antibody-derived binding domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen. Examples include, without limitation, a complementarity determining region (CDR), a variable region (Fv), a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain, a light chain, a single chain variable region (scFv), a Fab, a single domain camel antibody (camelid VHH), and single domain antibodies (dAb).

[0094] The present disclosure provides precursor bispecific antibody constructs comprising a first binding domain binding to a cell surface tumor associated antigen (TAA), for example but not limited to a TAA being an epidermal growth factor receptor (EGFR) antigen; and a second binding domain binding to an immune effector molecule, for example but not limited to a CD3 epsilon chain (CD3e) extracellular epitope; and a regulatory domain, for example but not limited to a regulatory domain comprising a cleavable half-life prolonging domain (Figures 1A-1C and 2A-2D).

[0095] In some embodiments, the first binding domain comprises a single chain variable fragment (ScFv). A skilled artisan would appreciate that a ScFv is not actually a fragment of an antibody, but instead is a fusion polypeptide comprising the variable heavy chain (VH) and variable light chain (VF) regions of an immunoglobulin, connected by a short linker peptide of ten to about 25 amino acids (Figures 1A-1C and Figures 2A-2D).

[0096] In some embodiments, the second binding domain comprises a Fab fragment, wherein the first binding domain is attached to the N-terminal end of the VF chain and the regulatory domain is attached at the N-terminal end of the VH chain. In some embodiments, the second binding domain comprises a Fab fragment, wherein the first binding domain is attached to the N-terminal end of the VH chain and the regulatory domain is attached at the N-terminal end of the VC chain. In some embodiments, between the scFv of the first binding domain and the VF of the second binding domain there may be a linker sequence. In some embodiments, between the scFv of the first binding domain and the VH of the second binding domain there may be a linker sequence. In some embodiments, between the regulatory domain and the VH chain of the second binding domain, there may be a linker sequence which is cleavable. In some embodiments, between the regulatory domain and the VF chain of the second binding domain, there may be a linker sequence which is cleavable. These general formats are the basic structure that can be built upon to construct the precursor bispecific antibody constructs described herein (Figures 1A-1C, and Figures 2A-2E).

[0097] In some embodiments, a regulatory domain comprises a protease cleavable linker component and a human serum albumin polypeptide (HSA) sequence component (Figures 1A-1C and 2A-2D). In some embodiments, a regulatory domain consists essentially of a protease cleavable linker component and a human serum albumin polypeptide (HSA) sequence component (Figure 2D). In some embodiments, the regulatory domain comprises a protease cleavable linker component, a human serum albumin polypeptide sequence component, and a CAP amino acid component (Figures 1A-1C, 2A— 2C, and 2E). In some embodiments, the regulatory domain consists essentially of a protease cleavable linker component, a human serum albumin polypeptide sequence component, and a CAP amino acid component (Figures 1A-1C, 2A-2C). In some embodiments, the regulatory domain comprises a protease cleavable linker component and a CAP amino acid component (Figures 1A-1C and 2E). In some embodiments, the regulatory domain consists essentially of a protease cleavable linker component and a CAP amino acid component (Figures 1C and 2E). The skilled artisan would appreciate that the presence of linkers, for example anyone of linkers L1-L7 in Figures 1A-1B and 2A-E, provide flexibility to a polypeptide while not necessarily providing essential regulatory feature to the regulatory domain, such as is provided by a CAP (a masking activity) or by an HSA component (increased half-life).

[0098] In some embodiments, the second binding domain anti-immune effector molecule, for example but not limited to an anti-CD3 epsilon chain (CD3e) extracellular epitope, binds specifically to the CAP amino acid component. In some embodiments, the CAP component effectively blocks binding of the precursor bispecific antibody construct with an immune effector target molecule, for example a T-cell. In some embodiments, activation of cytotoxicity to a target is specifically masked by the CAP component. In some embodiments, wherein the regulatory domain comprises a cleavable CAP component activation of cytotoxicity is limited to a tumor milieu (Figures 3A-3B). One embodiment of the origin of a first binding domain and the origin of a second binding domain is presented in Figure 3A (left hand-side).

[0099] In some embodiments, the CAP component comprises an amino acid sequence present within the human CD3 epsilon polypeptide chain. In some embodiments, the CAP component comprises an amino acid sequence present as part of the extracellular portion of the human CD3 epsilon chain. In some embodiments, the CAP component comprises an amino acid sequence selected from the amino acid sequence of the N-terminal end of human CD3 epsilon precursor polypeptide. In some embodiments, the CAP component comprises an amino acid sequence selected from the amino acid sequence of the N-terminal end of human CD3 epsilon mature polypeptide.

[00100] The amino acid sequence of the precursor human CD3 epsilon is set for in SEQ ID NO:

1.

MQS GTHWRVLGLCLLS V G VW GQDGNEEMGGITQTPYKVSIS GTT VILTCPQYPGSEILW QHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARV CEN CMEMD VMS VATI VIVDICIT GGLLLLV YYW S KNRKAKAKPVTRGAGAGGRQRGQ NKERPPP VPNPD YEPIRKGQRDLY S GLN QRRI (SEQ ID NO: 1). Human CD3 epsilon is expressed in a precursor form, wherein amino acids 1-21 form the signal peptide. The amino acid sequence of the mature human CD3 epsilon is set forth in amino acids 22-207 of SEQ ID NO: 2 as set forth herein in SEQ ID NO: 2.

QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSD EDH LS LKEFSELEQS GY YV C YPRGS KPED ANFYLYLRARV CEN CMEMD VMS V ATIVI VDICIT GGLLLLV YYW S KNRKAKAKP VTRGAGAGGRQRGQNKERPPP VPNPD YEPIRKGQRDL YSGLNQRRI (SEQ ID NO: 2) In some embodiments, the extracellular epitope of human CD3 epsilon is located within amino acids 1-26 of the precursor sequence, as set forth in SEQ ID NO: 3.MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEIL W QHNDKNIGGDEDDKNIGS DEDHLS LKEFSELEQS GY YV C YPRGS KPED ANF YLYLRA RVCENCMEMD (SEQ ID NO: 3). In some embodiments, the extracellular epitope of a mature human CD3 epsilon is set forth in amino acids 22-26 of the precursor sequence, and is set forth in SEQ ID NO: 103:

QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDH LS LKEFSELEQS GY YV C YPRGS KPED ANFYLYLRARV CEN CMEMD (SEQ ID NO: 103). In some embodiments, the extracellular epitope of human CD3 epsilon is located within amino acids QDGNEEMGGITQTP YKVSIS GTT VILT (SEQ ID NO: 4; AA1-27).

[00101] In some embodiments, the amino acid sequence of a CAP component is set forth in SEQ ID NO: 4, or a homolog thereof. In some embodiments, the amino acid sequence of a CAP component is a selected contiguous sequence within SEQ ID NO: 3, or a homolog thereof.

[00102] In some embodiments, homologues of SEQ ID NO: 4 or of a CAP sequence selected from SEQ ID NO: 3, comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence.

[00103] In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a human CD3 epsilon polypeptide or a portion thereof, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[00104] A skilled artisan would appreciate that the term“homology”, and grammatical forms thereof, encompasses the degree of similarity between two or more structures. The term “homologous sequences” refers to regions in macromolecules that have a similar order of monomers. When used in relation to nucleic acid sequences, the term“homology” refers to the degree of similarity between two or more nucleic acid sequences (e.g., genes) or fragments thereof. Typically, the degree of similarity between two or more nucleic acid sequences refers to the degree of similarity of the composition, order, or arrangement of two or more nucleotide bases (or other genotypic feature) of the two or more nucleic acid sequences. The term“homologous nucleic acids” generally refers to nucleic acids comprising nucleotide sequences having a degree of similarity in nucleotide base composition, arrangement, or order. The two or more nucleic acids may be of the same or different species or group. The term“percent homology” when used in relation to nucleic acid sequences, refers generally to a percent degree of similarity between the nucleotide sequences of two or more nucleic acids.

[00105] When used in relation to polypeptide (or protein) sequences, the term“homology” refers to the degree of similarity between two or more polypeptide (or protein) sequences (e.g., genes) or fragments thereof. Typically, the degree of similarity between two or more polypeptide (or protein) sequences refers to the degree of similarity of the composition, order, or arrangement of two or more amino acid of the two or more polypeptides (or proteins). The two or more polypeptides (or proteins) may be of the same or different species or group. The term“percent homology” when used in relation to polypeptide (or protein) sequences, refers generally to a percent degree of similarity between the amino acid sequences of two or more polypeptide (or protein) sequences. The term“homologous polypeptides” or“homologous proteins” generally refers to polypeptides or proteins, respectively, that have amino acid sequences and functions that are similar. Such homologous polypeptides or proteins may be related by having amino acid sequences and functions that are similar, but are derived or evolved from different or the same species using the techniques described herein.

[00106] In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a polypeptide or a portion thereof disclosed herein, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. In some embodiments, homologues comprise a nucleotide sequences which is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a nucleotide sequence or a portion thereof disclosed herein, as determined using BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters.

[00107] In some embodiments, homology also encompasses deletion, insertion, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. In one embodiment, the variant comprises conservative substitutions, or deletions, insertions, or substitutions that do not significantly alter the three-dimensional structure of the polypeptide component of interest described herein. In some embodiments, the deletion, insertion, or substitution does not alter the function of interest of the polypeptide component of interest disclosed herein.

[00108] In some embodiments, homology also encompasses deletion, insertion, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. In one embodiment, the variant comprises conservative substitutions, or deletions, insertions, or substitutions that do not significantly alter the three-dimensional structure of the CAP component, e.g., the portion of a human CD3 epsilon polypeptide present in the CAP component, particularly in the areas of the epitope recognized and bound by the second binding domain. In some embodiments, the deletion, insertion, or substitution does not alter the function of interest of the CAP component, which in some embodiment, is binding to the second binding domain, or reducing T-cell binding, or reducing T-cell activation, or any combination thereof.

[00109] In some embodiments, the CAP component is 6-110 amino acids long. In some embodiments, the CAP component is between about 6-10 amino acids long. In some embodiments, the CAP component is between about 10-20 amino acids long. In some embodiments, the CAP component is between about 20-30 amino acids long. In some embodiments, the CAP component is between about 20-40 amino acids long. In some embodiments, the CAP component is between about 30-40 amino acids long. In some embodiments, the CAP component is between about 40-60 amino acids long. In some embodiments, the CAP component is between about 60-80 amino acids long. In some embodiments, the CAP component is between about 80-100 amino acids long. In some embodiments, the CAP component is between about 80-110 amino acids long.

[00110] In some embodiments, the CAP component is 6 amino acids long. In some embodiments, the CAP component is 7 amino acids long. In some embodiments, the CAP component is 8 amino acids long. In some embodiments, the CAP component is 9 amino acids long. In some embodiments, the CAP component is 10 amino acids long. In some embodiments, the CAP component is 11 amino acids long. In some embodiments, the CAP component is 12 amino acids long. In some embodiments, the CAP component is 13 amino acids long. In some embodiments, the CAP component is 14 amino acids long.

[00111] In some embodiments, the CAP component is 15 amino acids long. In some embodiments, the CAP component is 16 amino acids long. In some embodiments, the CAP component is 17 amino acids long. In some embodiments, the CAP component is 18 amino acids long. In some embodiments, the CAP component is 19 amino acids long. In some embodiments, the CAP component is 20 amino acids long. In some embodiments, the CAP component is 21 amino acids long. In some embodiments, the CAP component is 22 amino acids long. In some embodiments, the CAP component is 23 amino acids long. In some embodiments, the CAP component is 24 amino acids long. In some embodiments, the CAP component is 25 amino acids long. In some embodiments, the CAP component is 26 amino acids long. In some embodiments, the CAP component is 27 amino acids long. In some embodiments, the CAP component is 28 amino acids long. In some embodiments, the CAP component is 29 amino acids long. In some embodiments, the CAP component is 30 amino acids long. In some embodiments, the CAP component is 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids long.

[00112] In some embodiments, the CAP component specifically binds to the second binding region, thereby reducing T-cell binding of the precursor construct. In some embodiments, the CAP component specifically binds to the second binding region, thereby inhibiting T-cell binding of the precursor construct. In some embodiments, the CAP component specifically binds to the second binding region, thereby reducing T-cell activation the precursor construct. In some embodiments, the CAP component specifically binds to the second binding region, thereby inhibiting T-cell activation by the precursor construct.

[00113] In some embodiments, a regulatory domain comprises a cleavable half-life prolonging domain. In some embodiments, a cleavable half-life prolonging domain comprises an HSA polypeptide. In some embodiments, a cleavable half-life prolonging domain comprises a CAP component that reduces the ability of the second binding domain to bind the extracellular epitope of human CD3 epsilon. In some embodiments, a cleavable half-life prolonging domain comprises an HSA polypeptide and a CAP component that reduces the ability of the second binding domain to bind the extracellular epitope of human CD3 epsilon.

[00114] In some embodiments, there is a linker between the components of the regulatory domain. In some embodiments, there is a linker between the regulatory domain and the N-terminus of the VH chain of the Fab fragment. In some embodiments, a linker between components of the regulatory domain is a cleavable linker. In some embodiments, any of the linkers between components of the regulatory domain is a cleavable linker. In some embodiments, a linker between components of the regulatory domain is not cleavable. In some embodiments, a linker between components of the regulatory domain is not cleavable (Figures 1A-1B (non-CP linkers), 2A-2E (non-CP linkers), and 4C).

[00115] In some embodiments, the regulatory domain comprises a cleavable half-life prolonging domain comprising a protease cleavable domain and a human serum albumin polypeptide (HSA). In some embodiments, the order of components in the regulatory domain is (N-terminal to C- terminal) HSA-L-protease cleavable domain, wherein L is a possible linker amino acid sequence (Figure 2D). In some embodiments, wherein the protease cleavable domain is C-terminal to an HSA polypeptide sequence, the precursor construct has a regulatable enhance half-life wherein the precursor construct has an enhanced half-life in circulation in vivo and in the absence of a tumor microenvironment. In some embodiments, the order of components in the regulatory domain is (N- terminal to C-terminal) protease cleavable domain-L-HSA, wherein L is a possible linker amino acid sequence. Thus, in some embodiments, the HSA is located N-terminal to the protease cleavable domain, and in other embodiments, the HSA is located C-terminal to the protease cleavable domain. In some embodiments, there is a linker between the regulatory domain and the N-terminus of the VH chain of the Fab fragment (Figures 1A-1C and 2A-2E).

[00116] In some embodiments, the regulatory domain comprises a cleavable half-life prolonging domain comprising a protease cleavable domain, a human serum albumin polypeptide (HSA), and a CAP amino acid component that binds with the anti-immune effector molecule second binding domain (Figures 1A-1C and 2A-2C). In some embodiments, the order of components in the regulatory domain is (N-terminal to C-terminal) CAP-L-HSA-L-protease cleavable domain, wherein L is a possible linker amino acid sequence (Figures 1A-1B and 2A-2B). In some embodiments, wherein the protease cleavable domain is C-terminal to an HSA polypeptide sequence and to a CAP component, the precursor construct has a regulatable enhance half-life wherein the precursor bispecific antibody construct has an enhanced half-life and is effectively blocked from binding with an immune effector target molecule. In some embodiments, wherein the protease cleavable domain of a regulatory domain is C-terminal to an HSA polypeptide sequence and to a CAP component, activation of cytotoxicity to target is specifically masked by the CAP component of the precursor construct and the precursor construct comprises an enhance half-life in circulation in vivo and in the absence of a tumor milieu. In some embodiments, wherein the regulatory domain comprises a protease cleavable domain, a CAP component, and an HSA component, and the protease cleavable domain of a regulatory domain is C-terminal to the HSA polypeptide sequence and to the CAP component, activation of cytotoxicity is limited to the tumor milieu.

[00117] In some embodiments, wherein the protease cleavable domain is N-terminal to an HSA polypeptide sequence, the precursor construct has an enhance half-life in circulation in vivo, and in the absence or presence of a tumor microenvironment (Figure 2C).

[00118] In some embodiments, the order of components in the regulatory domain is (N-terminal to C-terminal) CAP-L-protease cleavable domain-L-HSA, wherein L is a possible linker amino acid sequence (Figure 2C). In some embodiments, wherein the protease cleavable domain is N-terminal to an HSA polypeptide sequence, the precursor construct maintains an enhanced half-life in circulation in vivo and in the presence of a tumor microenvironment. In some embodiments, wherein the protease cleavable domain is C-terminal to the CAP component and N-terminal to an HSA polypeptide sequence, the precursor construct maintains an enhance half-life in circulation in vivo and in the presence of a tumor microenvironment and is effectively blocked from binding with an immune effector target molecule in circulation in vivo and within a non-tumor milieu. In some embodiments, wherein the protease cleavable domain is C-terminal to the CAP component and N- terminal to an HSA polypeptide sequence, activation of cytotoxicity to target is specifically masked by the CAP component of the precursor construct in circulation and in the absence of a tumor milieu, and the precursor construct comprises an enhance half-life in circulation in vivo and in the absence or presence of a tumor milieu. In some embodiments, wherein the regulatory domain comprises a protease cleavable domain, a CAP component, and an HSA component, and the protease cleavable domain of a regulatory domain is N-terminal to the HSA polypeptide sequence and C-terminal to the CAP component, the precursor construct has an extended half-life while activation of cytotoxicity is limited to the tumor milieu. In some embodiments, there is a linker between the regulatory domain and the N-terminus of the VH chain of the second binding domain. In some embodiments, there is a linker between the regulatory domain and the N-terminus of the VL chain of the second binding domain. In some embodiments, there is a linker between the regulatory domain and the N-terminus of the VH chain of the Fab fragment. In some embodiments, there is a linker between the regulatory domain and the N-terminus of the VL chain of the Fab fragment.

[00119] In some embodiments, the regulatory domain comprises a protease cleavable domain and a CAP component that binds to the anti-immune effector molecule that effectively blocks binding of the precursor construct with an immune effector target molecule (Figure 2E). In some embodiments, activation of cytotoxicity to target is specifically masked by the CAP component in circulation and in a non-tumor milieu. In some embodiments, wherein the regulatory domain comprises a cleavable CAP component as for Figures 1A-1C, 2A, 2B, 2C, and 2E, activation of cytotoxicity is limited to the tumor milieu.

[00120] In some embodiments, the amino acid sequence of the HSA component is set forth in SEQ ID NO: 5.

SEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAEN C DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEV DVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACL LPKLDELRDEGKAS S AKQRLKC AS LQKFGERAFKAW A V ARLS QRFPKAEF AE V S KLVT DLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEN DEMP ADLPS LAADF VES KD V CKNY AE AKD VFLGMFLYE Y ARRHPD Y S VVLLLRLAKT YETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRY TKKVPQVSTPTLVEVSRNLGKV GSKCCKHPE AKRMPC AED YLS VVLN QLC VLHEKTPV SDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTAL VELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAA (SEQ ID NO: 5) In some embodiments, the amino acid sequence of the HSA component is set forth in SEQ ID NO: 116.

E VAHRFKDLGEENFKALVLI AFAQYLQQCPFEDHVKLVNE VTEF AKT C V ADES AEN CD KSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVD VMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDE MPADLPSLAADFVES KD VCKNY AE AKDVFLGMFLYEY ARRHPD YS VVLLLRLAKT YE TTLEKCC A AADPHEC Y AKVFDEFKPLVEEPQNLIKQN CELFEQLGE YKF QN ALLVRYTK KVPQVSTPTLVE V S RNLGKV GS KCCKHPE AKRMPC AED YLS VVLN QLC VLHEKTPV S D RVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVE LVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAA (SEQ ID NO: 116).

[00121] In some embodiments, the amino acid sequence of the HSA components is any HSA polypeptide sequence known in the art or a portion thereof, or a homolog thereof. In some embodiments, the HSA component of a precursor bispecific antibody construct comprises, for example but not limited to, any human albumin protein sequence disclosed in a known database such as the protein data base the is part of National Center of Biotechnology Information (NCBI) or Swiss-Prot, wherein the sequence might be identified specifically as human or may be identified as a synthetic construct.

[00122] In some embodiments, the HSA component is encoded by the nucleotide sequence set forth in SEQ ID NO: 119. gaggtggcccacaggttcaaggatctgggcgaggagaacttcaaggccctggtgctgatc gccttcgcccagtatctgcagcagtgcccctt tgaggaccacgtgaagctggtgaacgaggtgaccgagttcgccaagacatgcgtggccga cgagtccgccgagaattgtgataagtctctg cacaccctgtttggcgataagctgtgcaccgtggccacactgagggagacatacggcgag atggccgactgctgtgccaagcaggagccc gagcgcaacgagtgcttcctgcagcacaaggacgataaccctaatctgccacggctggtg agacctgaggtggacgtgatgtgcaccgcct tccacgataatgaggagacatttctgaagaagtacctgtatgagatcgcccggagacacc cttacttttatgccccagagctgctgttctttgcc aagcggtacaaggcagccttcaccgagtgctgtcaggcagcagataaggcagcatgcctg ctgccaaagctggacgagctgagggatga gggcaaggcaagctccgccaagcagcgcctgaagtgtgcaagcctgcagaagttcggaga gagggcctttaaggcatgggcagtggca aggctgtcccagcggttcccaaaggccgagtttgccgaggtgtctaagctggtgaccgac ctgacaaaggtgcacaccgagtgctgtcacg gcgacctgctggagtgcgcagacgatagagccgatctggccaagtacatctgtgagaacc aggactctatctctagcaagctgaaggagtg ctgtgagaagcccctgctggagaagtcccactgcatcgccgaggtggagaacgacgagat gccagcagatctgccaagcctggcagcag acttcgtggagtccaaggacgtgtgcaagaattacgccgaggccaaggacgtgttcctgg gcatgtttctgtacgagtatgccaggcgccac cctgactactccgtggtgctgctgctgcggctggccaagacctatgagaccacactggag aagtgctgtgccgccgccgacccccacgagt gctatgcaaaggtgttcgacgagtttaagcccctggtggaggagcctcagaacctgatca agcagaattgtgagctgtttgagcagctgggc gagtacaagttccagaacgccctgctggtgagatataccaagaaggtgccacaggtgtct acccccacactggtggaggtgagccggaatc tgggcaaggtcggctccaagtgctgtaagcaccctgaggccaagagaatgccatgcgccg aggattacctgtccgtggtgctgaaccagct gtgcgtgctgcacgagaagacccccgtgagcgacagggtgaccaagtgctgtacagagtc tctggtgaaccggagaccatgctttagcgc cctggaggtggatgagacatatgtgcccaaggagttcaatgccgagaccttcacatttca cgccgacatctgtaccctgagcgagaaggagc gccagatcaagaagcagacagccctggtggagctggtgaagcacaagccaaaggccacca aggagcagctgaaggccgtgatggacg atttcgccgcctttgtggagaagtgctgtaaggccgacgataaggagacatgcttcgcag aggagggcaagaagctggtggcagcaagcc aggcagca (SEQ ID NO: 119).

[00123] In some embodiments, the nucleic acid sequence of the HSA components is any HSA nucleotide sequence known in the art or a portion thereof, or a homolog thereof. In some embodiments, the HSA component of a precursor bispecific antibody construct comprises a nucleic acid sequence that encodes, for example but not limited to, any human albumin protein sequence disclosed in a known database such as the protein data base the is part of National Center of Biotechnology Information (NCBI) or Swiss-Prot, wherein the sequence might be identified specifically as human or may be identified as a synthetic construct

[00124] In some embodiments, homologues of an HSA component comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence. In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to an HSA polypeptide or a portion thereof, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. In some embodiments, homologues encoding an HSA component comprise nucleotides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the nucleic acid sequence. In some embodiments, homologues encode polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to an HSA polypeptide or a portion thereof, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[00125] In some embodiments, homology also encompasses deletion, insertion, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. In one embodiment, the variant comprises conservative substitutions, or deletions, insertions, or substitutions that do not significantly alter the three-dimensional structure of the HSA component. In some embodiments, the deletion, insertion, or substitution does not alter the function of interest of the HSA component, which in some embodiment, is providing half-life prolonging domain.

[00126] Linear representation of embodiments of regulatory domains of a precursor bispecific antibody construct disclosed herein include but are not limited to (N-terminal to C-terminal)

(1) CAP-L-HSA-L-protease cleavable domain-L, wherein the L may or may not be present;

(2) CAP-L-protease cleavable domain-L-HSA-L, wherein the L may or may not be present;

(3) HSA-L-protease cleavable domain-L, wherein the L may or may not be present; and

(4) CAP-L-protease cleavable domain-L, wherein the L may or may not be present.

[00127] In some embodiments, a precursor bispecific antibody construct disclosed herein comprises a precursor construct having an increased therapeutic window, wherein its restricted presence provides the ability to target a wide array of new targets or provide improved activities or a combination thereof, for example but not limited to, the ability to activate T-cells only in the cancer microenvironment and targeting cancer-specific TAAs depending on a cancer type and the specific TAAs that are uniquely expressed by this cancer type in conjunction with the proteases produced by this cancer type.

[00128] As used herein, the "C-terminal" of a polypeptide and the like, e.g., carboxyl- terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein is translated from messenger RNA, it is created from N-terminus to C-terminus. The convention for writing peptide sequences is to put the C-terminal end on the right and write the sequence from N- to C-terminus. In some embodiments, the C-terminal end of a polypeptide encompasses to the last amino acid residue of the polypeptide which donates its amine group to form a peptide bond with the carboxyl group of its adjacent amino acid residue.

[00129] As used herein, the "N-terminal" of a polypeptide and the like, e.g., amino- terminus, Nth-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide referring to the free amine group (-NH ₂ ) located at the end of a polypeptide. Normally the amine group is bonded to another carboxylic group in a protein to make it a chain, but since the end of a protein has only 1 out of 2 areas chained, the free amine group is referred to the N-terminus. As stated above, by convention, peptide sequences are written N- terminus to C-terminus, left to right in LTR languages. This correlates the translation direction to the text direction (because when a protein is translated from messenger RNA, it is created from N- terminus to C-terminus - amino acids are added to the carbonyl end). In some embodiments, the N- terminal end of a polypeptide encompasses the first amino acid of the polypeptide which donates its carboxyl group to form a peptide bond with the amine group of its adjacent amino acid residue.

[00130] A skilled artisan would appreciate that a linker component may encompass an amino acid peptide linking through one or more chemical bonds or indirect linking through one or more linkers. Any suitable chemical bonds can be used to make a direct link, including without limitation, covalent bonds such as peptide bond and disulfide bond, non-covalent bonds such as hydrogen bond, hydrophobic bond, ionic bond, and Van der Waals bond.

[00131] A "covalent bond" refers herein to a stable association between two atoms which share one or more electrons. Examples of the covalent bonds include, without limitation, a peptide bond and a disulfide bond. "Peptide bond" as used herein refers to the covalent bond formed between the carboxyl group of an amino acid and the amine group of the adjacent amino acid. "Disulfide bond" as used herein refers to a covalent bond formed between two sulfur atoms. A disulfide bond can be formed from oxidation of two thiol groups. In certain embodiments, the covalently link is direct link through a covalent bond. In certain embodiments, the covalently link is direct link through a peptide bond or a disulfide bond.

[00132] A "non-covalent bond" refers herein to an attractive interaction between two molecules or two chemical groups that does not involve sharing of electrons. Examples of non-covalent bonds include, without limitation, a hydrogen bond, a hydrophobic bond, an ionic bond, and a Van der Waals bond. A "hydrogen bond" refers herein to attractive force between a hydrogen atom of a first molecule/group and an electronegative atom of a second molecule/group. A "hydrophobic bond" refers herein to a force that causes hydrophobic or non-polar molecules/groups to aggregate or associate together in an aqueous environment. An "ionic bond" refers herein to an attraction between a positive ion and a negative ion. A "Van der Waals bond" refers herein to a non-specific attraction force between two adjacent molecules/groups which have momentary random fluctuations in the distribution of electrons. In certain embodiments, the covalently link is direct link through a non-covalent bond. In certain embodiments, the covalently link is direct link through a hydrogen bond, a hydrophobic bond, an ionic bond, or a Van der Waals bond.

[00133] A skilled artisan would appreciate that a protease cleavable domain described herein encompasses linker comprising a protease cleavage site. Thus, the terms“protease cleavable domain” and protease cleavable linker” may be used interchangeably herein having all the same meanings and qualities.

[00134] A skilled artisan would appreciate that the terms“tumor microenvironment”, cancer microenvironment” and“tumor milieu” may be used interchangeably having the same qualities and meanings and encompassing the microenvironment to tumor development. While the normal cellular microenvironment can inhibit malignant cell growth, the modifications that occur in the tumor microenvironment may synergistically support cell proliferation.

[00135] Tumors shape their microenvironment and support the development of both tumor cells and non-malignant cells. The tumor microenvironment affects angiogenesis by interfering with the signaling pathways required for cell recruitment and vascular construction. Endothelial progenitor cells (EPCs) that are recruited under hypoxic conditions for angiogenesis have been associated as well with metastasis. In some embodiments, TAA comprise cell surface antigens associated with angiogenesis. In some embodiments, a TAA is overexpressed by a cancer cell. In some embodiments, a TAA is expressed on an embryonic cell. In some embodiments, a TAA is expressed on an embryonic cell and on a cancer cell but has no or only minimal expression on normal adult cells. In some embodiments, a TAA is expressed on a solid tumor cell. In some embodiments, a TAA is expression on a non-solid cancerous cell. In some embodiments, a TAA is expressed on an angiogenic tissue cell.

[00136] In addition proteins secreted by the tumor modify the microenvironment by contributing growth factors and proteases that degrade the extracellular matrix and affect cell motility and adhesion. Stromal cells secrete ECM proteins, cytokines, growth factors, proteases, protease inhibitors, and endoglycosidases such as heparanase. Matrix metalloproteinases (MMP) are important secreted proteins closely associated with cancer development. MMP are expressed at higher levels by tumor-associated epithelial cells than by normal epithelial cells. In some embodiments, the microenvironment of a tumor comprises increased protease activity compared with a non-tumor environment.

[00137] Figures 3A and 3B provide non-limiting examples of a precursor bispecific antibody construct that may have an enhanced half-live in vivo in circulation and while present in a non tumor environment. Further, the anti-CD3 second binding domain of the precursor bispecific antibody construct is blocked and may not interact or bind with a target T-cell while the precursor construct is in a non-tumor environment. In some embodiments, as is shown in Figure 3B, the cancer microenvironment provides protease cleavage of a precursor bispecific antibody construct, which removes the half-life extending component (HSA) and the CD3e CAP component, leading to the presence of an activated EGFR x CD3e antibody (Figure 2F) and T-cell activation. Figures 2A - 2E present embodiments, wherein the TAA is EGFR (first binding domain is an anti-EGFR scFv). A skilled artisan would appreciate that in other embodiments, the TAA may be another TAA known in the art, for example but not limited to a TAA comprising a 5T4 or an ROR1, wherein the first binding domain would comprise an anti-5T4 or anti-RORl scFv, respectively. In other embodiments, the TAA may be any TAA known in the art, for example but not limited to FcyRI, FcyRIIa FcyRIIb FcyRIIIa FcyRIIIb, CD28, CD137, CTLA-4, FAS, fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, glucocorticoid-induced TNFR-related (GITR) protein, lymphotoxin-beta receptor (LTpR), toll -like receptors (TER), tumor necrosis factor-related apoptosis-inducing ligand-receptor 1 (TRAIF receptor 1) and TRAIF receptor 2, pro state- specific membrane antigen (PSMA) protein, prostate stem cell antigen (PSCA) protein, tumor-associated protein carbonic anhydrase IX (CAIX), epidermal growth factor receptor 1 (EGFR1), EGFRvIII, human epidermal growth factor receptor 2 (Her2/neu; Erb2), ErbB3 also known as HER3, Folate receptor, ephrin receptors, PDGFRa, ErbB-2, CD20, CD22, CD30, CD33, CD40, CD37, CD38, CD70, CD74, CD56, CD40), CD80, CD86, CD2, p53, cMet also known as tyrosine-protein kinase Met or hepatocyte growth factor receptor (HGFR), MAGE-A1, MAGE-A2, MAGE-A3, MAGE- A4, MAGE-A6, MAGE- A 10, MAGE-A12, BAGE, DAM-6, -10, GAGE-l, -2, -8, GAGE-3, -4, - 5, -6, -7B, NA88-A, NY-ESO-l, BRCA1, BRCA2, MART-l, MC1R, GplOO, PSA, PSM, Tyrosinase, Wilms' tumor antigen (WT1), TRP-l, TRP-2, ART -4, CAMEL, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, P-cadherin, Myostatin (GDF8), Cripto (TDGF1), MUC5AC, PRAME, P15, RU1, RU2, SART-l, SART-3, WT1, AFP, b-catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-l, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin P, CDC27/m, TPEmbcr-abl, ETV6/AML, LDLR/FUT, Pml/RARa, TEL/AML 1, CD28, CD137, CanAg, Mesothelin, DR5, PD-l, PD1L, IGF-1R, CXCR4, Neuropilin 1, Glypicans, EphA2, CD138, B7-H3, gpA33, GPC3, SSTR2, ROR1, 5T4, or a VEGF-R2. In some embodiments, a TAA comprises a PSMA, CD30, B7-H3, gpA33, HER2, P- cadherin, gplOO, DR5, GPC3, SSTR2, Mesothelin, ROR1, 5T4, Folate receptor, or an EGFR. In some embodiments, a TAA comprises an EGFR. In some embodiments, a TAA comprises a ROR1. In some embodiments, a TAA comprises an PSMA. In some embodiments, a TAA comprises an 5T4.

[00138] In some embodiments, the amino acid sequence of a first binding region comprises a Tumor Associated Antigen (TAA) binding activity, comprising any anti-TAA sequence known in the art. In some embodiments, the amino acid sequence of a first binding region comprises a Tumor Associated Antigen (TAA) binding activity, comprising an EGFR anti-TAA sequence known in the art. In some embodiments, the amino acid sequence of a first binding region comprises a Tumor Associated Antigen (TAA) binding activity, comprising a 5T4 anti-TAA sequence known in the art. In some embodiments, the amino acid sequence of a first binding region comprises a Tumor Associated Antigen (TAA) binding activity, comprising a ROR1 anti-TAA sequence known in the art.

[00139] In some embodiments, a protease cleavable domain comprises a protease cleavable amino acid sequence (cleavable peptide/cleavable linker; CP) comprises a peptide cleavable by a serine protease, a cysteine protease, an aspartate protease, or a matrix metalloprotease (MMP) cleavable sequence. In some embodiments, the serine protease, cysteine protease, aspartate protease, or matrix metalloprotease (MMP) is expressed at higher levels in a tumor microenvironment. In some embodiments, the matrix metalloprotease is expressed at higher levels in a tumor microenvironment.

[00140] In some embodiments, the protease cleavable sequence is an MMP cleavable sequence. In some embodiments, the matrix metalloprotease cleavable sequence may be a matrix metalloprotease 1 (MMP-l), a matrix metalloprotease 2 (MMP-2), a matrix metalloprotease 9 (MMP-9), or a matrix metalloprotease 14 (MMP-14) cleavable sequence.

[00141] In some embodiments, the protease cleavable domain comprises an amino acid sequence 1 to 10 amino acids long. In some embodiments, the protease cleavable domain is 1 to 20 amino acids long.

[00142] In some embodiments, a protease cleavable domain comprises a protease substrate cleavage sequence, for example but not limited to, an MMP substrate cleavage sequence. A well- known peptide sequence of PLGLAG (SEQ ID NO: 6) in a substrate can be cleaved by most MMPs. Substrate sequences that can be cleaved by MMPs have been extensively studied. A protease substrate cleavage sequence refers to a peptide sequence that can be cleaved by protease treatment. An MMP substrate sequence refers to a peptide sequence that can be cleaved by incubation with an MMP. PLGLAG (SEQ ID NO: 6) is a commonly used MMP substrate cleavage sequence (see e.g., Jiang, PNAS (2004) 101:17867-72; Olson, PNAS (2010) 107:4311-6). In another embodiment, the protease cleavage site is recognized by MMP-2, MMP-9, or a combination thereof. In yet another embodiment, the protease site comprises the sequence GPLGMLSQ (SEQ ID NO: 7), GPLGLWAQ (SEQ ID NO: 8), GPLGLAG (SEQ ID NO: 101), KKNPAELIGPVD (SEQ ID NO: 104), or KKQPAANLVAPED (SEQ ID NO: 105). In some embodiments, the protease cleavage site comprises any protease cleavage site (protease cleavable peptide; CP) known in the art to be susceptible to proteases present in a tumor environment, for example by not limited to the protease cleavage sites disclosed in Eckhard, U, et al., (2016) Matrix Biol. Jan;49:37-60.

[00143] In some embodiments, a cleavable linker comprises multiple cleavage sites for at least two different proteases. In some embodiments, a cleavable linker comprises multiple cleavage sites for at least an MMP protease and a Matriptase/uPA/Legumain protease. In some embodiments, a cleavable linker comprising multiple cleavage sites for different proteases, comprises the amino acid sequence set forth in SEQ ID NO: 146.

[00144] In some embodiments, the term“protease cleavage peptides” and grammatical versions thereof, is interchangeable with the term“linker”, as a linker may in certain embodiments comprise a cleavable linker. In other embodiments, linkers are non-cleavable, for example but not limited to those linkers between a VH and VL domain in an scFv.

[00145] In some embodiments, a cleavable peptide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 121.

[00146] A stable linker or a protease non-cleavable linker refers to a linker peptide sequence that does not belong to the known protease substrate sequences and thus does not lead to significant cleavage product formation upon incubation with a protease.

[00147] In some embodiments, the cleavage substrate (or cleavage sequence) of the linker may include an amino acid sequence that can serve as a substrate for a protease, usually an extracellular protease. In other embodiments, the cleavage sequence comprises a cysteine-cysteine pair capable of forming a disulfide bond, which can be cleaved by action of a reducing agent. In other embodiments the cleavage sequence comprises a substrate capable of being cleaved upon photolysis.

[00148] The cleavage substrate is positioned within the protease cleavable domain such that when the cleavage substrate is cleaved by a cleaving agent (e.g., a cleavage substrate of a linker is cleaved by the protease and/or the cysteine-cysteine disulfide bond is disrupted via reduction by exposure to a reducing agent) or by light-induced photolysis, in the presence of a target, resulting in cleavage products having various functional properties as described herein. In some embodiments, cleavage products have decreased half-life. In some embodiments, cleavage product has the ability to activate T-cell (Figures 3A-3B).

[00149] The cleavage substrate of a cleavage domain may be selected based on a protease that is co-localized in the diseased tissue, or on the surface of the cell that expresses the target antigen of interest of a binding domain of a fusion moiety. A variety of different conditions are known in which a target of interest is co-localized with a protease, where the substrate of the protease is known in the art. In the example of cancer, the target tissue can be a cancerous tissue, particularly cancerous tissue of a solid tumor. There are reports in the literature of increased levels of proteases having known substrates in a number of cancers, e.g., solid tumors. See, e.g., [La Rocca et al, (2004) British J. of Cancer 90(7): 1414-1421. Radisky ES, Front Biosci (Landmark Ed). 2015 Jun l;20: 1144-63; Miao C, et ah, Oncotarget. 2017 May 9;8(l9):32309-3232l]. Non-limiting examples of disease include: all types of cancers (breast, lung, colorectal, prostate, head and neck, pancreatic, etc), rheumatoid arthritis, Crohn's disease, melanomas, SLE, cardiovascular damage, ischemia, etc. Furthermore, anti-angiogenic targets, such as VEGF, are known.

[00150] In some embodiments, where the TAA of the first binding domain is selected such that it is capable of binding a tumor antigen, a suitable cleavage substrate sequence for the linker will be one which comprises a peptide substrate that is cleavable by a protease that is present at the cancerous treatment site, tumor microenvironment that is particularly present at elevated levels at the cancer treatment site as compared to non-cancerous tissues.

[00151] In some embodiments, the first binding domain of a precursor construct disclosed herein can bind a TAA, e.g., EGFR and the cleavage substrate sequence can be a matrix metalloprotease (MMP) substrate, and thus is cleavable by an MMP. In some embodiments, the first binding domain of a precursor construct disclosed herein can bind a 5T4 TAA and the cleavage substrate sequence can be a matrix metalloprotease (MMP) substrate, and thus is cleavable by an MMP. In some embodiments, the first binding domain of a precursor construct disclosed herein can bind a ROR1 TAA and the cleavage substrate sequence can be a matrix metalloprotease (MMP) substrate, and thus is cleavable by an MMP. In other embodiments, a TAA comprises EGFR and the cleavage substrate sequence can be a matripase (MT-SP1, TADG-15, epithin, ST14) substrate, and thus is cleavable by a matriptase. In other embodiments, a TAA comprises 5T4 and the cleavage substrate sequence can be a matripase (MT-SP1, TADG-15, epithin, ST 14) substrate, and thus is cleavable by a matriptase. In other embodiments, a TAA comprises ROR1 and the cleavage substrate sequence can be a matripase (MT-SP1, TADG-15, epithin, ST 14) substrate, and thus is cleavable by a matriptase. In other embodiments, a TAA comprises EGFR and the cleavage substrate sequence comprises an MMP substrate and a matripase (MT-SP1, TADG-15, epithin, ST 14) substrate, and thus is cleavable by an MMP or a matriptase. In other embodiments, a TAA comprises 5T4 and the cleavage substrate sequence comprises an MMP substrate and a matripase (MT-SP1, TADG-15, epithin, ST14) substrate, and thus is cleavable by an MMP or a matriptase. In other embodiments, a TAA comprises ROR1 and the cleavage substrate sequence comprises an MMP substrate and a matripase (MT-SP1, TADG-15, epithin, ST14) substrate, and thus is cleavable by an MMP or a matriptase. In other embodiments, a TAA comprises any TAA disclosed herein, and the cleavage substrate sequence comprises an MMP substrate and a matripase (MT- SP1, TADG-15, epithin, ST14) substrate, and thus is cleavable by an MMP or a matriptase.

[00152] In other embodiments, the first binding domain of a precursor construct can bind a target of interest and the cleavage substrate present in the cleavable domain can be, for example, any of a legumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, or PSA, or a combination thereof. In other embodiments, the cleavage domain is cleaved by other disease-specific proteases, in diseases other than cancer, such as multiple sclerosis or rheumatoid arthritis.

[00153] In some embodiments, a precursor bispecific antibody construct may bind to a TAA by way of the first binding domain, wherein the cleavable domain of the regulatory arm remains uncleaved and therefore the second binding domain is specifically unavailable to a target CD3e antigen due to the presence of the CAP component. In some embodiments, a precursor bispecific antibody construct may bind to a TAA by way of the first binding domain, wherein the cleavable domain of the regulatory arm is uncleaved, wherein the precursor construct has enhanced half-life due to a half-life prolonging domain (e.g., an HSA polypeptide sequence) and the second binding domain remains specifically unavailable to a target CD3e antigen due to the presence of the CAP component.

[00154] In some embodiments, there are linkers (L) between any of the component parts of the precursor bispecific antibody construct (Figures 1A-1C and 2A-2E). In some embodiments, the linkers of the precursor construct (e.g., the linker between the VH of the Fab and the regulatory domain) comprises a cleavable domain linker. In some embodiments, the linkers between components of the regulatory domain (e.g., HSA polypeptide, CAP component) may be cleavable or non-cleavable, wherein the ability to be cleaved is independently selected for each linker. In some embodiments, the ability to be cleaved by a protease is independently selected for each linker. In some embodiments, a linker is cleavable by a protease. In some embodiments, a linker is not cleavable by a protease. In some embodiments, the linker between the VL of the Fab and the ScFv of the first binding domain comprises a non-cleavable linker. In some embodiments, the linker between the VH and VL chains of the ScFv comprises a non-cleavable linker.

[00155] A skilled artisan would appreciate that in some embodiments, a linker comprises a spacer between two active components or between two regions of an active component.

[00156] A skilled artisan would appreciate that the cleavable domain comprises a linear amino acid sequence comprising an enzyme cleavage site and may, in certain embodiments, be termed a “cleavable linker” or a“linker” or a“cleavable peptide” or a“CP”, wherein linkers disclosed herein may be cleavable or non-cleavable.

[00157] In some embodiments, a linker is present C-terminal to the Constant Heavy chain (CH1) of the Fab fragment. In some embodiments, a linker is present C-terminal to the Constant Light chain (CL) of the Fab fragment. In some embodiments, the linker C-terminal to the CH1 is cleavable. In some embodiments, the linker C-terminal to the CH1 is non-cleavable. In some embodiments, the linker C-terminal to the CL is cleavable. In some embodiments, the linker C- terminal to the CL is non-cleavable.

[00158] In some embodiments, a linker is a single amino acid. In some embodiments, a linker comprises the amino acid sequence set forth in any of SEQ ID NOs: 80-93, 95, 98-101, 104, 105, 145, and 146. In some embodiments, a linker between a VL-VH or a VH-VL comprises the amino acid sequence set forth in any of SEQ ID NOs: 80, 81, 82, 83, 84, 85, 86, 87, 8891-93, 95, 98, 99, 100, and 145.

[00159] In some embodiments, a linker is encoded by the nucleic acid sequence set forth in any of SEQ ID NOs: 118, 120-122, 125, 127, and 129.

[00160] For specific cleavage by an enzyme protease, contact between the enzyme and the cleavage substrate is made. When the precursor construct comprising a first binding domain binding to a TAA, a second binding domain binding to an extracellular epitope of CD3e, and a regulatory domain comprising a cleavable domain is in the presence sufficient enzyme activity, the cleavable domain can be cleaved. Sufficient enzyme activity can refer to the ability of the enzyme to make contact with the protease cleavable domain having the cleavage site and effect cleavage. In some embodiments, an enzyme may be in the vicinity of the precursor construct but unable to cleave because of other cellular factors or protein modification of the enzyme.

[00161] In some embodiments, cleavable domain substrates can include but are not limited to substrates cleavable by one or more of the following enzymes or proteases: ADAM10; Caspase 8, Cathepsin S, MMP 8, ADAM12, Caspase 9, FAP, MMP 9, ADAM17, Caspase 10, Granzyme B, MMP 13, AD AMTS, Caspase 11, Guanidinobenzotase (GB), MMP 14, ADAMTS5. Caspase 12, Hepsin, MT-SP1, BACE, Caspase 13, Human Neutrophil Elastase Neprilysin (HNE), Caspases, Caspase 14, Legumain, NS3/4A, Caspase 1, Cathepsins, Matriptase 2, Plasmin, Caspase 2, Cathepsin A, Meprin, PSA, Caspase 3, Cathepsin B, MMP 1, PSMA, Caspase 4, Cathepsin D, MMP 2, TACE, Caspase 5, Cathepsin E, MMP 3, TMPRSS 3/4, Caspase 6, Cathepsin K, MMP 7, uPA, Caspase 7, Matripase (MT-SP1, TADG-15, epithin, STM) and MT1-MMP.

[00162] In another embodiment, the cleavage substrate can involve a disulfide bond of a cysteine pair, which is thus cleavable by a reducing agent such as, for example, but not limited to a cellular reducing agent such as glutathione (GSH), thioredoxins, NADPH, flavins, ascorbate, and the like, which can be present in large amounts in tissue of or surrounding a solid tumor.

[00163] Other appropriate protease cleavage sites for use in the cleavable linkers herein are known in the art or may be identified using methods such as those described by Turk et al., 2001 Nature Biotechnology 19, 661-667.

[00164] In some embodiments, both the first binding domain and the second binding domain of the precursor bispecific antibody constructs can bind to their respective human and non-chimpanzee primate target molecules. The first binding domain, thus, binds to a human cell surface tumor associated antigen (TAA) and to the corresponding homolog of the cell surface TAA in a non chimpanzee primate. The identification and determination of homologs of human cell surface TAA in non-chimpanzee primates is well known to the person skilled in the art and can be carried out e.g. by sequence alignments. The second binding domain can bind to an antigen comprising an human CD3e extracellular epitope, and can bind to the corresponding homolog of the CD3e in a non-chimpanzee primate. In some embodiments, the first or second binding domains, or both also bind to their respective chimpanzee target molecules.

[00165] A skilled artisan would appreciate that in some embodiments, a cell surface tumor associated antigen (TAA) encompasses a molecule which is displayed on the surface of a cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a non-tumor cell present in the milieu of a tumor, for example but not limited to a cell present within vasculature tissue associated with a tumor or cancer.

[00166] A skilled artisan would appreciate that the terms "antigen" or "immunogen" encompass a peptide, protein, polypeptide which is immunogenic. In some embodiments, an antigen is capable of eliciting an immune response in a mammal, and therefore contains at least one and may contain multiple epitopes. An "antigen" molecule or a portion of a molecule is capable of being bound by a selective binding agent, such as an antigen-binding portion of a Fab fragment or an antigen binding portion of an scFv fragment. Additionally, an“antigen” is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. In some embodiments, a CAP component comprises the portion of an antigen to which the second binding domain binds.

[00167] The term "epitope" includes any determinant, in certain embodiments, a polypeptide determinant, capable of specific binding to a TAA or an immunoglobulin or T-cell receptor. An epitope is a region of an antigen that is bound by an antibody or an antigen-binding fragment thereof. In some embodiments, a CAP component comprises the epitope to which the second binding domain binds.

[00168] In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and may in certain embodiments have specific three-dimensional structural characteristics, and/or specific charge characteristics. In certain embodiments, a precursor bispecific antibody construct is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. A precursor bispecific antibody construct is said to specifically bind an antigen when the equilibrium dissociation constant is < 10 ⁵ , 10 ⁶ or 10 ⁷ M. In some embodiments, the equilibrium dissociation constant may be < 10 ⁸ M or 10 ⁹ M. In some further embodiments, the equilibrium dissociation constant may be < 10 ¹⁰ M or 10 ¹¹ M. Antigens disclosed herein included but are not limited to TAA, CAP components, and immuno-effector molecules such as a human CD3 epsilon polypeptide.

[00169] In some embodiments, the tumor associated antigen (TAA) is a tumor antigen. In some embodiments, tumor antigens comprise those antigens are presented on tumor cells. In some embodiments, the tumor antigen is present on a cell of solid tumor. In some embodiments, the tumor antigen is a cancer antigen, present on a cell of a non-solid tumor.

[00170] In some embodiments, when the TAA is a tumor cell antigen, the tumor cell comprises a cell from a solid tumor. Solid tumors may be benign (not cancer), or malignant (cancer). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. In some embodiments, solid tumors are neoplasms (new growth of cells) or lesions (damage of anatomic structures or disturbance of physiological functions) formed by an abnormal growth of body tissue cells other than blood, bone marrow or lymphatic cells. In some embodiments, a solid tumor consists of an abnormal mass of cells which may stem from different tissue types such as liver, colon, breast, or lung, and which initially grows in the organ of its cellular origin. However, such cancers may spread to other organs through metastatic tumor growth in advanced stages of the disease.

[00171] In some embodiments, the solid tumor comprises a sarcoma or a carcinoma, a fibrosarcoma, a myxosarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endothelio sarcoma, a lymphangiosarcoma, a lymphangioendotheliosarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor, an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilm's tumor, a cervical cancer or tumor, a uterine cancer or tumor, a testicular cancer or tumor, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodenroglioma, a schwannoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma. In some embodiments, the solid tumor comprises an Adrenocortical Tumor (Adenoma and Carcinoma), a Carcinoma, a Colorectal Carcinoma, a Desmoid Tumor, a Desmoplastic Small Round Cell Tumor, an Endocrine Tumor, an Ewing Sarcoma, a Germ Cell Tumor, a Hepatoblastoma a Hepatocellular Carcinoma, a Melanoma, a Neuroblastoma, an Osteosarcoma, a Retinoblastoma, a Rhabdomyosarcoma, a Soft Tissue Sarcoma Other Than Rhabdomyosarcoma, and a Wilms Tumor. In some embodiments, the solid tumor is a breast tumor. In another embodiment, the solid tumor is a prostate cancer. In another embodiment, the solid tumor is a colon cancer. In some embodiments, the tumor is a brain tumor. In another embodiment, the tumor is a pancreatic tumor. In another embodiment, the tumor is a colorectal tumor.

[00172] In some embodiments, the tumor cell comprises a cell from a non-solid tumor, that is a non-solid cancer. In some embodiments, a cancer may be a diffuse cancer, wherein the cancer is widely spread; not localized or confined. In some embodiments, a diffuse cancer may comprise a non-solid tumor. Examples of diffuse cancers include leukemias. Leukemias comprise a cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream.

[00173] In some embodiments, a diffuse cancer comprises a B-cell malignancy. In some embodiments, the diffuse cancer comprises leukemia. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is large B-cell lymphoma.

[00174] In some embodiments, the diffuse cancer or tumor comprises a hematological tumor. In some embodiments, hematological tumors are cancer types affecting blood, bone marrow, and lymph nodes. Hematological tumors may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages, and masT-cells, whereas the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas (e.g. Hodgkin's Lymphoma), lymphocytic leukemias, and myeloma are derived from the lymphoid line, while acute and chronic myelogenous leukemia (AML, CML), myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

[00175] In some embodiments, a non-solid (diffuse) cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cell myeloma), an acute lymphoblastic leukemia, an acute myelogenous leukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, a non-Hodgkin lymphoma, or plasma cell leukemia.

[00176] In some embodiments, the tumor or cancer comprises a metastasis of a tumor or cancer. [00177] In some embodiments a cell surface TAA is located in or on the plasma membrane of the cell, such that at least part of this molecule remains accessible from outside the cell in tertiary form. In some embodiments, a cell surface TAA that is located in the plasma membrane is a transmembrane protein comprising, in its tertiary conformation, regions of hydrophilicity and hydrophobicity.

[00178] These antigens can be presented on the cell surface with an extracellular part which is often combined with a transmembrane and cytoplasmic part of the molecule. These antigens can sometimes be presented only by tumor cells and never by the normal ones. Tumor antigens can be exclusively expressed on tumor cells or might represent a tumor specific mutation compared to normal cells. In this case, they are called tumor- specific antigens. More common are antigens that are presented by tumor cells and normal cells. In some embodiments, TAA include antigens exclusively expressed on a tumor cell. In some embodiments, TAA include antigens expressed on both tumor and normal cells.

[00179] In some embodiments, TAA can be overexpressed on tumor cells compared to normal cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to normal tissue.

[00180] In some embodiments, a first binding domain binding to a cell surface TAA comprises an amino acid sequence that binds to a human TAA. In some embodiments, an anti-scFv includes both a heavy chain variable region and a light chain variable region, wherein each region further comprises complementary -determining regions (CDR). In some embodiments, a first binding domain binding to a cell surface TAA comprises a linker between a scFv variable light chain (VH) region and a scFv variable heavy chain (VH) region. In some embodiments, a first binding domain binding to a cell surface TAA comprises a linker between a scFv fragment and the N-terminal of the VH region of the second binding domain. In some embodiments, a first binding domain binding to a cell surface TAA comprises a linker between a scFv fragment and the N-terminal of the VL region of the second binding domain. In some embodiments, a first binding domain binding to a cell surface TAA comprises a linker between a scFv fragment and the N-terminal of a Fab fragment comprising the second binding domain. In some embodiments, a first binding domain binding to a cell surface TAA comprises a linker between a scFv fragment and the N-terminal of a Fab fragment comprising the second binding domain.

[00181] In some embodiments, a first binding domain binding to a cell surface TAA, binds to a polypeptide target comprising a FcyRI, FcyRIIa FcyRIIb FcyRIIIa FcyRIIIb, CD28, CD 137, CTLA- 4, FAS, fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, glucocorticoid- induced TNFR-related (GITR) protein, lymphotoxin-beta receptor (LTpR), toll-like receptors (TLR), tumor necrosis factor-related apoptosis-inducing ligand-receptor 1 (TRAIL receptor 1; multiple malignancies including ovarian and colorectal carcinomas) and TRAIL receptor 2, pro state- specific membrane antigen (PSMA; prostate carcinoma) protein, prostate stem cell antigen (PSCA) protein (prostate adenocarcinoma), CA125 (multiple cancers including Ovarian carcinoma), tumor-associated protein carbonic anhydrase IX (CAIX; multiple cancers including renal cell carcinoma), epidermal growth factor receptor 1 (EGFR1; epithelial malignancies), EGFR (non-small cell lung cancer, epithelial ovarian cancer, colorectal cancer, head & neck cancer, breast cancer, lung cancer, esophageal cancer), EGFRvIII, human epidermal growth factor receptor 2 (Her2/neu; Erb2; epithelial malignancies), ErbB3 also known as HER3 (epithelial malignancies), Folate receptor, ephrin receptors, PDGFRa (epithelial malignancies), ErbB-2, CD20 (B cells, autoimmune, allergic or malignant), CD22 (B cells, autoimmune or malignant), CD30 (B cell malignancies), CD33 (myeloid malignancies), CD40, CD37, CD38, CD70 (B cells, autoimmune, allergic or malignant), CD74 (B cells, autoimmune, allergic or malignant), CD56 (T cell or NK cell lymphomas), CD40 (B cells, autoimmune, allergic or malignant); CD80 (B cells, autoimmune, allergic or malignant), CD86 (B cells, autoimmune, allergic or malignant), CD2 (T cell or NK cell lymphomas), p53, cMet also known as tyrosine-protein kinase Met or hepatocyte growth factor receptor (HGFR; Gastrointestinal tract and hepatic malignancies), MAGE-A1, MAGE-A2, MAGE -A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE -A 12, BAGE, DAM-6, -10, GAGE-l, - 2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, NY-ESO-l, BRCA1, BRCA2, MART-l, MC1R, GplOO, PSA, PSM, Tyrosinase, Wilms' tumor antigen (WT1), TRP-l, TRP-2, ART-4, CAMEL, Cyp-B, hTERT, hTRT, iCE, MUC1 (epithelial malignancies), MUC2, P-cadherin (Epithelial malignancies, including breast adenocarcinoma), Myostatin (GDF8) (many tumors including sarcoma and ovarian and pancreatic adenocarcinoma), Cripto (TDGF1) (Epithelial malignancies including colon, breast, lung, ovarian, and pancreatic cancers), ACVRL1/ALK1 (multiple malignancies including leukemias and lymphomas), MUC5AC (Epithelial malignancies, including breast adenocarcinoma), PRAME, P15, RU1, RU2, SART-l, SART-3, WT1, AFP, b-catenin/m, Caspase- 8/m, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-l, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr- abl, ETV6/AML, LDLR/FUT, Pml/RARa, TEL/AML1, CD28, CD137 (B cells or T cells, autoimmune, allergic or malignant), CanAg (tumors such as carcinomas of the colon and pancreas), Mesothelin (many tumors including mesothelioma and ovarian and pancreatic adenocarcinoma), DR5 (multiple malignancies including ovarian and colorectal carcinoma), PD-l (B cells, autoimmune, allergic or malignant), PD1L (Multiple malignancies including epithelial adenocarcinoma), IGF-1R (Most malignancies including epithelial adenocarcinoma), CXCR4 (B cells or T cells, autoimmune, allergic or malignant), Neuropilin 1 (Epithelial malignancies, including lung cancer), Glypicans (multiple cancers including liver, brain and breast cancers), EphA2 (multiple cancers including neuroblastoma, melanoma, breast cancer, and small cell lung carcinoma), CD138 (Myeloma), B7-H3 (CSC, stroma, NSCLC, Bladder tumors, mesothelioma, melanoma), gpA33 (colorectal cancers), GPC3 (liver, lung, esophageal, gastric, head and neck cancers), SSTR2 (Neuroendocrine tumors, GIST), ROR1 (Hematological, pancreatic, ovarian, renal cell carcinoma, NSCLC, and triple negative breast cancer), 5T4 (mesothelioma, gastic, ovarina, renal cancer, cancer stem cells in NSCLC, head and neck cancer), or a VEGF-R2 (The vasculature associated with the majority of malignancies including epithelial adenocarcinomas.) Examples of the unwanted target cells associated with the TAA presented are included in italics in parenthesis.

[00182] In some embodiments, the TAA is EGFR. In some embodiments, a first binding domain comprises an scFv that binds to human EGFR (anti-hEGFR). In some embodiments, the amino acid sequence of an anti-hEGFR- scFv light chain variable region (VL1) is set forth in SEQ ID NO: 9. In some embodiments, an anti-hEGFR scFv VL sequence comprises a homolog of SEQ ID NO: 9.

[00183] In some embodiments, the amino acid sequence of an anti-hEGFR- scFv light chain variable region (VL1) is set forth in SEQ ID NO: 12. In some embodiments, an anti-hEGFR scFv VL sequence comprises a homolog of SEQ ID NO: 12.

[00184] In some embodiments, the anti-hEGFR- scFv light chain variable region (VL1) is encoded by the nucleic acid sequence set forth in SEQ ID NO: 128. In some embodiments, the anti- hEGFR-scFv light chain variable region (VL1) is encoded by a homolog of the nucleic acid sequence set forth in SEQ ID NO: 128.

[00185] In some embodiments, the amino acid sequence of an anti-hEGFR- scFv heavy chain variable region (VH1) set forth in SEQ ID NO: 10. In some embodiments, an anti-hEGFR scFv VH sequence comprises a homolog of SEQ ID NO: 10.

[00186] In some embodiments, the anti-hEGFR- scFv heavy chain variable region (VH1) is encoded by the nucleic acid sequence set forth in SEQ ID NO: 126. In some embodiments, the anti- hEGFR-scFv heavy chain variable region (VH1) is encoded by a homolog of the nucleic acid sequence set forth in SEQ ID NO: 126.

[00187] In some embodiments, an anti-EGFR scFV comprises a linker between a VL and a VH region. In some embodiments, the linker between a VL and a VH region comprises any linker disclosed herein. In some embodiments, the amino acid sequence of a linker between a VL and VH region of an anti-EGFR scFV is set forth by SEQ ID NO: 100 (GGGGSGGGGSGGGGS). In some embodiments, the linker between a VL and a VH region of an anti-EGFR scFV comprises a homolog of SEQ ID NO: 100. In some embodiments, a linker between a VL and VH region of an anti-EGFR scFV is encoded by the nucleic acid sequence set forth in SEQ ID NO: 127. In some embodiments, a linker between a VL and VH region of an anti-EGFR scFV is encoded by a homolog by the nucleic acid sequence set forth in SEQ ID NO: 127.

[00188] In some embodiments, components of an anti-EGFR scFv comprises a VL-linker-VH order (N-terminal to C-terminal) (Figure 2A). In some embodiments, components of an anti-EGFR scFv comprises a VH-linker-VL order (N-terminal to C-terminal) (Figure 2B).

[00189] In some embodiments, an anti-EGFR scFV sequence comprises the sequence QV QLQES GPGLVKPSETLS LTCT V S GGS VSS GD YYWTWIRQSPGKGLE WIGHIY Y S GNT N YNPS LKS RLTIS IDTS KT QF S LKLS S VT A ADT AI Y Y C VRDRVT G AFDIW GQGTM VT V S S GGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKA PKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTK V EIK (SEQ ID NO: 94). In some embodiments, an anti-EGFR scFv comprises a homolog of SEQ ID NO: 94.

[00190] In some embodiments, an anti-EGFR scFV sequence comprises the sequence DIQMTQS PS S LS AS VGDRVTITCQ AS QDIS NYLNW Y QQKPGKAPKLLI YD ASNLETG VPS RFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKGGGGSGGGGSGGG GS Q V QLQES GPGLVKPSETLS LTCT V S GGS VS S GD YYWTWIRQSPGKGLE WIGHIYY S G NTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTV SS (SEQ ID NO: 96). In some embodiments, an anti-EGFR scFv comprises a homolog of SEQ ID NO: 96.

[00191] In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence of of an anti-EGFR scFv. In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence of an anti-EGFR scFv.

[00192] In some embodiments, homologues comprise nucleotides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the nucleic acid sequence of an anti-EGFR scFv. In some embodiments, homologues comprise nucleotides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of an anti-EGFR scFv.

[00193] In some embodiments, disclosed herein are homologues of an anti-hEGFR scFv VF (SEQ ID NO: 9) or anti-hEGFR scFv VH (SEQ ID NO: 10) or an anti-hEGFR scFv (SEQ ID NO: 94) or an anti-hEGFR scFv (SEQ ID NO: 96), respectively, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. In some embodiments, disclosed herein are homologues of nucleotide sequences encoding an anti-hEGFR scFv VL (SEQ ID NO: 9) or anti-hEGFR scFv VH (SEQ ID NO: 10) or an anti-hEGFR scFv (SEQ ID NO: 94) or an anti-hEGFR scFv (SEQ ID NO: 96), respectively, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[00194] In some embodiments, homology also encompasses deletion, insertion, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. In one embodiment, the variant comprises conservative substitutions, or deletions, insertions, or substitutions that do not significantly alter the three-dimensional structure of the polypeptide of interest, e.g., VL or VH region of the first binding domain, particularly in the areas of the CDR epitope binding regions. In some embodiments, the deletion, insertion, or substitution does not alter the function of interest of the anti-hEGFR VL or anti-hEGFR VH or an anti-EGFR ScFv, present in the first binding domain of a precursor construct, which in some embodiment, is binding to an EGFR on a target tumor cell.

[00195] In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 9, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 10, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 12, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 9 or a homolog thereof, and the sequence set forth in SEQ ID NO: 10 or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 12 or a homolog thereof, and the sequence set forth in SEQ ID NO: 10 or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the amino acid sequence set forth in SEQ ID NO: 94, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the amino acid sequence set forth in SEQ ID NO: 96, or a homolog thereof. [00196] In some embodiments, the nucleotide sequences encoding a precursor bispecific antibody construct polypeptide is optimized for mammalian transcription and translation. In some embodiments, the nucleotide sequences encoding a first binding domain of a precursor bispecific antibody construct polypeptides is optimized for mammalian transcription and translation. In some embodiments, the nucleotide sequence of a VL or VH, or of both a VL and VH regions of a first binding domain are optimized for mammalian transcription and translation.

[00197] In some embodiments, a first binding domain VL region comprising a polypeptide region encoded by the nucleotide sequence set forth in SEQ ID NO: 128. In some embodiments, a first binding domain VL region is encoded by a homolog of SEQ ID NO: 128.

[00198] In some embodiments, a first binding domain VH region comprising a polypeptide encoded by the nucleotide sequence set forth in SEQ ID NO: 126. In some embodiments, a first binding domain VH region is encoded by a homolog of SEQ ID NO: 126.

[00199] In some embodiments, a first binding domain binding to a cell surface tumor associated antigen comprising an anti-EGFR scFv is encoded by the nucleotide sequence set forth in SEQ ID NO: 131. In some embodiments, an anti-EGFR scFV first binding domain is encoded by a homolog of SEQ ID NO: 131. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen comprising an anti-EGFR scFv is encoded by the nucleotide sequence set forth in SEQ ID NO: 132. In some embodiments, an anti-EGFR scFV first binding domain is encoded by a homolog of SEQ ID NO: 132.

[00200] In some embodiments, the TAA is 5T4. In some embodiments, a first binding domain comprises an scFv that binds to human 5T4 (anti-h5T4). In some embodiments, the amino acid sequence of an anti-h5T4-scFv light chain variable region (VL1) is set forth in SEQ ID NO: 138 (DIQMTQS PS S LS AS VGDRVTITCRAS QGIS NYLA WF QQKPGKAPKS LIYRANRLQS GVPS RFS GSGS GTDFTLTIS SLQPEDVAT YY CLQYDDFPWTFGQGTKLEIK; SEQ ID NO: 138). In some embodiments, an anti-h5T4 scFv VL sequence comprises a homolog of SEQ ID NO: 138.

[00201] In some embodiments, the amino acid sequence of an anti-h5T4-scFv heavy chain variable region (VH1) set forth in SEQ ID NO: 139

(DIQMTQS PS S LS AS VGDRVTITCRAS QGIS NYLA WF QQKPGKAPKS LIYRANRLQS GVPS RFS GS GS GTDFTLTIS SLQPEDVAT YY CLQYDDFPWTF GQGTKLEIKGGGGS GGGGS GG GGS GGGGS QV QLV QS G AE VKKPGAS VKV S CKAS GYTFTSFWMHWVRQAPGQGLEWM GRIDPNRGGTEYNEKAKSRVTMTADKSTSTAYMELSSLRSEDTAVYYCAGGNPYYPMD YWGQGTTVTVSS; SEQ ID NO: 139). In some embodiments, an anti-h5T4 scFv VH sequence comprises a homolog of SEQ ID NO: 139.

[00202] In some embodiments, an anti-5T4 scFV comprises a linker between a VL and a VH region. In some embodiments, the linker between a VL and a VH region comprises any linker disclosed herein. In some embodiments, the amino acid sequence of a linker between a VL and VH region of an anti-5T4 scFV is set forth by SEQ ID NO: 145 (GGGGS GGGGS GGGGS GGGGS ) . In some embodiments, the linker between a VL and a VH region of an anti-5T4 scFV comprises a homolog of SEQ ID NO: 145.

[00203] In some embodiments, components of an anti-5T4 scFv comprises a VL-linker-VH order (N-terminal to C-terminal). In some embodiments, components of an anti-5T4 scFv comprises a VH-linker-VL order (N-terminal to C-terminal).

[00204] In some embodiments, an anti-5T4 scFV (VL-linker-VH) sequence comprises the sequence set forth in SEQ ID NO: 137.

DIQMTQS PS S LS AS VGDRVTITCRAS QGIS NYLAWF QQKPGKAPKS LI YRANRLQS GVPS RFS GS GS GTDFTLTIS SLQPED VAT YY CLQ YDDFPWTF GQGTKLEIKGGGGS GGGGS GG GGS GGGGS QV QLV QS G AE VKKPGAS VKV S CKAS GYTFTSFWMHWVRQAPGQGLEWM GRIDPNRGGTEYNEKAKSRVTMTADKSTSTAYMELSSLRSEDTAVYYCAGGNPYYPMD YWGQGTTVTVSS SEQ ID NO: 137). In some embodiments, an anti-5T4 scFv comprises a homolog of SEQ ID NO: 137.

[00205] In some embodiments, an anti-5T4 scFV (VH-linker-VL) sequence comprises the sequenceQV QLV QS GAE VKKPGAS VKV S CKAS GYTFTSFWMHWVRQ APGQGLE WMGRI DPNRGGTE YNEKAKS RVTMT ADKSTST A YMELS S LRSEDT A V YY C AGGNP YYPMD YW GQGTT VT V S S GGGGS GGGGS GGGGS GGGGS DIQMTQS PS S LS AS VGDRVTITCRAS QGI SNYLAWF QQKPGKAPKS LI YRANRLQS GVPS RFS GS GS GTDFTLTIS SLQPED VAT YY CL QYDDFPWTFGQGTKLEIK (SEQ ID NO: 140). In some embodiments, an anti-5T4 scFv comprises a homolog of SEQ ID NO: 140.

[00206] In some embodiments, an anti-5T4 scFV (VL-linker-VH) amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 147.

(gacatacagatgacccagtcccccagctccctctcagcctctgtgggcgacagggt gaccatcacctgcagagcttcccaaggcatctcca actacctggcctggttccagcagaagccaggcaaggctcctaagagcctgatctacaggg ctaaccgtctgcagtccggcgtgccctccag gttctctgggtccggcagcggcacagacttcacccttaccatctcctccctgcagcccga ggacgtagccacatattactgcctgcagtacga tgacttcccctggactttcggacagggcaccaagctggagataaagggaggtggtggctc agggggtgggggctccggcggaggggggt ctggcggtggcgggtcccaggtgcagctggttcagtctggtgctgaggtgaagaagcctg gcgcctcagttaaagtgtcatgcaaggcctc cggctacaccttcaccagtttctggatgcactgggtgaggcaggcacccggtcagggcct ggagtggatgggccgaatcgatcccaaccgt ggcggcactgagtacaacgagaaggccaagagcagggtcacaatgaccgccgacaagtcc acctcaactgcttatatggagctgtcctcct tgcgttctgaggacactgccgtgtactactgtgcaggcggcaacccctactaccccatgg actattgggggcagggtaccactgtcaccgtgt catct SEQ ID NO: 147). In some embodiments, a nucleotide sequence encoding anti-5T4 scFv comprises of homolog of SEQ ID NO: 147.

[00207] In some embodiments, an anti-5T4 scFV (VH-linker-VL) amino acid sequence is encoded by the nucleotide sequence set forth in the sequence caggtgcagctggttcagtctggtgctgaggtgaagaagcctggcgcctcagttaaagtg tcatgcaaggcctccggctacaccttcaccagt ttctggatgcactgggtgaggcaggcacccggtcagggcctggagtggatgggccgaatc gatcccaaccgtggcggcactgagtacaac gagaaggccaagagcagggtcacaatgaccgccgacaagtccacctcaactgcttatatg gagctgtcctccttgcgttctgaggacactgc cgtgtactactgtgcaggcggcaacccctactaccccatggactattgggggcagggtac cactgtcaccgtgtcatctggaggtggtggct cagggggtgggggctccggcggaggggggtctggcggtggcgggtccgacatacagatga cccagtcccccagctccctctcagcctct gtgggcgacagggtgaccatcacctgcagagcttcccaaggcatctccaactacctggcc tggttccagcagaagccaggcaaggctccta agagcctgatctacagggctaaccgtctgcagtccggcgtgccctccaggttctctgggt ccggcagcggcacagacttcacccttaccatct cctccctgcagcccgaggacgtagccacatattactgcctgcagtacgatgacttcccct ggactttcggacagggcaccaagctggagata aag (SEQ ID NO: 148). In some embodiments, a nucleotide sequence encoding an anti-5T4 scFv comprises a homolog of SEQ ID NO: 148.

[00208] In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 137%, at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence of an anti-5T4 scFv. In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 137%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence of an anti-5T4 scFv.

[00209] In some embodiments, homologues comprise nucleotides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 137%, at least 97%, at least 98%, or at least 99% homologous to the nucleic acid sequence of an anti-5T4 scFv. In some embodiments, homologues comprise nucleotides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 137%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of an anti-5T4 scFv.

[00210] In some embodiments, disclosed herein are homologues of an anti-h5T4 scFv VL (SEQ ID NO: 138) or anti-h5T4 scFv VH (SEQ ID NO: 139) or an anti-h5T4 scFv (SEQ ID NO: 137) or an anti-h5T4 scFv (SEQ ID NO: 140), respectively, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. In some embodiments, disclosed herein are homologues of nucleotide sequences encoding an anti-h5T4 scFv VL (SEQ ID NO: 138) or anti-h5T4 scFv VH (SEQ ID NO: 139) or an anti-h5T4 scFv (SEQ ID NO: 137) or an anti-h5T4 scFv (SEQ ID NO: 140), respectively, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[00211] In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 138, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 139, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 138 or a homolog thereof, and the sequence set forth in SEQ ID NO: 139 or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the amino acid sequence set forth in SEQ ID NO: 140, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the amino acid sequence set forth in SEQ ID NO: 137, or a homolog thereof.

[00212] In some embodiments, the TAA is ROR1. In some embodiments, a first binding domain comprises an scFv that binds to human ROR1 (anti-hRORl). In some embodiments, the amino acid sequence of an anti-hRORl -scFv light chain variable region (VL1) is set forth in SEQ ID NO: 142. (DIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWYQQKPGKPPKLLIYRASNLASGVP S RFS GSGS GTDFTLTIS SLQPEDVAT YY CLGGV GNVS YRTSFGGGTKVEIK; SEQ ID NO: 142) In some embodiments, an anti-hRORl scFv VL sequence comprises a homolog of SEQ ID NO: 142.

[00213] In some embodiments, the amino acid sequence of an anti-hRORl -scFv heavy chain variable region (VH1) set forth in SEQ ID NO: 143.

(QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFINSGGST WYA S WVKGRFTIS RTSTT VDLKMT S LTTDDT AT YFC ARG YST Y Y GDFNIW GPGTLVTIS ; SEQ ID NO: 143). In some embodiments, an anti-hRORl scFv VH sequence comprises a homolog of SEQ ID NO: 143.

[00214] In some embodiments, an anti-RORl scFV comprises a linker between a VL and a VH region. In some embodiments, the linker between a VL and a VH region comprises any linker disclosed herein. In some embodiments, the amino acid sequence of a linker between a VL and VH region of an anti-RORl scFV is set forth by SEQ ID NO: 145. In some embodiments, the linker between a VL and a VH region of an anti-RORl scFV comprises a homolog of SEQ ID NO: 145.

[00215] In some embodiments, components of an anti-RORl scFv comprises a VL-linker-VH order (N-terminal to C-terminal). In some embodiments, components of an anti-RORl scFv comprises a VH-linker-VL order (N-terminal to C-terminal). [00216] In some embodiments, an anti-RORl scFV (VL-linker-VH) sequence comprises the sequence set forth in SEQ ID NO: 141.

(DIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWYQQKPGKPPKLLIYRASNLAS GVPS RFS GSGS GTDFTLTIS SLQPEDVAT YY CLGGV GNVS YRTSFGGGTKVEIKGGGGSGGGG SGGGGS GGGGS QS VKESEGDLVTPAGNLTLTCTAS GSDIND YPIS WVRQAPGKGLEWIG FINSGGSTWYASWVKGRFTISRTSTTVDLKMTSLTTDDTATYFCARGYSTYYGDFNIWG PGTLVTIS; SEQ ID NO: 141). In some embodiments, an anti-RORl scFv comprises a homolog of SEQ ID NO: 141.

[00217] In some embodiments, an anti-RORl scFV (VH-linker-VL) sequence comprises the sequence

QSVKESEGDLVTPAGNLTLTCTASGSDINDYPISWVRQAPGKGLEWIGFINSGGSTWYA S WVKGRFTIS RTSTT VDLKMT S LTTDDT AT YFC ARG YST Y Y GDFNIW GPGTLVTIS GGG GSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTINCQASQSIDSNLAWYQQKPGK PPKLLIYRASNLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGGVGNVSYRTSFG G GTKVEIK; SEQ ID NO: 144. In some embodiments, an anti-RORl scFv comprises a homolog of SEQ ID NO: 144.

[00218] In some embodiments, an anti-RORl scFV (VL-linker-VH) amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 149.

(gacatccagatgactcagtcccccagttccctgtccgcctccgtgggcgacagggt gacaatcaactgccaggcctcacagtctatcgaca gtaaccttgcctggtatcaacagaagcccgggaagccccccaagctgctgatctacaggg cctccaatctggcatccggcgtgccctccag gttctccggttccggctcaggcaccgattttaccctgaccatatcctccttgcagcccga ggacgtggctacctactactgtctgggcggtgtg ggcaacgtgtcctacaggacctcctttggtggcggcaccaaggtggagatcaagggcgga ggagggtccggtggagggggcagtggtg ggggaggatcaggaggtggtggctcccagagtgtgaaggagtccgagggcgacctggtga cccctgctggcaatctgaccctcacctgc accgcttccggtagcgacatcaacgactaccccatatcatgggtgagacaggctcccggc aagggcctggagtggatcggcttcatcaata gcggtgggtccacatggtacgcaagttgggtgaagggcaggttcaccatctctcgaacct caactaccgtcgacctgaaaatgacctccctg acaaccgacgacaccgcaacctatttctgcgccaggggctactccacatactatggcgac ttcaacatctgggggccagggaccctggtca ctatctca; SEQ ID NO: 149). In some embodiments, a nucleotide sequence encoding an anti-RORl scFv comprises a homolog of SEQ ID NO: 149.

[00219] In some embodiments, an anti-RORl scFV (VH-linker-VL) amino acid sequence is encoded by the sequence set forth in SEQ ID NO: 150

(cagagtgtgaaggagtccgagggcgacctggtgacccctgctggcaatctgaccct cacctgcaccgcttccggtagcgacatcaacgac taccccatatcatgggtgagacaggctcccggcaagggcctggagtggatcggcttcatc aatagcggtgggtccacatggtacgcaagtt gggtgaagggcaggttcaccatctctcgaacctcaactaccgtcgacctgaaaatgacct ccctgacaaccgacgacaccgcaacctatttct gcgccaggggctactccacatactatggcgacttcaacatctgggggccagggaccctgg tcactatctcaggcggaggagggtccggtg gagggggcagtggtgggggaggatcaggaggtggtggctccgacatccagatgactcagt cccccagttccctgtccgcctccgtgggcg acagggtgacaatcaactgccaggcctcacagtctatcgacagtaaccttgcctggtatc aacagaagcccgggaagccccccaagctgct gatctacagggcctccaatctggcatccggcgtgccctccaggttctccggttccggctc aggcaccgattttaccctgaccatatcctccttgc agcccgaggacgtggctacctactactgtctgggcggtgtgggcaacgtgtcctacagga cctcctttggtggcggcaccaaggtggagat caag; SEQ ID NO: 150.) In some embodiments, a nucleotide sequence ecoding an anti-RORl scFv comprises a homolog of SEQ ID NO: 150.

[00220] In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 141%, at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence of an anti-RORl scFv. In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 141%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence of an anti-RORl scFv.

[00221] In some embodiments, homologues comprise nucleotides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 141%, at least 97%, at least 98%, or at least 99% homologous to the nucleic acid sequence of an anti-RORl scFv. In some embodiments, homologues comprise nucleotides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 141%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence of an anti-RORl scFv.

[00222] In some embodiments, disclosed herein are homologues of an anti-hRORl scFv VL (SEQ ID NO: 142) or anti-hRORl scFv VH (SEQ ID NO: 143) or an anti-hRORl scFv (SEQ ID NO: 141) or an anti-hRORl scFv (SEQ ID NO: 144), respectively, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters. In some embodiments, disclosed herein are homologues of nucleotide sequences encoding an anti- hRORl scFv VL (SEQ ID NO: 142) or anti-hRORl scFv VH (SEQ ID NO: 143) or an anti-hRORl scFv (SEQ ID NO: 141) or an anti-hRORl scFv (SEQ ID NO: 144), respectively, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[00223] In some embodiments, homology also encompasses deletion, insertion, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. In one embodiment, the variant comprises conservative substitutions, or deletions, insertions, or substitutions that do not significantly alter the three-dimensional structure of the polypeptide of interest, e.g., VL or VH region of the first binding domain, particularly in the areas of the CDR epitope binding regions. In some embodiments, the deletion, insertion, or substitution does not alter the function of interest of: the anti-hEGFR VL or anti-hEGFR VH or an anti-EGFR ScFv, present in the first binding domain of a precursor construct, which in some embodiment, is binding to an EGFR on a target tumor cell; or the anti-h5T4 VL or anti-h5T4 VH or an anti-5T4 ScFv, present in the first binding domain of a precursor construct, which in some embodiment, is binding to an ROR1 on a target tumor cell; or the anti-hRORl VL or anti-hRORl VH or an anti-RORl ScFv, present in the first binding domain of a precursor construct, which in some embodiment, is binding to an ROR1 on a target tumor cell. In some embodiments, a deletion, insertion, or substitution does not alter the function of interest of an anti-TAA VL or anti-TAA VH or an anti-TAA ScFv, present in the first binding domain of a precursor construct, which in some embodiment, is binding to a TAA on a target tumor cell;

[00224] In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 142, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 143, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the sequence set forth in SEQ ID NO: 142 or a homolog thereof, and the sequence set forth in SEQ ID NO: 143 or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the amino acid sequence set forth in SEQ ID NO: 144, or a homolog thereof. In some embodiments, a first binding domain binding to a cell surface tumor associated antigen includes the amino acid sequence set forth in SEQ ID NO: 141, or a homolog thereof.

[00225] In another embodiment, the TAA provided herein is an angiogenic antigen which is expressed on both activated pericytes and pericytes in tumor angiogenic vasculature, which is associated with neovascularization in vivo. Angiogenic antigens are known in the art see for example W02010/102140, which is incorporated by reference herein. For example, an angiogenic antigen may be selected from; Angiopoietin-l (Angl), Angiopoietin 3, Angiopoietin 4, Angiopoietin 6; Del-l; Fibroblast growth factors: acidic (aFGF) and basic (bFGF); Follistatin; Granulocyte colony-stimulating factor (G-CSF); Hepatocyte growth factor (HGF) /scatter factor (SF); Interleukin-8 (IL-8); Leptin; Midkine; Placental growth factor; Platelet-derived endothelial cell growth factor (PD-ECGF); Platelet-derived growth factor-BB (PDGF-BB); Pleiotrophin (PTN); Progranulin; Proliferin; survivin; Transforming growth factor-alpha (TGF-alpha); Transforming growth factor-beta (TGF-beta); Tumor necrosis factor-alpha (TNF-alpha); Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF).

[00226] As described above and throughout, in some embodiments the first binding domain (TAA binding domain) comprises a single chain variable fragment (scFv). In some embodiments, the second binding domain (CD3e extracellular epitope binding domain) comprises a Fab fragment. The specific structural order of components of a precursor bispecific antibody construct, for example comprised of a polypeptide A and a polypeptide B, is described throughout in more detail.

[00227] In some embodiments, a precursor bispecific antibody construct comprises at its core, an Fab fragment, which in some embodiments, comprises the second binding domain. As would be understood by the skilled person, a Fab fragment is the antigen-binding fragment of an antibody. The Fab is composed of one constant and one variable region of an immunoglobulin heavy and an immunoglobulin light chain. The heavy chain constant (CH1) and variable (VH) regions heterodimerize with the light chain variable (VF) and constant (CF) regions and are usually covalently linked by a disulfide bond between the heavy and light chain constant regions (see e.g., diagrams in Figures 1A-1B and 2A-2E, and the amino acid sequences presented in Figures 4A, 4C, 4E, 4G, and 41, wherein the cysteine residues that may form a disulfide bond (Cys-S-S-Cys bond) between polypeptides A and B of a precursor construct are indicated (highlighted in green and red). The codons encoding these Cys residues are indicated in the nucleic acid sequences presented in Figures 4B, 4D, 4F, 4H, and 4K (highlighted in green and red). Thus, a skilled artisan would appreciate that the term "Fab" with regard to an antibody generally encompasses that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.

[00228] As would be recognized by the skilled person, a disulfide bond between the heavy and light chain is preferable, but not essential for function (Orcutt, et al. (2010), PEDS, 23:221-228). Thus, in certain embodiments the Fab fragment disclosed herein may not comprise a disulfide bond. In this regard, the heavy and light chains may be engineered in such a way so as to stably interact without the need for disulfide bond. For example, in certain embodiments, the heavy or light chain can be engineered to remove a cysteine residue and wherein the heavy and light chains still stably interact and function as a Fab. In some embodiments, mutations are made to facilitate stable interaction between the heavy and light chains. For example, a "knobs into holes" engineering strategy can be used to facilitate dimerization between the heavy and light chains of a Fab (see e.g., 1996 Protein Engineering, 9:617-621). Using this strategy, "knobs" are created by replacing small amino acid side chains at the interface between interacting domains with larger ones. Corresponding "holes" are made at the interface between interacting molecules by replacing large side chains with smaller ones. Thus, also contemplated for use herein are variant Fab fragments designed for a particular purpose, for example, amino acid changes in the constant domains of CH1 and or CL, and removal of a disulfide bond or addition of tags for purification.

[00229] In some embodiments, the configuration of the variable and constant regions within the Fab fragment may be different from what is found in a native Fab. In other words, in one embodiment, the orientation of the variable and constant regions may be VH-CL in one chain and in another VL-CH1 (Shaefer et al. (2011), PNAS, 108:111870-92). Such modified Fab fragments still function to bind their particular target antigen and are contemplated for use in the precursor construct disclosed herein. Thus, in this regard the variable regions and constant regions that make up the Fab are considered modular.

[00230] In certain embodiments, the Fab fragments of this disclosure are derived from monoclonal antibodies and may be derived from antibodies of any type, including IgA, IgM, IgD, IgG, IgE and subtypes thereof, such as IgGl, IgG2, IgG3, and IgG4. The light chain domains may be derived from the kappa or lambda chain. The Fab fragments for use herein may be made recombinantly.

[00231] As is well known in the art, an antibody is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one epitope recognition site, located in the variable region of the immunoglobulin molecule. A skilled artisan would appreciate that the term“antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also humanized antibodies, chimeric antibodies, antibody fragments including antibody fragments lacking an Fc region, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity, including scFv fragments and Fab fragments. In some embodiments, the precursor antibody constructs described herein lack an Fc region.

[00232] The Fab fragment as disclosed herein, comprises an antigen-binding portion (second binding domain) comprised of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region (VH and VL, respectively). Similarly, the scFv fragment described above (first binding domain), comprises an antigen-binding portion comprised of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region (VH and VL, respectively). More specifically, the term "antigen-binding portion" as used herein refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chains that binds to the target antigen of interest, such as the TAA of the first binding region, or a CD3 molecule of the second binding region. In this regard, an antigen-binding portion of the herein described precursor constructs may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence of a parent antibody that binds to a target antigen of interest. In certain embodiments, the antigen-binding portion of the scFv fragment (second binding domain) of a precursor bispecific antibody construct binds to a TAA, for example but not limited to a human EGFR. In certain embodiments, the antigen-binding portion of the Fab fragment of a precursor bispecific antibody construct binds to CD3.

[00233] In certain embodiments, a specific VH and/or VL of the precursor bispecific antibody construct described herein may be used to screen a library of the complementary variable region to identify VH/VL with desirable properties, such as increased affinity for a target antigen of interest. Such methods are described, for example, in Portolano et al., J. Immunol. (1993) 150:880-887; Clarkson et al., Nature (1991) 352:624-628.

[00234] Other methods may also be used to mix and match CDRs to identify Fab having desired binding activity (such as binding to CD3, or other target antigen of interest as described herein for other binding domains present in the precursor bispecific antibody construct). For example: Klimka et al., British Journal of Cancer (2000) 83: 252-260, describe a screening process using a mouse VL and a human VH library with CDR3 and FR4 retained from the mouse VH. After obtaining antibodies, the VH was screened against a human VL library to obtain antibodies that bound antigen. Beiboer et al., J. Mol. Biol. (2000) 296:833-849 describe a screening process using an entire mouse heavy chain and a human light chain library. After obtaining antibodies, one VL was combined with a human VH library with the CDR3 of the mouse retained. Antibodies capable of binding antigen were obtained. Rader et al., PNAS (1998) 95:8910-8915 describe a process similar to Beiboer et al above.

[00235] These just-described techniques are, in and of themselves, known as such in the art. The skilled person will, however, be able to use such techniques to obtain antigen-binding fragments of antibodies according to several embodiments of the disclosure described herein, using routine methodology in the art.

[00236] Also disclosed herein is a method for obtaining an antibody antigen binding domain specific for a target antigen (e.g., CD3 or any target antigen described elsewhere herein for targets of binding domains described herein), the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH domain set out herein a VH domain which is an amino acid sequence variant of the VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations to identify a specific binding member or an antibody antigen binding domain specific for a target antigen of interest (e.g., CD3) and optionally with one or more desired properties. The VL domains may have an amino acid sequence which is substantially as set out herein. An analogous method may be employed in which one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.

[00237] A skilled artisan would appreciate that an epitope that "specifically binds" or "preferentially binds" (used interchangeably herein) to an antibody or a polypeptide is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody, or Fab or scFv thereof, "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a CD3 epitope is an antibody that binds one CD3 epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other CD3 epitopes or non-CD3 epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[00238] In certain embodiments, antigen-binding portions of the Fab fragment (second binding domain) as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as "CDR1," "CDR2," and "CDR3" respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.

[00239] As used herein, the term "FR set" refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain "canonical" structures— regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non- covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.

[00240] The structures and locations of immunoglobulin variable regions may be determined by reference to Rabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (immuno.bme.nwu.edu).

[00241] A skilled artisan would recognize that the term "monoclonal antibody" encompasses a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') ₂ , Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. as described herein.

[00242] The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab') ₂ fragment which comprises both antigen-binding sites. An Fv fragment for use according to certain embodiments as disclosed herein, can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096. [00243] In some embodiments of the present disclosure, the Fab fragment comprising a second binding domain binds to CD3. In some embodiments of the present disclosure, the Fab fragment comprising a second binding domain binds to CD3epsilon. "T-cell receptor" (TCR) is a molecule found on the surface of T-cells that, along with CD3, is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It consists of a disulfide- linked heterodimer of the highly variable (alpha) and (beta) chains in most T-cells. In other T-cells, an alternative receptor made up of variable Y and (delta) chains is expressed. Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one N-terminal immunoglobulin variable region, one immunoglobulin constant region, a transmembrane region, and a short cytoplasmic tail at the C-terminal end (see, Abbas and Lichtman, Cellular and Molecular Immunology (5th Ed.), Editor: Saunders, Philadelphia, 2003; Janeway et ai, Immunobiology: The Immune System in Health and Disease, 4th Ed., Current Biology Publications, p 148, 149, and 172, 1999). TCR as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.

[00244] "Anti-TCR Fab" or "Anti-TCR precursor bispecific antibody construct", refers to a Fab or a precursor bispecific antibody construct comprising an Fab that specifically binds to a TCR molecule or one of its individual chains (e.g., TCR (alpha), TCR (beta), TCRY or TCR (delta) chain). In certain embodiments, an anti-TCR Fab binds to a TCR (alpha), a TCR (beta), or both. A skilled person would appreciate that the term "Anti-TCR Fab", may in some embodiments encompass the second binding domain of a precursor bispecific antibody construct described herein. In some embodiments, the term "Anti-TCR Fab" may encompass the precursor construct, wherein reference is being made the to binding attributes of the second binding domain.

[00245] "CD3" is known in the art as a multi-protein complex of six chains (see, Smith-Garvin et al., Annu Rev Immunol. 2009;27:591-619 )). In mammals, the complex comprises a CD3(gamma) chain, a CD3(delta) chain, two CD3(epsilon; e) chains, and a homodimer of CD3(zeta) chains. The CD3(gamma), CD3(delta), and CD3(epsilon) chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3(gamma), CD3(delta), and CD3(epsilon) chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T-cell receptor chains. The intracellular tails of the CD3(gamma), CD3(delta), and CD3(epsilon) chains each contain a single conserved motif known as an immunoreceptor tyrosine- based activation motif or IT AM, whereas each CD3(zeta) chain has three. Without wishing to be bound by theory, it is believed the ITAMs are important for the signaling capacity of a TCR complex. CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.

[00246] "Anti-CD3 Fab" as used herein, refers to a Fab comprising a second binding domain that specifically binds to individual CD3 chains (e.g., CD3(gamma) chain, CD3(delta) chain, or CD3(epsilon; e) chain) or a complex formed from two or more individual CD3 chains (e.g., a complex of more than one CD3(epsilon) chains, a complex of a CD3(gamma) and CD3(epsilon) chain, a complex of a CD3(delta) and CD3(epsilon) chain). In certain embodiments, an anti-CD3 Fab specifically binds to a CD3(gamma), a CD3(delta), or a CD3(epsilon), or any combination thereof, and in certain embodiments, a CD3(epsilon). In some embodiments, an anti-CD3 Fab binds to the N-terminus of CD3 epsilon. In some embodiments, an anti-CD3 Fab binds to an extracellular epitope of CD3 epsilon.

[00247] In some embodiments, the anti-CD3 Fab binds to an epitope with amino acids 1-27 of CD3 epsilon. In some embodiments, the anti-CD3 Fab binds to amino acids 1-27 of CD3 epsilon. In some embodiments, the anti-CD3 Fab binds to amino acids 1-27 of a human CD3 epsilon. Amino acids 1-27 of CD3 epsilon are set forth in SEQ ID NO: 4.

[00248] A skilled person would appreciate that the term "Anti-CD3 Fab", may in some embodiments encompass the second binding domain of a precursor bispecific antibody construct described herein. In some embodiments, the term "Anti-CD3 Fab" may encompass the precursor construct, wherein reference is being made the to binding attributes of the second binding domain.

[00249] In some embodiments, a second binding domain of a precursor construct comprises a Fab. In some embodiments, when referring to a second binding domain of a precursor construct the term“Fab” will be used, wherein the term encompasses a second bind domain of a precursor construct. In some embodiments, the term“Fab” may be used interchangeably with the phrase “second binding domain” having all the same qualities and meanings. Fab comprise an antigen binding domain and include a variable region of heavy chain (VH), a variable region of light chain (VL), a constant region of heavy chain 1 (CHi) and a constant region of light chain (CL). In addition, the skilled artisan would recognize that the“Fd” region of the Fab is an essential component of the antigen-binding Fab fragment. The Fd region, similar to light chains, contains a C-terminal constant (CH1) and N-terminal variable (VH) domain. The hypervariable regions in both the light chain and Fd determine the specificity of the Fab binding domain.

[00250] In some embodiments, a precursor bispecific antibody construct comprises a second binding domain that binds to an extracellular epitope of CD3 epsilon. In some embodiments, a precursor bispecific antibody construct comprises a second binding domain that binds to the N- terminus of CD3 epsilon. In some embodiments, a precursor bispecific antibody construct comprises a second binding domain that binds to an epitope with amino acids 1-27 of CD3 epsilon. In some embodiments, the anti-CD3 Fab binds to amino acids 1-27 of CD3 epsilon. In some embodiments, the anti-CD3 Fab binds to amino acids 1-27 of a human CD3 epsilon. Amino acids 1-27 of CD3 epsilon are set forth in SEQ ID NO: 4.

[00251] "TCR complex," as used herein, refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can be composed of a CD3(gamma) chain, a CD3(delta) chain, two CD3(epsilon) chains, a homodimer of CD3(zeta) chains, a TCR( alpha) chain, and a TCR(beta) chain. Alternatively, a TCR complex can be composed of a CD3(gamma) chain, a CD3(delta) chain, two CD3(epsilon) chains, a homodimer of CD3(zeta) chains, a TCRY chain, and a TCR(delta) chain.

[00252] "A component of a TCR complex," as used herein, refers to a TCR chain (i.e., TCR(alpha), TCR(beta), TCRY or TCR(delta)), a CD3 chain (i.e., CD3(gamma), CD3(delta), CD3(epsilon) or CD3(zeta)), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCR( alpha) and TCR(beta), a complex of TCRY and TCR(delta), a complex of CD3(epsilon) and CD3(delta), a complex of CD3(gamma) and CD3(epsilon), or a sub-TCR complex of TCR( alpha), TCR(beta), CD3(gamma), CD3(delta), and two CD3(epsilon) chains).

[00253] By way of background, the TCR complex is generally responsible for initiating a T-cell response to antigen bound to MHC molecules. It is believed that binding of a peptide:MHC ligand to the TCR and a co-receptor (i.e., CD4 or CD8) brings together the TCR complex, the co-receptor, and CD45 tyrosine phosphatase. This allows CD45 to remove inhibitory phosphate groups and thereby activate Lck and Fyn protein kinases. Activation of these protein kinases leads to phosphorylation of the IT AM on the CD3(zeta) chains, which in turn renders these chains capable of binding the cytosolic tyrosine kinase ZAP-70. The subsequent activation of bound ZAP-70 by phosphorylation triggers three signaling pathways, two of which are initiated by the phosphorylation and activation of PLC-(gamma), which then cleaves phosphatidylinositol phosphates (PIPs) into diacylglycerol (DAG) and inositol trisphosphate (IP3). Activation of protein kinase C by DAG leads to activation of the transcription factor NFKB. The sudden increase in intracellular free Ca ²⁺ as a result of IP3 action activates a cytoplasmic phosphatase, calcineurin, which enables the transcription factor NFAT (nuclear factor of activated T-cells) to translocate form the cytoplasm to the nucleus. Full transcriptional activity of NFAT also requires a member of the AP-l family of transcription factors; dimers of members of the Fos and Jun families of transcription regulators.

[00254] A third signaling pathway initiated by activated ZAP-70 is the activation of Ras and subsequent activation of a MAP kinase cascade. This culminates in the activation of Fos and hence of the AP-l transcription factors. Together, NFKB, NFAT, and AP-l act on the T-cell chromosomes, initiating new gene transcription that results in the differentiation, proliferation and effector actions of T-cells. See, Pitcher et al., 2003., TRENDS in Immunol. 24, 554-560; Smith- Garvin et al., Annu Rev Immunol. 2009;27:591-619 .

[00255] In certain embodiments, the Fab specifically binds to an individual human CD3 chain (e.g., human CD3(gamma) chain, human CD3(delta) chain, or human CD3(epsilon) chain) or a combination of two or more of the individual human CD3 chains (e.g., a complex of human CD3(gamma) and human CD3(epsilon) or a complex of human CD3(delta) and human CD3(epsilon)). In certain embodiments, the Fab specifically binds to a human CD3(epsilon) chain. In certain embodiments, the Fab specifically binds to an extracellular epitope of a human CD3(epsilon) chain. In certain embodiments, the Fab specifically binds to an epitope within SEQ ID NO: 3.

[00256] In certain embodiments, the second binding domain specifically binds to an individual human CD3 chain (e.g., human CD3(gamma) chain, human CD3(delta) chain, or human CD3(epsilon) chain) or a combination of two or more of the individual human CD3 chains (e.g., a complex of human CD3(gamma) and human CD3(epsilon) or a complex of human CD3(delta) and human CD3(epsilon)). In certain embodiments, the second binding domain specifically binds to a human CD3(epsilon) chain. In certain embodiments, the second binding domain specifically binds to an extracellular epitope of a human CD3(epsilon) chain. In certain embodiments, the second binding domain specifically binds to an epitope within SEQ ID NO: 3.

[00257] In certain other embodiments, a Fab of the present disclosure comprising a second binding domain specifically binds to TCR(alpha), TCR(beta), or a heterodimer formed from TCR( alpha) and TCR(beta). In certain embodiments, a Fab specifically binds to one or more of human TCR(alpha), human TCR(beta), or a heterodimer formed from human TCR( alpha) and human TCR(beta).

[00258] In certain embodiments, a Fab of the present disclosure comprising a second binding domain binds to a complex formed from one or more CD3 chains with one or more TCR chains, such as a complex formed from a CD3(gamma) chain, a CD3(delta) chain, a CD3(epsilon) chain, a TCR( alpha) chain, or a TCR(beta) chain, or any combination thereof. In other embodiments, a Fab of the present disclosure binds to a complex formed from one CD3(gamma) chain, one CD3(delta) chain, two CD3(epsilon) chains, one TCR(alpha) chain, and one TCR(beta) chain. In further embodiments, a Fab of the present disclosure binds to a complex formed from one or more human CD3 chains with one or more human TCR chains, such as a complex formed from a human CD3(gamma) chain, a human CD3(delta) chain, a human CD3(epsilon), a human TCR(alpha) chain, or a human TCR(beta) chain, or any combination thereof. In certain embodiments, a Fab of the present disclosure binds to a complex formed from one human CD3(gamma) chain, one human CD3(delta) chain, two human CD3(epsilon) chains, one human TCR(alpha) chain, and one human TCR(beta) chain.

[00259] Fabs of this disclosure can be generated as described herein or by a variety of methods known in the art (see, e.g., U.S. Pat. Nos. 6,291,161; 6,291,158). Sources of Fabs include monoclonal antibody nucleic acid sequences from various species (which can be formatted as antibodies, Fvs, scFvs or Fabs, such as in a phage library), including human, camelid (from camels, dromedaries, or llamas; Hamers-Casterman et al. (1993) Nature, 363:446 and Nguyen et al. (1998) J. Mol. Biol., 275:413), shark (Roux et al. (1998) Proc. Nat'l. Acad. Sci. (USA) 95: 11804), fish (Nguyen et al. (2002) Immunogenetics, 54:39), rodent, avian, or ovine.

[00260] An anti-human CD3 antibody with cross reactivity to monkey CD3 is particularly desirable, such as the SP34 mouse monoclonal antibody, which binds specifically to human CD3 in denatured form (Western blot or dot blot) and in native form (on T-cells) (Pressano, S. The EMBO J. 4:337-344, 1985; Alarcon, B. EMBO J. 10:903-912, 1991). SP34 mouse monoclonal antibody also binds to CD3c singly transfected COS cells as well as CD3e/y or CD3.e/5 double transfectants (Salmeron A. et al., J. Immunol. 147:3047-52, 1991). SP34 antibody also cross reacts non-human primates (Yoshino N. et al., Exp. Anim 49:97-110, 2000; Conrad M L. et al., Cytometry 7lA:925-33, 2007). In addition, SP34 activates T-cell when cross-linked (Yang et al., J. Immunol. 137: 1097-1100, 1986). Cross-reactivity to monkey CD3 is important as this allows toxicity studies to be carried out in non-human primates using the clinical candidate directly, rather than in chimpanzee or using a surrogate molecule. Thus, toxicity studies using such cross-reactive anti- CD3 Fab in a precursor bispecific antibody construct of the present disclosure provide more relevant safety assessments.

[00261] Other illustrative anti-CD3 antibodies include the Cris-7 monoclonal antibody (Reinherz, E. L. et al. (eds.), Leukocyte typing II., Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172: 1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and derivatives thereof such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), visilizumab (Carpenter et al. (2002) Blood 99:2712), and 145- 2C11 monoclonal antibody (Hirsch et al. (1988) J. Immunol. 140: 3766). Further CD3 binding molecules contemplated for use herein include UCHT-l (Beverley, P C and Callard, R. E. (1981) Eur. J. Immunol. 11: 329-334) and CD3 binding molecules described in W02004/106380; W02010/037838; W02008/119567; W02007/042261; W02010/0150918, which are incorporated herein in their entirety.

[00262] In some embodiments, the amino acid sequence of a second binding region comprising an anti-CD3 epsilon binding activity comprises any anti-CD3 epsilon sequence known in the art. In some embodiments, the amino acid sequence of a second binding region comprising binding activity to an anti-CD3 epsilon or a derivative thereof or an antibody fragment thereof, comprises any anti-CD3epsilon sequence known in the art. Examples of known anti-CD3 epsilon amino acid sequences may be found for example but no limit to United States Patents Nos: 9,822,180; 9,493,563; 9,587,021; 9,562,073; United States Published Application Nos: 2013/0129729; 2017/0247476; 2016/0194399; 2010/0150918; 2018/0112011; and WO2017/162587, which all included herein in their entirety.

[00263] An exemplary anti-TCR antibody is H57 monoclonal antibody (Lavasani et al. (2007) Scandinavian Journal of Immunology 65:39-47).

[00264] Antigen binding fragment sequences (e.g., heavy and light chain variable region sequences) for Fab fragments may be available in public databases or using traditional strategies for hybridoma development using a CD3 chain, TCR component, or other Fab binding target as an immunogen in convenient systems (e.g., mice, HuMAb Mouse.RTM., TC Mouse.TM., KM- Mouse.RTM., llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop Fabs for use herein. As would be understood by the skilled person, Fab fragments may be generated using various technologies known in the art, including antibody display technologies such as phage, yeast, ribosome and mRNA display technologies; B cell culture technology such as SEAM technology; or using high throughput gene sequencing technologies on B cells or plasma B cells isolated from an immunized animal subject or immunized human subject.

[00265] In some embodiments, a second binding domain (an Fab) disclosed herein, comprises humanized FR amino acid sequence and native sequence of a mouse monoclonal antibody for the CDR amino acid sequences. Examples of anti-CD3 epsilon amino acid sequences wherein the FR sequences have been humanized while the CDR amino acid sequences remain those of the SP34 mouse monoclonal antibody, are disclosed in International Application Publication No. WO 2007/042261, which is incorporated here in its entirety.

[00266] Illustrative second binding domains (for example but not limited to anti-CD3 epsilon Fabs) sequences comprised within a precursor bispecific antibody construct of the present disclosure include the VH, CH1, VF, and CF amino acid sequences, and the polynucleotides encoding them, as set forth in Tables 1 and 2 below, wherein the amino acid sequences include SEQ ID NOs: 13-39, 42-79, 124, and 133-136 including CDRs thereof, such as those set forth in SEQ ID NOs: 71-79. In some embodiments, second binding domains (e.g., Fabs) sequences comprised within a precursor bispecific antibody constmct of the present disclosure include the VH, CH1, VF, and CF amino acid sequences, as set forth in Table 1, or a homolog thereof. In some embodiments, homologs of SEQ ID NOs: 13-39 42-70, 124, 133 andl35, maintain CDR regions, for example as set for the in SEQ ID NOs: 71-79.

[00267] Table 1: Amino Add Sequences of Anti-CD3 VH, VL, HC, LC, and CDR, and Combinations Thereof.

[00268] In some embodiments, a second binding domain binds a CD3 epsilon polypeptide. In some embodiments, a second binding domain binds an extracellular domain of a human CD3 epsilon polypeptide. In some embodiments, a second binding domain comprises an Fab fragment comprising a variable heavy chain region (VH) comprising a CDR-H1, a CDR-H2, and a CDR-H3, and a variable light chain region (VL) comprising a CDR-L1, a CDR-L2, and a CDR-L3, wherein the second binding domain binds an extracellular domain of a human CD3 epsilon polypeptide. In some embodiments, a second binding domain binds to an epitope within SEQ ID NO: 3. In some embodiments, a second binding domain binds SEQ ID NO: 4.

[00269] In some embodiments, the amino acid sequence of an anti-human CD3 epsilon CDR-H1 is set forth in SEQ ID NO: 71. In some embodiments, the amino acid sequence of an anti-human CD3 epsilon CDR-H2 is set forth in SEQ ID NO: 72. In some embodiments, the amino acid sequence of an anti-human CD3 epsilon CDR-H3 is set forth in SEQ ID NO: 73. In some embodiments, the amino acid sequence of an anti-human CD3 epsilon CDR-L1 is set forth in any one of SEQ ID NOs: 74-76. In some embodiments, the amino acid sequences of an anti-human CD3 3epsilon CDR-L1 is set forth in SEQ ID NO: 74. In some embodiments, the amino acid sequences of an anti-human CD3 3epsilon CDR-L1 is set forth in SEQ ID NO: 75. In some embodiments, the amino acid sequences of an anti-human CD3 3epsilon CDR-L1 is set forth in SEQ ID NO: 76. In some embodiments, the amino acid sequence of an anti-human CD3 epsilon CDR-L2 is set forth in SEQ ID NO: 77. In some embodiments, the amino acid sequence of an anti human CD3 epsilon CDR-L3 is set forth in any one of SEQ ID NOs: 78-79. In some embodiments, the amino acid sequences of an anti-human CD3 3epsilon CDR-L3 is set forth in SEQ ID NO: 78. In some embodiments, the amino acid sequences of an anti-human CD3 3epsilon CDR-L3 is set forth in SEQ ID NO: 79. In some embodiments, the amino acid sequences of an anti-human CD3 3epsilon CDR-L1, CDR-L2, andCDR-L3 is set forth in SEQ ID NOs: 74, 77, and 78.

[00270] In some embodiments, a second binding domain comprises an Fab fragment comprising a variable heavy chain region (VH) and a variable light chain region (VL) that binds an extracellular domain of a human CD3 epsilon polypeptide. In some embodiments, the amino acid sequence of VH and VL for an anti-human CD3 epsilon are selected from the amino acid sequences set forth in any of SEQ ID NO: 13-39, 42-79, 133, and 135. In some embodiments, the amino acid sequence of VH and VL for an anti-human CD3 epsilon comprises a homolog of sequences selected from the amino acid sequences set forth in any of SEQ ID NO: 13-39, 42-79, 133, and 135.

[00271] In some embodiments, the amino acid sequence of a VH for a human CD3 epsilon second binding domain (VH2) are selected from the amino acid sequences set forth in any of SEQ ID NOs: 13-39 and 133, or a homolog thereof. In some embodiments, the amino acid sequence of a VL for a human CD3 epsilon second binding domain (VL2) are selected from the amino acid sequences set forth in any of SEQ ID NOs: 42-70 and 135, or a homolog thereof. In some embodiments, the amino acid sequence of an anti-human CD3 epsilon VH2 is set forth in SEQ ID NO: 133. In some embodiments, an anti-human CD3 epsilon VH2 amino acid sequence comprises a homolog of SEQ ID NO: 133. In some embodiments, the amino acid sequence of an anti-human CD3 epsilon VL2 is set forth in SEQ ID NO: 135. In some embodiments, an anti-human CD3 epsilon VL2 amino acid sequence comprises a homolog of SEQ ID NO: 135.

[00272] In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence of variable light or variable heavy chains of anti-CD3epsilon. In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of variable light or variable heavy chains of anti-CD3epsilon. In some embodiments, homologues comprise polypeptides which are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to an anti-human CD3 epsilon VH or an anti-human CD3 epsilon VL, respectively, as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters.

[00273] In some embodiments, homology also encompasses deletion, insertion, or substitution variants, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof. In one embodiment, the variant comprises conservative substitutions, or deletions, insertions, or substitutions that do not significantly alter the three-dimensional structure of the polypeptide of interest, e.g., VL or VH region, particularly in the areas of the CDR epitope binding regions. In some embodiments, the deletion, insertion, or substitution does not alter the function of interest of the anti-human CD3 epsilon Fab, which in some embodiments, is binding to a CD3 epsilon sequence on a target T-cell.

[00274] In some embodiments, a second binding domain binding to a CD3 epsilon extracellular epitope VH2 includes the sequences set forth in SEQ ID NOs: 13-39 or 133, or a homolog thereof. In some embodiments, a second binding domain binding to a CD3 epsilon extracellular epitope VL2 includes the sequences set forth in SEQ ID NO: 42-70, or 135, or a homolog thereof. In some embodiments, a second binding domain binding to a CD3 epsilon extracellular epitope comprises a sequence selected from the sequences set forth in SEQ ID NOs: 13-39, 124, 133, and 134 or a homolog thereof, and a sequence selected from the sequences set forth in SEQ ID NOs: 41-70, 135, and 136, or a homolog thereof. In some embodiments, a second binding domain binding to a CD3 epsilon extracellular epitope comprises the sequence set forth in SEQ ID NO: 133 or a homolog thereof, and the sequence set forth in SEQ ID NO: 135 or a homolog thereof. In some embodiments, a second binding domain binding to a CD3 epsilon extracellular epitope comprises the sequence set forth in SEQ ID NO: 124 or a homolog thereof, and the sequence set forth in SEQ ID NO: 41 or a homolog thereof.

[00275] In some embodiments, the second binding domain VL region comprises amino acid sequences as set forth for CDR-L1 (selected from SEQ ID NO: 74-76), CDR-L2 (SEQ ID NO: 77), and CDR-L3 (selected from SEQ ID NOs: 78,79), and the second binding domain VH region comprises CDR-H1 (SEQ ID NO: 71), CDR-H2 (SEQ ID NO: 72), and CDR-H3 (SEQ ID NO: 73).

[00276] In some embodiments of a precursor bispecific antibody construct, the VL region of the second binding comprises the amino acid sequence set forth in any of SEQ ID NO: 42-70 and 135, or an amino acid sequence having at least 80% homology thereto. In some embodiments, a VL region of the second binding comprising an amino acid sequence having at least 80% homology thereto, comprises framework sequences having at least 80% homology, wherein the CDR regions are“as is” in the selected amino acid sequence (SEQ ID NO: 74-79).

[00277] In some embodiments of a precursor bispecific antibody construct, the VH region of the second binding comprises the amino acid sequence set forth in any of SEQ ID NO: 13-39 and 133, or an amino acid sequence having at least 80% homology thereto. In some embodiments, a VH region of the second binding comprising an amino acid sequence having at least 80% homology thereto, comprises framework sequences having at least 80% homology, wherein the CDR regions are“as is” in the selected amino acid sequence (SEQ ID NO: 71-73).

[00278] In some embodiments, a first binding domain comprises a humanized binding domain. In some embodiments, a second binding domain comprises a humanized binding domain.

[00279] As would be understood by the skilled person and as described herein, in some embodiments, a complete antibody comprises two heavy chains and two light chains (See for example Figure 3A - at left wherein it is shown illustrative source sequences for scFv and Fab comprised within an embodiments of a precursor bispecific antibody construct). Each heavy chain consists of a variable region and a first, second, and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as a, d. e, g, and m, and mammalian light chains are classified as l or K. Immunoglobins comprising the a, d. e, g, and m, heavy chains are classified as immunoglobin (Ig)A, IgD, IgE, IgG, and IgM. The complete antibody forms a "Y" shape. The stem of the Y consists of the second and third constant regions (and for IgE and IgM, the fourth constant region) of two heavy chains bound together and disulfide bonds (inter-chain) are formed in the hinge. Heavy chains g, a, and d have a constant region composed of three tandem (in a line) Ig domains, and a hinge region for added flexibility; heavy chains m and e have a constant region composed of four immunoglobulin domains. The second and third constant regions are referred to as "CH2 domain" and "CH3 domain", respectively. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. [00280] "Complementarity determining region" or "CDR" with regard to an antibody refers to a highly variable loop in the variable region of the heavy chain or the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs. The CDRs can be defined or identified by conventional methods, such as by sequence according to Rabat et al (Wu, T T and Rabat, E. A., J Exp Med. 132(2) :211-50, (1970); Borden, P. and Rabat E. A., PNAS, 84: 2440- 2443 (1987); Rabat, E. A. et al, Sequences of proteins of immunological interest, Published by DIANE Publishing, 1992), or by structure according to Chothia et al (Choithia, C. and Lesk, A. M., J. Mol. Biol., 196(4): 901-917 (1987), Choithia, C. et al, Nature, 342: 877-883 (1989)).

[00281] "Heavy chain variable region" or "VH" with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs.

[00282] "Light chain variable region" or "VL" with regard to an antibody refers to the fragment of the light chain that contains three CDRs interposed between framework regions.

[00283] "Fv" with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[00284] "Single-chain Fv antibody" or "scFv" with regard to an antibody refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence.

[00285] "Single domain camel antibody" or "camelid VHH" as used herein refers to the smallest known antigen-binding unit of a heavy chain antibody (Roch-Nolte, et al, FASEB J., 21: 3490-3498 (2007)). A "heavy chain antibody" or a "camelid antibody" refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al, J. Immunol. Methods 231:25-38 (1999); WO94/04678; W094/25591; U.S. Pat. No. 6,005,079).

[00286] "Single domain antibody" or "dAb" refers to an antibody fragment that consists of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL domain) (Holt, L., et al, Trends in Biotechnology, 21(11): 484-490).

[00287] The term "disulfide bond" as used herein refers to the binding of a heavy chain fragment and a light chain fragment through one or more disulfide bonds. The one or more disulfide bonds can be formed between the two fragments by linking the thiol groups in the two fragments. In certain embodiments, the one or more disulfide bonds can be formed between one or more cysteine residues in the heavy chain fragment and the light chain fragment, respectively.

[00288] A "variable region linking sequence" is an amino acid sequence that connects a heavy chain variable region to a light chain variable region and provides a linker function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody that comprises the same light and heavy chain variable regions. In certain embodiments, a hinge useful for linking a binding domain to an immunoglobulin CH2 or CH3 region polypeptide may be used as a variable region linking sequence.

[00289] In some embodiments, a second binding domain comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein said first binding domain is located N- terminally to said VL region or VH region of said second binding domain. In some embodiments, a second binding domain comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein said first binding domain is located N-terminally to said VL region of said second binding domain, and a regulatory domain is located N-terminally to said VH region of said second binding domain. In some embodiments, a second binding domain comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein said first binding domain is located N-terminally to said VH region of the second binding domain, and the regulatory domain is located N-terminally to said VL region of the second binding domain. In some embodiments, a second binding domain of a precursor construct comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein said first binding domain is located N-terminally to said VL or VH region of the second binding domain, wherein when said first binding domain is located N-terminally to said VL region of said second binding domain, said regulatory domain is located N-terminally to said VH region of said second binding domain, and when said first binding domain is located N-terminally to said VH region of said second binding domain, said regulatory domain is located N-terminally to said VL region of said second binding domain.

[00290] An alternative source of binding domains may include sequences that encode random peptide libraries or sequences that encode an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds, such as fibrinogen domains (see, e.g., Weisel et al. (1985) Science 230: 1388), Kunitz domains (see, e.g., U.S. Pat. No. 6,423,498), lipocalin domains (see, e.g., WO 2006/095164), V-like domains (see, e.g., US Patent Application Publication No. 2007/0065431), C-type lectin domains (Zelensky and Gready (2005) FEBS J. 272:6179), or Fcab.TM. (see, e.g., PCT Patent Application Publication Nos. WO 2007/098934; WO 2006/072620), or the like.

[00291] As depicted in the Figures 1A-1C, 2A-2f and 3A-3B, scFv are particularly illustrative binding domains. A first binding domain, which in some embodiments comprises an scFv fragment, may bind to any of a variety of target molecules, including but not limited to a FcyRI, FcyRIIa FcyRIIb FcyRIIIa FcyRIIIb, CD28, CD137, CTLA-4, FAS, fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, glucocorticoid-induced TNFR-related (GITR) protein, lymphotoxin-beta receptor (LTpR), toll -like receptors (TLR), tumor necrosis factor-related apoptosis-inducing ligand-receptor 1 (TRAIL receptor 1) and TRAIL receptor 2, pro state- specific membrane antigen (PSMA) protein, prostate stem cell antigen (PSCA) protein, tumor-associated protein carbonic anhydrase IX (CAIX), epidermal growth factor receptor 1 (EGFR1), EGFRvIII, human epidermal growth factor receptor 2 (Her2/neu; Erb2), ErbB3 also known as HER3, Folate receptor, ephrin receptors, PDGFRa, ErbB-2, CD20, CD22, CD30, CD33, CD40, CD37, CD38, CD70, CD74, CD56, CD40), CD80, CD86, CD2, p53, cMet also known as tyrosine-protein kinase Met or hepatocyte growth factor receptor (HGFR), MAGE-A1, MAGE-A2, MAGE-A3, MAGE- A4, MAGE-A6, MAGE- A 10, MAGE-A12, BAGE, DAM-6, -10, GAGE-l, -2, -8, GAGE-3, -4, - 5, -6, -7B, NA88-A, NY-ESO-l, BRCA1, BRCA2, MART-l, MC1R, GplOO, PSA, PSM, Tyrosinase, Wilms' tumor antigen (WT1), TRP-l, TRP-2, ART -4, CAMEL, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, P-cadherin, Myostatin (GDF8), Cripto (TDGF1), MUC5AC, PRAME, P15, RU1, RU2, SART-l, SART-3, WT1, AFP, b-catenin/m, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-l, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin P, CDC27/m, TPEmbcr-abl, ETV6/AML, LDLR/FUT, Pml/RARa, TEL/AML 1, CD28, CD137, CanAg, Mesothelin, DR5, PD-l, PD1L, IGF-1R, CXCR4, Neuropilin 1, Glypicans, EphA2, CD138, B7-H3, gpA33, GPC3, SSTR2, ROR1, 5T4, or a VEGF-R2. In some embodiments, a TAA comprises a PSMA, CD30, B7-H3, gpA33, HER2, P- cadherin, gplOO, DR5, GPC3, SSTR2, Mesothelin, ROR1, 5T4, Folate receptor, or an EGFR. These and other tumor proteins or tumor associated proteins are known to the skilled artisan.

[00292] In certain embodiments, the first binding domain specifically binds to an antigen target that is associated with a disease condition. The disease condition may include a physiological condition, a pathological condition and a cosmetic condition. Examples of illustrative conditions include, without limitation, cancer, inflammatory disorders, allograft transplantation, type I diabetes, type II diabetes, and multiple sclerosis.

[00293] In some embodiments, the specific structural components of a precursor bispecific antibody construct comprise a first binding domain, for example but not limited to an scFv fragment, a second binding domain, for example but not limited to an Fab fragment, linker regions, and a regulatory domain, where said regulatory domain may comprise a protease cleavable domain, an HSA polypeptide sequence, a CAP component, and linkers, or any combination thereof, as have been described herein detail.

Precursor bispecific polypeptides

[00294] In some embodiments, a precursor bispecific antibody construct comprises two polypeptides. In some embodiments, polypeptide A comprises components having an order N- terminal to C-terminal: the regulatory domain (R), the CD3 second binding domain VH chain (VH2), a constant Heavy chain domain (CH1). In other embodiments, polypeptide A comprises components having an order N-terminal to C-terminal: the regulatory domain (R), the CD3 second binding domain VL chain (VL2), a constant Light chain domain (CL). In some embodiments, polypeptide B comprises components having an order N-terminal to C-terminal: the TAA first binding domain (VL1-L-VH1), the CD3 second binding domain VL region (VL2), a constant Light chain domain (CL). In some embodiments, polypeptide B comprises components having an order N-terminal to C-terminal: the TAA first binding domain (VL1-VH1), the CD3 second binding domain VH region (VH2), a constant Heavy chain domain (CH1).

[00295] A skilled artisan would appreciate that the first TAA comprised in a polypeptide of a precursor bispecific construct may comprise any TAA disclosed herein, for example but not limited to wherein the TAA is EGFR or 5T4 or ROR1.

[00296] A skilled artisan would appreciate that the designations “ Polypeptide A” and “ Polypeptide B” are merely names indicating two heterologous polypeptide chains, and as such the names themselves may be interchanged or change, e.g., Polypeptide 1 and Polypeptide 2. Further, encompassed by this terminology is two structurally different polypeptide chains that together form a precursor bispecific antibody construct, as described herein.

[00297] In some embodiments, a precursor bispecific antibody construct described herein comprises two polypeptides as follows (order is N-terminal to C-terminal)

Polypeptide A: R-L-VH2-L-CH1,

Polypeptide B VL 1 -L- VH 1 -L- VL2-L-CL,

wherein“L” is a linker, which may or may not be present in each embodiment (Figure 1A).

[00298] In some embodiments, a precursor bispecific antibody construct comprises two polypeptides as follows (order is N-terminal to C-terminal)

Polypeptide A\ R-L-VL2-L-CL

Polypeptide B VL 1 -L- VH 1 -L- VH2-L-CH 1 ,

wherein“L” is a linker, which may or may not be present in each embodiment (not shown).

[00299] As described above in detail, in some embodiments, the order of components comprising a regulatory domain is as follows (order is N-terminal to C-terminal):

CAP-L-HSA-L-Protease cleavage domain-L (Figures 1A, IB, 2A, and 2B); or CAP-L-Protease cleavage domain-L-HSA-L (Figure 2C); or

HSA-L-Protease cleavage domain-L (Figure 2D); or

CAP-L-Protease cleavage domain-L (Figure 2E);

wherein“L” is a linker, which may or may not be present in each embodiment.

[00300] A skilled artisan would appreciate that different regulatory domains may be included with a precursor construct depending on the desired functionality of the precursor construct. Thus, embodiments of a precursor bispecific antibody construct comprising two polypeptides, as described herein includes but is not limited to (order is N-terminal to C-terminal):

(i) Polypeptide A: CAP-L-HSA-L-Pro tease cleavage domain-L-VH2-L-CHl,

Polypeptide B: VL 1 -L- VH 1 -L- VL2-L-CL,

wherein“L” is a linker, which may or may not be present in each embodiment (Figures 1A and 2B);

(ii) Polypeptide A: CAP-L-Protease cleavage domain-L-HSA-L-VH2-L-CHl,

Polypeptide B: VL1 -L- VH 1 -L- VL2-L-CL,

wherein“L” is a linker, which may or may not be present in each embodiment (Figure 2C);

(iii) Polypeptide A: HSA-L-Protease cleavage domain-L-VH2-L-CHl,

Polypeptide B: VL1 -L- VH 1 -L- VL2-L-CL,

wherein“L” is a linker, which may or may not be present in each embodiment (Figure 2D);

(iv) Polypeptide A: CAP-L-Protease cleavage domain-L-VH2-L-CHl,

Polypeptide B: VL1 -L- VH1 -L- VL2-L-CL,

wherein“L” is a linker, which may or may not be present in each embodiment (Figure 2E);

(v) Polypeptide A: CAP-L-HSA-L-Protease cleavage domain-L- VL2-L-CL

Polypeptide B: VL 1 -L- VH 1 -L- VH2-L-CH 1 ,

wherein“L” is a linker, which may or may not be present in each embodiment;

(vi) Polypeptide A: CAP-L-Protease cleavage domain-L-HSA-L- VL2-L-CL

Polypeptide B: VL 1 -L- VH 1 -L- VH2-L-CH 1 ,

wherein“L” is a linker, which may or may not be present in each embodiment;

(vii) Polypeptide A: HSA-L-Protease cleavage domain-L- VL2-L-CL

Polypeptide B: VL 1 -L- VH 1 -L- VH2-L-CH 1 ,

wherein“L” is a linker, which may or may not be present in each embodiment; and

(viii) Polypeptide A: CAP-L-Protease cleavage domain-L- VL2-L-CL

Polypeptide B: VL 1 -L- VH 1 -L- VH2-L-CH 1 ,

wherein“L” is a linker, which may or may not be present in each embodiment.

[00301] In certain embodiments, a first binding domain and a regulatory domain are linked directly to the N-terminus of the VH or VL of the second binding domain, e.g., an Fab (i.e., with no additional amino acids added between). In other embodiments, a first binding domain and a regulatory domain are linked to the N-terminus of the VH or VL of the second binding domain, e.g., an Fab, using a linker as described above (with additional amino acids as described below). In some embodiments, it may be necessary to delete several amino acids (e.g., from 1-3 amino acids or from 1-10 amino acids; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) from the C-terminus of a given first binding domain and/or a regulatory domain, depending on the second binding domain target and the surrounding space of the second domain target on the cell surface (i.e., for example, accessibility of the CD3 epsilon target on the cell surface of a T-cell).

[00302] In other embodiments, it may be necessary to delete several amino acids (e.g., from 1-3 amino acids or from 1-10 amino acids; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) from the N- terminus of the heavy and/or light chain of the second binding domain. In yet further embodiments, it may be necessary to delete several amino acids (e.g., from 1-3 amino acids or from 1-10 amino acids; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) from the C-terminus of the first binding domain and/or the regulatory domain, and at the same time, to delete several amino acids (e.g., from 1-3 amino acids or from 1-10 amino acids; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) from the N- terminus of a second binding domain chain (VH or VL). The length and the sequence of the junction between a first binding domain, a regulatory domain, and the second binding domain VH and VL chains can be the same or different.

[00303] The junction between the first binding domain and /or the regulatory domain, and the second binding domain VH and VL chains may make use of a combination of deletions and linkers as needed. As would be understood by the skilled artisan, the junction between the second binding domain VH and VL chains and the first binding domain and/or the regulatory domain can be adjusted accordingly and tested for desired functionality (e.g., binding affinity, T-cell activity) using methods known in the art and described herein.

[00304] As described herein, junctions between domain or between components within domains comprises linkers. In some embodiments, a linker is present between domains. In some embodiments, there is not a linker between domains. In some embodiments, a linker is present between components that comprise a domain. In some embodiments, there is not a linker between components that comprise a domain.

[00305] Figures 1A-1C and 2A-2E show graphically where linkers may exist in an embodiment of precursor bispecific antibody constructs disclosed herein.

[00306] In some embodiments, the linker between a first binding domain and a second binding domain VH or VL is 1-10 amino acids long. In other embodiments, the linker between a first binding domain and a second binding domain VH or VL is 1-20 or 20 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long.

[00307] In some embodiments, the linker between a regulatory domain and a second binding domain VH or VL is 1-10 amino acids long. In other embodiments, the linker between a regulatory domain and a second binding domain VH or VL is 1-20 or 20 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In further embodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. In some embodiments, the linker between components within a first binding domain is 1-10 amino acids long. In other embodiments, the linker between components within a first binding domain is 1-20 or 20 amino acids long. In this regard, the linker between components may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In further embodiments, the linker between components may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long.

[00308] In some embodiments, the linker between components within a regulatory domain is 1- 10 amino acids long, wherein it should be understood that a linker between different components need not be the same length. In other embodiments, the linker between components within a regulatory domain is 1-20 or 20 amino acids long, wherein it should be understood that a linker between different components need not be the same length. In this regard, the linker between each set of components may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In further embodiments, the linker between each set of components may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long.

[00309] In certain embodiments, linkers suitable for use in the precursor constructs described herein are flexible linkers. Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[00310] In some embodiments, flexible linkers include glycine polymers (G) _n , glycine- serine polymers (including, for example, (GS) _n , (GSGGS) _n (SEQ ID NO: 81) and (GGGS) _n (SEQ ID NO: 82), where n is an integer of at least one, for example but not limited to a flexible linker comprising (GS) ₂ wherein the amino acid sequence is GSGS (SEQ ID NO: 80)), glycine-alanine polymers, alanine- serine polymers, and other flexible linkers known in the art. Glycine and glycine- serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173- 142 (1992)). In some embodiments, flexible linkers include, but are not limited to Gly-Gly-Ser-Gly (GGSG; SEQ ID NO: 83), Gly-Gly-Ser-Gly-Gly (GGSGG; SEQ ID NO: 84), Gly-Ser-Gly-Ser- Gly (GSGSG; SEQ ID NO: 85), Gly-Ser-Gly-Gly-Gly (GSGGG; SEQ ID NO: 86), Gly-Gly-Gly- Ser-Gly (GGGSG; SEQ ID NO: 87), Gly-Ser-Ser-Ser-Gly (GSSSG; SEQ ID NO: 88), and the like. The ordinarily skilled artisan will recognize that design of a precursor bispecific antibody construct can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired precursor bispecific antibody construct structure.

[00311] In some embodiments, a flexible linker used in a precursor construct comprises any flexible linker known in the art. In some embodiments, a flexible linker comprises a flexible unstructured linker. Linkers known in the art have been describe at least in Chengcheng Liu, Ju Xin Chin, Dong-Yup Lee; SynLinker: an integrated system for designing linkers and synthetic fusion proteins, Bioinformatics, Volume 31, Issue 22, 15 November 2015, Pages 3700-3702; Fusion protein linkers: property, design and functionality Chen X et al, Adv Drug Deliv Rev. 2013 Oct;65(l0): 1357-69; The Linker Data base provided by The Centre for Integrative Bioinformatics vrije Universiteit Amsterdam (http://www.ibi.vu.nl/programs/linkerdbwww); and the CSD Linker Database provided by The Cambridge Crystallographic Data Centre (https://www.ccdc.cam.ac.uk/solutions/partnersoftware/csdlin kerdatabase/).

[00312] In certain embodiments, the linker between the second binding domain and the first binding domain or the regulatory domain, or both is a stable linker (not cleavable by protease, especially MMPs). In certain embodiments, the linker is a peptide linker. In certain embodiments, the precursor construct comprises a stable peptide linker, and the N-terminal of the peptide linker is covalently linked to the C-terminal of the fusion moiety, and the C terminal of the peptide linker is covalently linked to the N-terminal of the antigen-binding domain.

[00313] In certain embodiments, the linker between the second binding domain and the first binding domain or the regulatory domain, or both is a protease cleavable linker (not cleavable by protease, especially MMPs). In some embodiments, the linker between the second binding domain VH or VL chains and a first binding domain or a regulatory domain or both, comprises a protease substrate cleavage sequence, for example, an MMP substrate cleavage sequence. A well known peptide sequence of PLGLAG (SEQ ID NO: 6) in a substrate can be cleaved by most MMPs. Substrate sequences that can be cleaved by MMPs have been extensively studied. A protease substrate cleavage sequence refers to a peptide sequence that can be cleaved by protease treatment. An MMP substrate sequence refers to a peptide sequence that can be cleaved by incubation with a MMP. SEQ ID NO: 6 is a commonly used MMP substrate cleavage sequence (see e.g., Jiang, PNAS (2004) 101:17867-72; Olson, PNAS (2010) 107:4311-6). In another embodiment, the protease cleavage site is recognized by MMP-2, MMP-9 or a combination thereof. In yet another embodiment, the protease site comprises the sequence selected from the group consisting of GPLGMLSQ (SEQ ID NO: 89) and GPLGLWAQ (SEQ ID NO: 90). A stable linker or a protease non-cleavable linker refers to a linker peptide sequence that does not belong to the known protease substrate sequences and thus does not lead to significant cleavage product formation upon incubation with a protease.

[00314] In some embodiments, the cleavage substrate (or cleavage sequence or protease cleavage domain) of the linker may include an amino acid sequence that can serve as a substrate for a protease, usually an extracellular protease. In other embodiments, the cleavage sequence comprises a cysteine-cysteine pair capable of forming a disulfide bond, which can be cleaved by action of a reducing agent. In other embodiments the cleavage sequence comprises a substrate capable of being cleaved upon photolysis.

[00315] The cleavage substrate is positioned in the linker such that when the cleavage substrate is cleaved by a cleaving agent (e.g., a cleavage substrate of a linker is cleaved by the protease and/or the cysteine-cysteine disulfide bond is disrupted via reduction by exposure to a reducing agent) or by light-induced photolysis, in the presence of a target, resulting in cleavage products having various functional properties as described herein.

[00316] The cleavage substrate of a linker may be selected based on a protease that is co-localized in the diseased tissue, or on the surface of the cell that expresses the target antigen of interest of a binding domain of a fusion moiety. A variety of different conditions are known in which a target of interest is co-localized with a protease, where the substrate of the protease is known in the art. In the example of cancer, the target tissue can be a cancerous tissue, particularly cancerous tissue of a solid tumor. There are reports in the literature of increased levels of proteases having known substrates in a number of cancers, e.g., solid tumors. (See, e.g., La Rocca et al, (2004) British J. of Cancer 90(7): 1414-1421). Non-limiting examples of disease include: all types of cancers (breast, lung, colorectal, prostate, head and neck, pancreatic, etc), rheumatoid arthritis, Crohn's disease, melanomas, SLE, cardiovascular damage, ischemia, etc. Furthermore, anti-angiogenic targets, such as VEGF, are known. As such, where the binding domain of a fusion moiety of the precursor bispecific antibody construct of the present disclosure is selected such that it is capable of binding a tumor antigen, a suitable cleavage substrate sequence for the linker will be one which comprises a peptide substrate that is cleavable by a protease that is present at the cancerous treatment site, particularly that is present at elevated levels at the cancer treatment site as compared to non- cancerous tissues. In one exemplary embodiment, the binding domain of a precursor bispecific antibody construct can bind, e.g., Her2 and the cleavage substrate sequence can be a matrix metalloprotease (MMP) substrate, and thus is cleavable by an MMP. In other embodiments, the binding domain of a fusion moiety in the precursor bispecific antibody construct can bind a target of interest and the cleavage substrate present in the linker can be, for example, legumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, or PSA. In other embodiments, the cleave substrate is cleaved by other disease-specific proteases, in diseases other than cancer such as multiple sclerosis or rheumatoid arthritis.

[00317] The unmodified or uncleaved linker can allow for tethering the first, second, and regulatory domains.

[00318] The linkers of the precursor bispecific antibody construct (e.g., the linker between the VH of the second binding domain and a first binding domain and the linker between the VL of the second binding domain and a regulatory domain, or vice versa with regard to the VH and VL of the second binding domain) can comprise the same cleavage substrate or may comprise different cleavage substrates, e.g., the first linker may comprise a first cleavage substrate and the second linker may comprise a second cleavage substrate. The first and second cleavage substrates can be different substrates for the same enzyme (for example exhibiting different binding affinities to the enzyme), or different substrates for different enzymes, or the first cleavage substrate can be an enzyme substrate and the second cleavage substrate can be a photolysis substrate, or the first cleavage substrate can be an enzyme substrate and the second cleavage substrate can be a substrate for reduction, and the like. Or, in some embodiments, one of the linkers may be non-cleavable while the other is a cleavable linker. Thus, in some embodiments, a linker between the second binding domain and a regulatory domain is cleavable while the linker between the second binding domain and the first binding domain is not cleavable. In some embodiments, a linker between the second binding domain and a first binding domain is cleavable while the linker between the second binding domain and the regulatory domain in not cleavable. In some embodiments, it may be that a linker between the second binding domain and a regulatory domain and the linker between the second binding domain and the first binding domain are not cleavable, but there is a cleavable linker within the regulatory domain (i.e., between regulatory domain components).

[00319] For specific cleavage by an enzyme, contact between the enzyme and the cleavage substrate is made. When the precursor bispecific antibody construct is present within a microenvironment comprising sufficient enzyme activity, the cleavage substrate can be cleaved. Sufficient enzyme activity can refer to the ability of the enzyme to make contact with the linker having the cleavage substrate and effect cleavage. It can readily be envisioned that an enzyme may be in the vicinity of the precursor bispecific antibody construct but unable to cleave because of other cellular factors or protein modification of the enzyme.

[00320] In some embodiments, substrates can include but are not limited to substrates cleavable by one or more of the following enzymes or proteases: ADAM10; Caspase 8, Cathepsin S, MMP 8, ADAM 12, Caspase 9, FAP, MMP 9, ADAM 17, Caspase 10, Granzyme B, MMP-13, ADAMTS, Caspase 11, Guanidinobenzotase (GB), MMP 14, ADAMTS5. Caspase 12, Hepsin, MT-SP1, BACE, Caspase 13, Human Neutrophil Elastase Neprilysin (HNE), Caspases, Caspase 14, Legumain, NS3/4A, Caspase 1, Cathepsins, Matriptase 2, Plasmin, Caspase 2, Cathepsin A, Meprin, PSA, Caspase 3, Cathepsin B, MMP 1, PSMA, Caspase 4, Cathepsin D, MMP 2, TACE, Caspase 5, Cathepsin E, MMP 3, TMPRSS 3/4, Caspase 6, Cathepsin K, MMP 7, uPA, Caspase 7, Matripase (MT-SP1, TADG-15, epithin, ST 14), and MT1-MMP.

[00321] In other embodiments, the cleavage substrate can involve a disulfide bond of a cysteine pair, which is thus cleavable by a reducing agent such as, for example, but not limited to a cellular reducing agent such as glutathione (GSH), thioredoxins, NADPH, flavins, ascorbate, and the like, which can be present in large amounts in tissue of or surrounding a solid tumor.

Other appropriate protease cleavage sites for use in the cleavable linkers herein are known in the art or may be identified using methods such as those described by Turk et al., 2001 Nature Biotechnology 19, 661-667.

[00322] In certain embodiments, the linker can be a peptide linker, a thiol residue-containing peptide linker, such as a cysteine residue, a polymer linker or a chemical linker. In certain embodiments, the precursor bispecific antibody construct comprises a linker where one end of the linker is covalently linked to the C-terminal of the fusion moiety, and the other end of the linker is covalently linked to the N-terminal of the VH or VL of the second binding domain.

[00323] In some embodiment, there is just one or a few amino acids between domains or components within domains. In certain embodiments, there may be one or a few amino acid residues between two domains of a precursor bispecific antibody construct, such as between a binding domain and a linker polypeptide, such as amino acid residues resulting from construct design of the precursor construct (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a the polypeptide chains (polypeptide A and polypeptide B). As described herein, such amino acid residues may be referred to "junction amino acids" or "junction amino acid residues", or "peptide linkers".

[00324] In certain illustrative embodiments, a peptide linker is between 1 to 5 amino acids, between 5 to 10 amino acids, between 5 to 25 amino acids, between 5 to 50 amino acids, between 10 to 25 amino acids, between 10 to 50 amino acids, between 10 to 100 amino acids, or any intervening range of amino acids. In other illustrative embodiments, a peptide linker comprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids in length.

[00325] Such junctional amino acids link any of the domains or components within domain of the precursor bispecific antibody construct. In certain embodiments, the junctional amino acid(s) is a hinge, or a part of a hinge as defined herein. In certain embodiments, a variable region linking sequence useful for connecting a heavy chain variable region to a light chain variable region may be used as a peptide linker.

[00326] In one illustrative embodiment, peptide linker sequences contain, for example, Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala, may also be included in the linker sequence.

[00327] Other amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180, incorporated herein in their entirety.

[00328] Other illustrative linkers may include, for example, Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly- Ser-Glu-Ser-Lys-Val-Asp (EGKSSGSGSESKVD; SEQ ID NO: 91) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 1066-1070) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala- Gln-Phe-Arg-Ser-Leu-Asp (KESGSVSSEQLAQFRSLD; SEQ ID NO: 92) (Bird et al., 1988, Science 242:423-426).

[00329] In some embodiments, linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. Two coding sequences or domains of the precursor bispecific antibody construct of the present disclosure can be fused directly without any junctional amino acids or by using a flexible polylinker composed, for example, of the pentamer Gly-Gly-Gly-Gly-Ser (GGGGS; SEQ ID NO: 93) repeated 1 to 3 times. Such a linker has been used in constructing single chain antibodies (scFv) by being inserted between VH and VL (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5979-5883).

[00330] A peptide linker, in certain embodiments, is designed to enable the correct interaction between two beta-sheets forming the variable region of the single chain antibody. Any suitable linkers can be used to make an indirect link, such as without limitation, peptide linker, polymer linker, and chemical linker. In certain embodiments, the covalent link is an indirect link through a peptide linker.

[00331] A distinguishing characteristic of the precursor bispecific antibody construct, as described herein, is that the precursor construct does not depend on steric hindrance to reduce or inhibit the binding affinity of the second binding domain. In the case of the precursor constructs described herein, reduction or inhibition of binding affinity is due to specific binding between a CAP component and a second binding domain of the precursor construct. On the contrary, the proteins described in US 2012/0321626 depend solely on three-dimensional structure for reduced specificity, wherein the polypeptides may or may not form such a three-dimensional structure, and thus they lack any specificity for the reduction or inhibition of binding to the antibody target of the second binding region. Further distinguishing characteristics in comparison with other multi- or bispecific antibodies that include a mask, is that the reduction or inhibition of binding to a target of the second binding domain by precursor bispecific antibody construct, may also be temporally controlled, wherein reduction or inhibition of binding to a target of the second binding domain may be maintained when the precursor construct is in circulation in vivo or within a non-tumor microenvironment. This may reduce negative side effects caused by the use of multi- or bispecific antibodies lacking this temporal regulation.

[00332] Illustrative precursor bispecific antibody construct of the present disclosure comprise a Polypeptide A comprising the amino acid sequence set forth in SEQ ID NO: 106, or a homologue thereof; and a Polypeptide B comprising any one of the amino acid sequences set forth in SEQ ID NOs: 112 and 114.

Functionality of Precursor Bispecific Antibody Constructs

[00333] In some embodiments, the precursor bispecific antibody constructs of this disclosure comprising a first binding domain binding to a cell surface tumor associated antigen (TAA), a second binding domain binding to an extracellular epitope of human CD3 epsilon, and a regulatory domain, possesses many unique features and these features can be utilized to develop human therapeutics with desirable attributes in drug safety, efficacy and manufacturability. These features have been described in detail above and will not necessarily be repeated herein. A skilled artisan would appreciate that the uses as described herein below, include use of the many embodiments of a precursor bispecific antibody construct as described above.

[00334] As described herein, the property of a precursor construct comprising a regulatable extended half-life, a regulatory reduction of T-cell binding (reduction of T-cell activation), or a combination thereof, may be used advantageously in the precursor bispecific antibody construct of the present disclosure to mask T-cell binding until the precursor bispecific antibody construct are in an appropriate microenvironment (e.g., in the vicinity of a tumor). In some embodiments, a pharmaceutical composition comprises a precursor bispecific antibody construct, as described herein, and a pharmaceutically acceptable carrier. [00335] A skilled artisan would recognize that in some embodiments, the term“precursor bispecific antibody construct” may be used interchangeably with the term“drug” having all the same meanings and qualities. In some embodiments, a drug comprising a precursor bispecific antibody construct comprises a pharmaceutical composition.

[00336] In some embodiments, a precursor bispecific antibody disclosed herein comprises a first binding domain binding to a TAA, a second binding domain binding to an extracellular epitope of human CD3e, and a regulatory domain. A precursor bispecific antibody comprising a first binding domain binding to a TAA, a second binding domain binding to an extracellular epitope of human CD3e, and a regulatory domain comprising for example, a cleavable half-life prolonging domain comprising a protease cleavable domain and a human serum albumin (HSA) polypeptide, provides unique properties as described throughout.

[00337] The precursor bispecific antibody construct of the present disclosure functions to enhance drug stability, specificity, selectivity, potency, and safety and the convenience of drug administration. In certain embodiments, the second binding domain, when expressed without the regulatory domain fused to its N-terminus (VH or VL chain), is able to bind to its target antigen in soluble recombinant form (usually the extracellular domain of a receptor protein, e.g., a T-cell receptor component such as CD3) as well as on the cell surface. In certain embodiments, the second binding domain, when expressed with a regulatory domain fused to its N-terminus (VL or VH chain) and a first binding domain fused to the N-terminus (VL or VH chain- alternative chain to which the regulatory domain is fused), has no binding or has reduced binding to its specific antigen presented on cell surface at pharmacological concentrations of the drug (concentration of the polypeptide in treated patients) compared with a second binding domain alone. Lack of binding or greatly reduced binding to a cell surface antigen in the absence of the target antigen binding by a second binding domain may be explained by the dramatically reduced affinity resulting from the specific blocking of the antigen binding site by a CAP component.

[00338] Lack of binding or greatly reduced binding to cell surface antigen, for example an antigen on a T-cell, in the absence of the TAA target antigen binding by a first binding domain may be viewed as a desirable property for use of a precursor bispecific antibody construct as a human therapeutic. It is important to note that lack of binding or significantly reduced binding of precursor bispecific antibody construct alone (in the absence of tumor target cells) to, e.g., T-cell can, 1) dramatically improve the undesirable systematic T-cell activation, therefore to dramatically improve the drug safety profile; 2) dramatically improves the feasibility of subcutaneous route of drug administration; and 3) dramatically increase the drug tolerability of high drug concentration in blood circulation. Further, the regulatable temporal regulation provided by a half-life prolonging component (e.g., an HSA polypeptide) of a precursor construct may ensure the extended presence of the precursor construct in circulation until such time as the drug is present in the environment of TAA target cells (e.g., tumor target cell microenvironment.

[00339] It is important to note that T-cell binding by antibodies such as OKT3 or UCHT-l via conformational epitopes may transduce partial signaling, leading either to unwanted T-cell activation (causing cytokine storm) or T-cell anergy (resulting in T-cells unable to kill tumor cells). Mu-lF3, hu-lF3 and its variants binding to a linear epitope of CD3 is conceivably less likely to induce T-cell signaling in the absence of cross linking of the CD3. This property may be advantageous for reducing systemic side effects that occur when using OKT3 and UCHT-l like antibodies.

[00340] It is also important to note that once a regulatory domain or a portion thereof in a precursor bispecific antibody construct is cleaved by protease, it functions such that the specific binding inhibition at the second antigen-binding site (e.g., CD3 epsilon binding site) is removed so that it can then bind to its target with high affinity, particularly target antigens expressed on the cell surface. Therefore, following cleavage at the cleavage substrate sequence in a protease cleavable linker (thereby releasing a fusion moiety) the precursor bispecific antibody construct is converted into a more potent cross linker between tumor and T-cells (Figure 3B).

[00341] Furthermore, it is important to note that once a TAA first binding domain binds to its target antigen, the precursor bispecific antibody construct molecules become highly concentrated on a tumor cell surface to create high avidity based binding toward the second binding domain target (e.g. CD3) on T-cells. Therefore, only in the presence of the TAA first binding domain is the second antigen-binding domain able to bind its target thus for precursor bispecific antibody construct to function as a cross-linker between tumor and T-cells.

[00342] The properties of the precursor bispecific antibody construct of the present disclosure allow for relatively high dose of the precursor bispecific antibody construct in circulation for an enhanced period of time, without unwanted side-effects (e.g., the precursor bispecific antibody construct does not bind to the second binding domain target antigen (e.g., CD3) when in circulation. This also allows for reduced dosing frequency and promotes tissue penetration by diffusion driven by concentration gradient.

[00343] The properties of the precursor bispecific antibody construct of the present disclosure also allow the potential for the subcutaneous administration, which can enhance access to the target. Further, although in certain embodiments the precursor bispecific antibody construct are permissive for cross linking without protease treatment, in certain embodiments, the binding activity and the tumor killing potency increase dramatically after protease treatment. [00344] In one embodiment, the second binding domain antigen binding domain formed by VH2 and VL2 is stabilized by the CH1 and CL heterodimerizing domain, and is further stabilized by the disulfide bond, or other stabilizing interaction (e.g., knobs/hole interaction), between CH1 and CL.

[00345] In some embodiments, the second binding domain in the precursor bispecific antibody construct is specifically blocked by the regulatory domain at its N-termini such that binding to the second binding domain target antigen (especially when cell surface target antigens are concerned) is specifically reduced or inhibited in a statistically significant manner (i.e., relative to an appropriate control as will be known to those skilled in the art; e.g., as compared to the same second binding domain in a format without a regulatory domain comprising a CAP component, at its N- termini (either VH and VL)). In a further embodiment, the second binding domain in the precursor bispecific antibody construct is specifically blocked so that binding to the desirable antigen (especially when cell surface target antigens are concerned) is reduced by at least 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 20 fold, 30 fold, or 100 fold, or 1000 fold, or 10,000 fold as compared to the same second binding domain in a format without a regulatory domain comprising a CAP component at its N-termini (both VH and VL).

[00346] In certain embodiments, the affinity of the second binding domain (e.g., CD3 epsilon antigen-binding domain) in the precursor construct is below 500 nM. In further embodiments, the affinity of the second binding domain antigen binding of a precursor construct demonstrates no significant detectible binding as measured using FACS or other binding measurement method (e.g., cell binding ELISA) at concentration ranges of the therapeutics used in humans. In one embodiment, less than 1% of a population of second binding domain target cells (e.g., CD3+ cells) will be bound by the second binding domain of a precursor construct at a therapeutic concentration (this is in the absence of a tumor cell microenvironment). In one embodiment, less than 5% population of the second binding domain target cells will be bound by the precursor bispecific antibody construct at a therapeutic concentration. In yet another embodiment, less than 10% population of the second binding domain target cells will be bound by the precursor bispecific antibody construct at a therapeutic concentration.

[00347] The elevated level of proteases, especially MMPs, present in tumor tissues (tumor microenvironment) will generate cleavage products at the MMP substrate cleavage site of a protease cleavable linker. Because the cleavage of the protease substrate sequence of a linker results in the release of a CAP component that may be bound at the second binding domain antigen-binding region, the binding to the second binding domain cell surface target will be fully restored or at least partially restored. The restored binding can be demonstrated using techniques of FACS, cell-based ELISA) or other cell binding techniques known to the skilled person.

[00348] A skilled artisan would appreciate that the term "dramatically reduced affinity" may encompass at least 30% reduction in the binding of the second binding domain antigen-binding domain, as compared to the binding in the absence of a CAP component of a regulatory domain present at the N-terminus of the second binding domain. The percentage of reduction can be, for example without limitation, 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% or greater. Methods for detecting binding are known to the skilled person and can be performed using FACS, cell binding ELISA or cell binding using radioisotype labeled antibodies.

[00349] The illustrative second binding domain for use in the precursor bispecific antibody construct of the present disclosure as described herein is an anti-CD3 epsilon domain. In this regard, the precursor bispecific antibody construct functions such that, when the TAA first binding domain binds to a tumor associated antigen, the precursor construct is present within a tumor microenvironment comprising proteases, wherein the a cleavable regulatory domain or a portion thereof is cleaved releasing a CAP anti-CD3 epsilon binding component such that the second binding domain is now able to bind to CD3 epsilon of passing T-cells, thereby redirecting the T- cells and activating them to kill the tumor cell or tumor associate cell. (Figure 3B) In another embodiment, an activated bispecific antibody construct (also referred to herein as activated bispecific antibody construct (Figure 2F) where the second binding domain fragment binds to an immune effector molecule, such as CD3 epsilon) can exhibit an avidity effect when clustered on tumor cell surface via TAA (e.g., tumor antigen) binding by the TAA first binding domain. As such, the apparent binding to immune cells by the second binding domain can increase due to avidity. As such, an activated bispecific antibody construct becomes capable of bridging immune and tumor cells thereby mediating anti-tumor activity.

[00350] In certain embodiments, a precursor bispecific antibody construct is separately bound to a TAA and not bound to a CD3 epsilon, and thus the T-cells will not be activated. In some embodiments, this lack of binding to a CD3 epsilon antigen may be a result of the TAA present on a non-tumor cell. In some embodiments, this lack of binding to a CD3 epsilon antigen may be the results of the TAA present on a cell in a non-tumor microenvironment. As such, the precursor construct bound to a TAA in a non-tumor environment and in certain embodiments, can therefore not be activated (i.e., there will be no cleavage of the regulatable domain or a portion thereof. This avoid significant side effect and tissue damage that may occur where the precursor construct to activate T-cells at a non-tumor cell environment. However, within a tumor microenvironment, when the CD3 and tumor surface antigen are simultaneously bound to the activated bispecific antibody construct and when multiple copies of the bound complexes are anchored and clustered on tumor cell surface, the T-cells are activated in the vicinity of cancer cells bearing the tumor surface antigen, and therefore significantly enhance the tumor killing efficiency of T-cells locally and avoid the side effects due to cytokine storm.

[00351] In certain embodiments, the combination of a second binding domain antigen target being CD3 epsilon and a first binding domain antigen target being a cell surface tumor associated antigen comprised within a precursor construct, which is temporally regulated by a half-life enhancing component of a regulatory domain and activity regulated by a CAP component or a regulatory domain, the combination of which provide for enhanced tumor killing effects by T-cells once the precursor construct has been located to a tumor microenvironment. In certain embodiments, the combination of the second binding domain antigen target and the first binding doamin antigen target can be FcyR and TAA, respectively, which combination can induce FcyR- expressing immune cells to kill tumor cells once the precursor construct has been located to a tumor microenvironment. In certain embodiments, the combination of the second binding domain antigen target and the first binding domain antigen target can be NKG2D and a TAA, respectively, which combination can induce natural killer (NK) cell to kill tumor cells once the precursor construct has been located to a tumor microenvironment.

[00352] Thus, in an illustrative embodiment, the precursor bispecific antibody construct of the present disclosure comprises a second binding domain that binds to the TCR or a component thereof, such as a CD3 polypeptide. As noted above, the precursor bispecific antibody construct of the present disclosure does not bind to the second binding domain target antigen except following a linker cleavage event, wherein a CAP component is release or in the absence of a CAP component comprised within the precursor construct.

[00353] Thus, in certain embodiments, a precursor bispecific antibody construct of the present disclosure does not activate T-cells in the absence of target antigen engagement at the second binding domain. A precursor bispecific antibody construct "does not or minimally or nominally activates T-cells" if the precursor bispecific antibody construct does not cause a statistically significant increase in the percentage of activated T-cells as compared to activation of T-cells in the presence of cells expressing TAA first binding domain target antigens (e.g., an appropriate tumor cel 1/cel 1 line; tumor micro-environment), as measured in at least one in vitro or in vivo assay. Such assays are known in the art and include, without limitation, proliferation assays, CTL chromium release assays (see e.g., Lavie et al., (2000) International Immunology l2(4):479-486), ELISPOT assays, intracellular cytokine staining assays, and others as described, for example, in Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009). In certain embodiments, T-cell activation is measured using an in vitro primed T-cell activation assay.

[00354] In a related aspect, therefore, the present disclosure provides a method for detecting T- cell activation induced by an activated precursor bispecific antibody construct that comprises a first binding domain that specifically binds to a TAA, a seoncd binding domain that specifically binds to a TCR complex, and a regulatory domain, wherein the precursor construct is activated in the presence of a tumor microenvironment. In some embodiments, activation of a precursor construct comprises cleavage of a complete regulatory domain. In some embodiments, activation of a precursor construct comprises cleavage of a portion of a regulatory domain. In some embodiments, activation of a precursor construct comprises cleavage of a complete regulatory domain comprising a CAP component. In some embodiments, activation of a precursor construct comprises cleavage of a portion of a regulatory domain, wherein the portion of the regulatory domain comprises a CAP component. In some embodiments, activation of a precursor construct comprises cleavage of a complete regulatory domain comprising an HSA component. In some embodiments, activation of a precursor construct comprises cleavage of a portion of a regulatory domain, wherein the portion of the regulatory domain comprises an HSA component. In some embodiments, activation of a precursor construct comprises cleavage of a complete regulatory domain comprising a CAP component and an HSA component. In some embodiments, activation of a precursor construct comprises cleavage of a portion of a regulatory domain, wherein the portion of the regulatory domain comprises a CAP component and an HSA component.

[00355] In some embodiments, a method for detecting T-cell activation comprises (a) providing antigen or mitogen-primed T-cells, (b) treating the primed T-cells of step (a) with the precursor bispecific antibody construct that comprises a second binding domain that specifically binds to a TCR complex or a component thereof (following exposure to a tumor microenvironment and cleavage of a regulatory domain or a portion thereof), and (c) detecting activation of the primed T- cells that have been treated in step (b).

[00356] The term "mitogen" as used herein refers to a chemical substance that induces mitosis in lymphocytes of different specificities or clonal origins. Exemplary mitogens that may be used to prime T-cells include phytohaemagglutinin (PHA), concanavalin A (ConA), lipopoly saccharide (LPS), poke weed mitogen (PWM), and phorbol myristate acetate (PMA). Antigen-loaded beads or PBMC can also be used to prime T-cells.

[00357] In certain embodiments of methods for detecting T-cell activation provided herein, the precursor bispecific antibody construct comprising a second binding domain that specifically binds to a TCR complex or a component thereof comprises a first binding domain that bind to tumor associated antigens, and a regulatory domain that provides enhanced half-life prolonging properties, reduction in T-cell binding properties, reduction in T-cell activation properties, or any combination thereof. In certain embodiments, methods for detecting T-cell activation provided herein, are performed in tumor and non-tumor microenvironments.

[00358] T-cell activation may be detected by measuring the expression of activation markers known in the art, such as CD25, CD40 ligand, and CD69. Activated T-cells may also be detected by cell proliferation assays, such as CFSE labeling and thymidine uptake assays (Adams (1969) Exp. Cell Res. 56:55). T-cell effector function (e.g., cell killing) can be measured, for example, by chromium release assays or FACS based assays using fluorescent dyes (e.g. TP3). In a related aspect, T-cell activation and cytolytic activity can be measured by lytic synapse formation between T-cell and tumor cell. Effector molecules such as Granzymes and porforin can be detected in the cytolytic synapse.

[00359] In another related aspect, T-cell activation may be measured by cytokine release. A method for detecting cytokine release induced by a precursor bispecific antibody construct that comprises a second binding domain that specifically binds to a TCR complex or a component thereof, may comprise: (a) providing primed T-cells, (b) treating the primed T-cells of step (a) with the precursor bispecific antibody construct that comprises a second that specifically binds to a TCR complex or a component thereof, (c) incubating the precursor construct in a tumor microenvironment e.g., with tumor cells associated with the antigen target of the TAA first binding domain, and (d) detecting release of a cytokine from the primed T-cells that have been treated in step (b). In particular embodiments, experiments are carried out in the presence or absence of appropriate cancer cells or cell lines expressing target tumor antigens bound by binding domains present in the first binding domain of the precursor bispecific antibody construct (step c).

[00360] In certain embodiments of methods for detecting cytokine release provided herein, the precursor bispecific antibody construct that comprises a second binding domain that specifically binds to a TCR complex or a component thereof is performed in the presence or absence of appropriate cancer cells or cell lines expressing target tumor antigens bound by binding domains present in the first binding domain of the precursor bispecific antibody construct

[00361] In certain embodiments, the precursor bispecific antibody construct of the present disclosure do not induce a cytokine storm or do not induce a cytokine release sufficient to induce toxic side-effects. A precursor bispecific antibody construct "does not induce a cytokine storm" (also referred to as "inducing an undetectable, nominal, or minimal cytokine release" or "does not induce or induces a minimally detectable cytokine release") if, in the absence of TAA target cells or appropriate linker cleavage agents (such as proteases), it does not cause a statistically significant increase in the amount of at least one cytokine including IFNy.; In certain embodiments at least two cytokines including IFNy and TNFa or IL-6 and TNFa.; in one embodiment three cytokines including IL-6, IFNy and TNFa; in another embodiment four cytokines including IL-2, IL-6, IFNy, and TNFa; and in yet a further embodiment at least five cytokines including IL-2, IL-6, IL-10, , IFNy, and TNFa; released from treated cells in the absence of TAA target cells (e.g., an appropriate cancer cell line) or appropriate linker cleavage agents, as compared to from treated cells in the presence of appropriate TAA target cells or linker cleavage agents, in at least one in vitro or in vivo assay known in the art or provided herein. Clinically, cytokine-release syndrome is characterized by fever, chills, rash, nausea, and sometimes dyspnea and tachycardia, which is in parallel with maximal release of certain cytokines, such as IFNy, as well as IL-2, IL-6, and TNFa. Cytokines that may be tested for release in an in vitro assay or in vivo include G-CSF, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IP-10, KC, MCP1, IFNy, and TNFa; and in another embodiment include IL-2, IL-6, IL-10, IFNy, and TNFa.

[00362] In further embodiments, a precursor bispecific antibody construct of the present disclosure causes an increase in calcium flux in cells, such as T-cells. A precursor bispecific antibody construct causes an "increase in calcium" if, when used to activate T-cells in the presence of an appropriate TAA target cell (e.g., cancer cell) or linker cleavage agents, it causes a statistically significant, rapid increase in calcium flux of the treated cells (within 300 seconds, or within 200 seconds, or within 100 seconds of treatment) as compared to cells treated in the absence of an appropriate TAA target cell or linker cleavage agents, as measured in an in vitro assay known in the art or provided herein.

[00363] In further embodiments, a precursor bispecific antibody construct of the present disclosure induces phosphorylation of a molecule in the TCR signal transduction pathway. The "TCR signal transduction pathway" refers to the signal transduction pathway initiated via the binding of a peptide:MHC ligand to the TCR and its co-receptor (CD4 or CD8). A "molecule in the TCR signal transduction pathway" refers to a molecule that is directly involved in the TCR signal transduction pathway, such as a molecule whose phosphorylation state (e.g., whether the molecule is phosphorylated or not), whose binding affinity to another molecule, or whose enzymatic activity, has been changed in response to the signal from the binding of a peptide:MHC ligand to the TCR and its co-receptor. Exemplary molecules in the TCR signal transduction pathway include the TCR complex or its components (e.g., CD3 chains), ZAP-70, Fyn, Lck, phospholipase c-g, protein kinase C, transcription factor NF.kappa.B, phasphatase calcineurin, transcription factor NFAT, guanine nucleotide exchange factor (GEF), Ras, MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK), MAP kinase (ERK1/2), and Fos.

[00364] A precursor bispecific antibody construct of this disclosure "induces phosphorylation of a molecule in the TCR signal transduction pathway" if it causes a statistically significant increase in phosphorylation of a molecule in the TCR signal transduction pathway (e.g., CD3 chains, ZAP- 70, and ERK1/2) only in the presence of cells expressing TAA antigen (e.g., cancer cells expressing tumor antigens bound by a first binding domains, or when the TAA is present on a non-tumor cell, tumor cells expressing proteases able to cleave the protease cleavable domain of a regulatory domain are present) or linker cleavage agents, in an in vitro or in vivo assay or receptor signaling assays known in the art. Results from most receptor signaling assays known in the art are determined using immunohistochemical methods, such as western blots or fluorescence microscopy.

[00365] Similarly, an activated precursor bispecific antibody construct of the present disclosure may induce killing of TAA target cell, such as tumor cells or vascular cells which support the growth and maintenance of tumor cells, by T-cells following exposure to a tumor cell microenvironment or exposure to a protease or proteases able to cleave the protease cleavable component of a regulatory domain. Such cell killing can be measured using a variety of assays known in the art, including chromium release assays.

[00366] The specificity and function of a precursor bispecific antibody construct of the present disclosure may be tested by contacting the precursor bispecific antibody construct with appropriate test sample and, in certain embodiments, treating the precursor bispecific antibody construct with an appropriate protease which is thought to be specific for the cleavage recognition site in the linker and assaying for cleavage products. Proteases may be isolated, for example from cancer cells or they may be prepared recombinantly, for example following the procedures in Darket et al. (J. Biol. Chem. 254:2307-2312 (1988)). The cleavage products may be identified for example based on size, antigenicity or activity. The toxicity of the precursor bispecific antibody construct may be investigated by subjecting the precursor bispecific antibody construct and cleavage products thereof to in vitro cytotoxicity, proliferation, binding, or other appropriate assays known to the skilled person. Toxicity of the cleavage products may be determined using a ribosomal inactivation assay (Westby et al., Bioconjugate Chem. 3:377-382 (1992)). The effect of the cleavage products on protein synthesis may be measured in standardized assays of in vitro translation utilizing partially defined cell free systems composed for example of a reticulocyte lysate preparation as a source of ribosomes and various essential cofactors, such as mRNA template and amino acids. Use of radiolabeled amino acids in the mixture allows quantitation of incorporation of free amino acid precursors into trichloroacetic acid precipitable proteins. Rabbit reticulocyte lysates may be conveniently used (O'Hare, FEBS Lett. 273:200-204 (1990)).

[00367] The ability of an activated precursor bispecific antibody construct as disclosed herein, to destroy cancer cells and/or activate T-cells may be readily tested in vitro using cancer cell lines, T- cell lines or isolated PBMC or T-cells. The effects of the precursor bispecific antibody construct of the present disclosure may be determined, for example, by demonstrating by selective lysis of cancer cells. In addition, the protease specificity can be tested by comparing the inhibition of cellular proliferation using a precursor bispecific antibody construct of the present disclosure alone or in the presence of protease- specific inhibitors. Such protease inhibitors may include MMP-2/MMP-9 inhibitors GM1489, GM6001 and GI-I to GI-IV.

[00368] Toxicity may also be measured based on cell viability, for example the viability of normal and cancerous cell cultures exposed to the precursor bispecific antibody construct may be compared. Cell viability may be assessed by known techniques, such as trypan blue exclusion assays. Toxicity may also be measured based on cell lysis, for example the lysis of normal and cancerous cell cultures exposed to the precursor bispecific antibody construct may be compared. Cell lysis may be assessed by known techniques, such as Chromium (Cr) release assays or dead cell indicator dyes (propidium Iodide, TO-PRO-3 Iodide).

Precursor Bispecific Antibody Construct Components

[00369] The present disclosure provides precursor bispecific antibody construct polypeptides, and fragments thereof. As described in detail above, in some embodiments a precursor bispecific antibody construct comprises two polypeptides: polypeptide A and polypeptide B. Illustrative polypeptides, and the polynucleotides encoding them, are provided in SEQ ID NOs:l06 (polypeptide sequence of polypeptide A), 112 and 114 (polypeptide sequences of polypeptide B), 107 (polynucleotide sequence encoding polypeptide A), and 113 and 115 (polynucleotide sequences encoding polypeptide B).

[00370] A skilled artisan would appreciate that terms "polypeptide" "protein" and "peptide" and "glycoprotein" are used interchangeably and encompass a polymer of amino acids not limited to any particular length. The term does not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms "polypeptide" or "protein" may encompass one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" may encompass a polypeptide A or a polypeptide B of a precursor bispecific antibody construct and heterodimers thereof of the present disclosure, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of a precursor bispecific antibody construct as disclosed herein. Thus, a "polypeptide" or a "protein" can comprise one (termed "a monomer") or a plurality (termed "a multimer") of amino acid chains.

[00371] The term "isolated protein" referred to herein encompasses a subject protein (1) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a protein with which the "isolated protein" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[00372] The term "polypeptide fragment" encompasses a polypeptide, which can be monomeric or multimeric, that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion or substitution of a naturally-occurring or recombinantly-produced polypeptide. In certain embodiments, a polypeptide fragment can comprise an amino acid chain at least 5 to about 500 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,

110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Particularly useful polypeptide fragments include functional domains, including antigen-binding domains or fragments of antibodies. In the case of an anti-CD3, or other antibody, useful fragments include, but are not limited to: a CDR region, especially a CDR3 region of the heavy or light chain; a variable region of a heavy or light chain; a portion of an antibody chain or just its variable region including two CDRs; and the like.

[00373] Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be fused in-frame or conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.

[00374] Amino acid sequence modification(s) of the precursor bispecific antibody constructs described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the precursor bispecific antibody construct. For example, amino acid sequence variants of a linker sequence, or a binding domain, or a regulatory component(s) thereof may be prepared by introducing appropriate nucleotide changes into a polynucleotide that encodes the precursor bispecific antibody construct polypeptides, or a domain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the precursor bispecific antibody construct polypeptides. Any combination of deletion, insertion, and substitution may be made to arrive at the final precursor bispecific antibody construct polypeptides, provided that the final construct possesses the desired characteristics, such as specific binding to a target antigen of interest by a first binding domain or a second binding domain, or enhanced half-life by an HSA polypeptide, or specific binding to a second binding domain by a CAP component, or protease cleavage by a protease cleavage domain (linker). The amino acid changes also may alter post-translational processes of the precursor bispecific antibody construct polypeptides, such as changing the number or position of glycosylation sites. Any of the variations and modifications described above for polypeptides as disclosed herein, may be included in precursor bispecific antibody constructs presented herein.

[00375] The present disclosure provides variants of the precursor bispecific antibody construct polypeptides disclosed herein. In certain embodiments, such variant precursor bispecific antibody construct polypeptides comprise variant binding domains or fragments thereof, or antigen-binding fragments, or TAA binding fragments, or CDRs of binding domains, bind to a target of interest at least about 50%, at least about 70%, and in certain embodiments, at least about 90% as well as a given reference or wild-type sequence, including any such sequences specifically set forth herein. In further embodiments, such variants bind to a target antigen with greater affinity the reference or wild-type sequence set forth herein, for example, that bind quantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110% as well as a reference sequence specifically set forth herein. In certain embodiments, such variant precursor bispecific antibody construct polypeptides comprise variant regulatory domains or fragments thereof, or HSA components, or CAP components or fragments thereof, wherein said variant has at least about 50%, at least about 70%, and in certain embodiments, at least about 90% of the activity of a reference or wild-type regulatory domain or component, including any such sequences specifically set forth herein.

[00376] In certain embodiments, the present disclosure provides variants of the precursor bispecific antibody constructs or polypeptides thereof, disclosed herein where such variants comprise second binding domains that have been modified with regard to the disulfide bond between the VH2 and V2L chains. As would be recognized by the skilled person, in certain embodiments the second binding domain, which in some embodiments comprises an Fab fragment, used in the precursor bispecific antibody construct described herein may not comprise a disulfide bond. In this regard, the heavy and light chains may be engineered in such a way so as to stably interact without the need for disulfide bond. For example, in certain embodiments, the heavy or light chain can be engineered to remove a cysteine residue and wherein the heavy and light chains still stably interact and function as a binding domain e.g. a Fab fragment. In some embodiments, mutations are made to facilitate stable interaction between the heavy and light chains. For example, a "knobs into holes" engineering strategy can be used to facilitate dimerization between the heavy and light chains of a Fab second binding domain (see e.g., 1996 Protein Engineering, 9:617-621). Thus, also contemplated for use herein are variant amino acid sequences of the second binding domain (e.g., Fab fragments) designed for a particular purpose, for example, removal of a disulfide bond addition of tax for purification, etc.

[00377] In particular embodiments, a subject precursor bispecific antibody construct polypeptide may have: an amino acid sequence that is at least 80% identical, at least 95% identical, at least 90%, at least 95% or at least 98% or 99% identical, to the precursor bispecific antibody construct polypeptides described herein.

[00378] Determination of the three-dimensional structures of representative polypeptides may be made through routine methodologies such that substitution, addition, deletion or insertion of one or more amino acids with selected natural or non-natural amino acids can be virtually modeled for purposes of determining whether a so derived structural variant retains the space-filling properties of presently disclosed species. See, for instance, Donate et al., 1994 Prot. Sci. 3:2378; Bradley et al., Science 309: 1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci. USA 103: 1244 (2006); Dodson et al., Nature 450: 176 (2007); Qian et al., Nature 450:259 (2007); Raman et al. Science 327: 1014-1018 (2010). Some additional non-limiting examples of computer algorithms that may be used for these and related embodiments, include VMD which is a molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3-D graphics and built-in scripting (see the website for the Theoretical and Computational Biophysics Group, University of Illinois at Urbana-Champagne, at ks.uiuc.edu/Research/vmd/). Many other computer programs are known in the art and available to the skilled person and which allow for determining atomic dimensions from space-filling models (van der Waals radii) of energy -minimized conformations; GRID, which seeks to determine regions of high affinity for different chemical groups, thereby enhancing binding, Monte Carlo searches, which calculate mathematical alignment, and CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER (Weiner et al (1981) J. Comput. Chem. 106: 765), which assess force field calculations, and analysis (see also, Eisenfield et al. (1991) Am. J. Physiol. 26EC376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ. Health Perspect. 61: 185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn. 9:475-488). A variety of appropriate computational computer programs are also commercially available, such as from Schrodinger (Munich, Germany).

Polynucleotides Encoding Precursor Bispecific Antibody Construct Components, Vectors, Host Cells, and Methods of Producing Precursor Bispecific Antibody Constructs

[00379] The present disclosure further provides in certain embodiments an isolated nucleic acid encoding the polypeptide precursor bispecific antibody construct as described herein. Illustrative polynucleotides and fragments thereof, are provided in Table 2 below. Nucleic acids include DNA and RNA. These and related embodiments may include polynucleotides encoding the precursor bispecific antibody construct as described herein. The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the isolated polynucleotide (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[00380] A skilled artisan would appreciate that the terms“polynucleotide” and“nucleic acid sequence” may in some embodiments be used interchangeably having all the same meanings and qualities.

[00381] In some embodiments, an isolated nucleic acid sequences encode polypeptide A and polypeptide B of a precursor bispecific antibody construct, said construct comprising (a) a first binding domain binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a cleavable half-life prolonging domain. In some embodiments, an isolated nucleic acid sequences encodes polypeptide A of a precursor bispecific antibody construct, as described above in detail. In some embodiments, an isolated nucleic acid sequences encodes polypeptide B of a precursor bispecific antibody construct, as described above in detail. In some embodiments, polypeptides A and B form a heterodimer comprising a precursor construct as described herein comprising (a) a first binding domain binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a cleavable half-life prolonging domain.

[00382] Table 2: Nucleotide Sequences of Encoding Anti-CD3 VH, VL, HC, LC, , Anti- EGFR, Regulatory components, and Combinations thereof

[00383] The term "operably linked" encompasses components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions. For example, a transcription control sequence "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[00384] The term "control sequence" as used herein encompasses polynucleotide sequences that can affect expression, processing or intracellular localization of coding sequences to which they are ligated or operably linked. The nature of such control sequences may depend upon the host organism. In particular embodiments, transcription control sequences for prokaryotes may include a promoter, ribosomal binding site, and transcription termination sequence. In other particular embodiments, transcription control sequences for eukaryotes may include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, transcription termination sequences and polyadenylation sequences. In certain embodiments, "control sequences" can include leader sequences and/or fusion partner sequences.

[00385] The term "polynucleotide" as used herein encompasses single- stranded or double- stranded nucleic acid polymers. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose and intemucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term "polynucleotide" specifically includes single and double stranded forms of DNA.

[00386] The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl. Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077; Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991, Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), Oxford University Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures of which are hereby incorporated by reference for any purpose. An oligonucleotide can include a detectable label to enable detection of the oligonucleotide or hybridization thereof.

[00387] In other related embodiments, polynucleotide variants may have substantial identity to a polynucleotide sequence encoding a precursor bispecific antibody construct, or domain thereof as described herein. For example, a polynucleotide may be a polynucleotide comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a reference polynucleotide sequence such as a sequence encoding a precursor bispecific antibody construct or domain thereof described herein, using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[00388] Typically, polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably such that the binding affinity of a binding domain, or binding affinity of a first or second binding domain, or function of the precursor bispecific antibody construct polypeptide encoded by the variant polynucleotide is not substantially diminished relative to the unmodified reference protein encoded by a polynucleotide sequence specifically set forth herein.

[00389] In certain other related embodiments, polynucleotide fragments may comprise or consist essentially of various lengths of contiguous stretches of sequence identical to or complementary to a sequence encoding a precursor bispecific antibody construct polypeptide or domain thereof as described herein. For example, polynucleotides are provided that comprise or consist essentially of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110,

120, 130, 140, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of a sequences the encodes a precursor bispecific antibody construct polypeptide or domain thereof, such as a first binding domain or a second binding domain or a regulatory domain, or components thereof, disclosed herein as well as all intermediate lengths there between. It will be readily understood that "intermediate lengths", in this context, means any length between the quoted values, such as 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200- 500; 500-1,000, and the like. A polynucleotide sequence as described here may be extended at one or both ends by additional nucleotides not found in the native sequence. This additional sequence may consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at either end of a polynucleotide encoding a precursor bispecific antibody construct polypeptide or domain or component part thereof described herein or at both ends of a polynucleotide encoding a precursor bispecific antibody construct polypeptide or domain or component part thereof described herein.

[00390] In another embodiment, polynucleotides are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence encoding precursor bispecific antibody construct polypeptide or domain or component part thereof, such as a first binding domain or a second binding domain or a regulatory domain, or component parts thereof, as provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide as provided herein with other polynucleotides include prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50.degree. C.-60.degree. C, 5.times.SSC, overnight; followed by washing twice at 65.degree. C. for 20 minutes with each of 2.times., 0.5.times. and 0.2.times.SSC containing 0.1% SDS. One skilled in the art will understand that the stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60°C-65°C or 65°C-70°C.

[00391] In certain embodiments, the polynucleotides described above, e.g., polynucleotide variants, fragments and hybridizing sequences, encode a precursor bispecific antibody construct polypeptide or domain thereof or component part thereof, such as a first binding domain, e.g., a scFv that binds to a human EGFR, or a second binding domain, e.g., a Fab fragment that binds CD3 epsilon, or an HSA polypeptide that extends half-life, or a CAP component that specifically binds to a second binding domain. In other embodiments, such polynucleotides encode precursor bispecific antibody construct polypeptides or domains or components thereof that bind to CD3 and/or a tumor associated antigen at least about 50%, at least about 70%, and in certain embodiments, at least about 90% as well as a precursor bispecific antibody construct polypeptide sequence specifically set forth herein. In other embodiments, such polynucleotides encode precursor bispecific antibody construct polypeptides or domains or components thereof that extend the half-life of the precursor construct at least about 50%, at least about 70%, and in certain embodiments, at least about 90% as well as a precursor bispecific antibody construct polypeptide sequence specifically set forth herein. In other embodiments, such polynucleotides encode precursor bispecific antibody construct polypeptides or domains or components thereof that specifically bind to the second binding site of the precursor construct at least about 50%, at least about 70%, and in certain embodiments, at least about 90% as well as a precursor bispecific antibody construct polypeptide sequence specifically set forth herein. In further embodiments, such polynucleotides encode a precursor bispecific antibody construct polypeptide or domain thereof, that, e.g., bind to CD3 and/or a tumor associated antigen with greater affinity than the precursor bispecific antibody construct polypeptide, or domain thereof, set forth herein, for example, that bind quantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110% as well as a precursor bispecific antibody construct polypeptide or domain thereof sequence specifically set forth herein.

[00392] As described elsewhere herein, determination of the three-dimensional structures of representative polypeptides (e.g., variant precursor bispecific antibody construct and polypeptides thereof, as provided herein, for instance, a precursor bispecific antibody construct having a first TAA binding domain and a second CD3 epsilon binding domain as provided herein) may be made through routine methodologies such that substitution, addition, deletion or insertion of one or more amino acids with selected natural or non-natural amino acids can be virtually modeled for purposes of determining whether a so derived structural variant retains the space-filling properties of presently disclosed species. A variety of computer programs are known to the skilled artisan for determining appropriate amino acid substitutions (or appropriate polynucleotides encoding the amino acid sequence) within, for example, an antibody or antigen-binding fragment thereof, such that, for example, affinity is maintained or better affinity is achieved.

[00393] The polynucleotides described herein, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful.

[00394] When comparing polynucleotide sequences, two sequences are said to be "identical" if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[00395] Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins— Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990); Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M., CABIOS 5:151- 153 (1989); Myers, E. W. and Muller W., CABIOS 4:11-17 (1988); Robinson, E. D., Comb. Theor 11:105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4:406-425 (1987); Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy— the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif. (1973); Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad., Sci. USA 80:726- 730 (1983).

[00396] Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman, Add. APL. Math 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection. [00397] One example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nucl. Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity among two or more the polynucleotides. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands.

[00398] In certain embodiments, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

[00399] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a precursor bispecific antibody construct as described herein. Some of these polynucleotides bear minimal sequence identity to the nucleotide sequence of the native or original polynucleotide sequence that encode precursor bispecific antibody construct polypeptides or domains or components thereof, for example forming a precursor bispecific antibody construct that binds to CD3 and or a tumor associated antigen. Nonetheless, polynucleotides that vary due to differences in codon usage are expressly contemplated by the present disclosure. In certain embodiments, sequences that have been codon- optimized for mammalian expression are specifically contemplated.

[00400] Therefore, in another embodiment as disclosed herein,, a mutagenesis approach, such as site-specific mutagenesis, may be employed for the preparation of variants and/or derivatives of the precursor bispecific antibody construct polypeptides described herein. By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques provides a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.

[00401] Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[00402] In certain embodiments, mutagenesis of the polynucleotide sequences that encode a precursor bispecific antibody construct polypeptide or domain thereof or component part thereof, as disclosed herein, is contemplated in order to alter one or more properties of the encoded polypeptide/domain/component, such as the binding affinity of a first binding domain or a second binding domain, or the function of a regulatory domain or component thereof. The techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter a specific portion of a DNA molecule. In such embodiments, a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.

[00403] As will be appreciated by those of skill in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art. Double- stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.

[00404] In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single- stranded vector or melting apart of two strands of a double- stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single- stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.

[00405] The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.

[00406] As used herein, the term "oligonucleotide directed mutagenesis procedure" encompasses template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" encompasses a process that involves the template-dependent extension of a primer molecule. The term“template dependent process” encompasses nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Pat. No. 4,237,224, specifically incorporated herein by reference in its entirety.

[00407] In another approach for the production of polypeptide variants, recursive sequence recombination, as described in U.S. Pat. No. 5,837,458, may be employed. In this approach, iterative cycles of recombination and screening or selection are performed to "evolve" individual polynucleotide variants having, for example, increased binding affinity. Certain embodiments also provide constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as described herein.

[00408] In certain embodiments, the isolated polynucleotide is inserted into a vector. The term "vector" as used herein encompasses a vehicle into which a polynucleotide encoding a protein may be covalently inserted so as to bring about the expression of that protein and/or the cloning of the polynucleotide. The isolated polynucleotide may be inserted into a vector using any suitable methods known in the art, for example, without limitation, the vector may be digested using appropriate restriction enzymes and then may be ligated with the isolated polynucleotide having matching restriction ends.

[00409] Examples of suitable vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).

[00410] For expression of the polypeptide, the vector may be introduced into a host cell to allow expression of the polypeptide within the host cell. The expression vectors may contain a variety of elements for controlling expression, including without limitation, promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, the promoter sequences may be selected to promote the transcription of the polynucleotide in the vector. Suitable promoter sequences include, without limitation, T7 promoter, T3 promoter, SP6 promoter, beta- actin promoter, EFla promoter, CMV promoter, and SV40 promoter. Enhancer sequences may be selected to enhance the transcription of the polynucleotide. Selectable markers may be selected to allow selection of the host cells inserted with the vector from those not, for example, the selectable markers may be genes that confer antibiotic resistance. Signal sequences may be selected to allow the expressed polypeptide to be transported outside of the host cell.

[00411] A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.

[00412] In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a polypeptide of a precursor construct, or encoding a domain within a polypeptide of the precursor construct, or encoding a component part of a domain within a polypeptide of the precursor construct. Binding domains and the components thereof have been described in detail above.

[00413] In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a polypeptide A. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a polypeptide B. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a part of a polypeptide A. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a part of a polypeptide B. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a first binding domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding an scFv of a first binding domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding part of an scFv of a first binding domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding an EGFR binding domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding an EGFR scFv binding domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a VH region of a CD3 epsilon binding domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a VL region of a CD3 epsilon binding domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a regulatory domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a component part of a regulatory domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a CAP component of a regulatory domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding an HSA component of a regulatory domain. In some embodiments, an expression vector comprises an isolated nucleic acid sequence encoding a CAP component, an HSA component of a regulatory domain, and a linker(s).

[00414] For cloning of the polynucleotide, the vector may be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify the copies of the polynucleotide contained therein. The cloning vectors may contain sequence components generally include, without limitation, an origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences, and selectable markers. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, the origin of replication may be selected to promote autonomous replication of the vector in the host cell.

[00415] In certain embodiments, the present disclosure provides isolated host cells containing the vector provided herein. The host cells containing the vector may be useful in expression or cloning of the polynucleotide(s) contained in the vector.

[00416] Suitable host cells can include, without limitation, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.

[00417] Suitable prokaryotic cells for this purpose include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa , and Streptomyces.

[00418] The expression of antibodies and antigen-binding fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of antibodies or antigen-binding fragments thereof, see recent reviews, for example Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

[00419] Suitable fungal cells for this purpose include, without limitation, filamentous fungi and yeast. Illustrative examples of fungal cells include, Saccharomyces cerevisiae, common baker's yeast, Schizosaccharomyces pombe, Kluyveromyces hosts such as, eg., K. lactis, K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus, yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[00420] Higher eukaryotic cells, in particular, those derived from multicellular organisms can be used for expression of glycosylated polypeptide provided herein. Suitable higher eukaryotic cells include, without limitation, invertebrate cells and insect cells, and vertebrate cells. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruiffly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the K-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein as described herein, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts. Examples of vertebrate cells include, mammalian host cell lines such as monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRK-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[00421] The vector can be introduced to the host cell using any suitable methods known in the art, including, without limitation, DEAE-dextran mediated delivery, calcium phosphate precipitate method, cationic lipids mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histone, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of a vector of interest are well known in the art.

[00422] In certain embodiments, the host cells comprise a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In certain embodiments, the first vector and the second vector may be the same or not the same. In certain embodiments, the first polypeptide and the second polypeptide may be the same or not the same.

[00423] In certain embodiments, the host cells comprise a first vector encoding a polypeptide A and a second vector encoding a polypeptide B. In certain embodiments, the first vector and the second vector may be the same or not the same. In certain embodiments, the polypeptide A and the polypeptide B may be encoded on the same vector.

[00424] In some embodiments, an isolated cell comprises an isolated nucleic acid sequence, as disclosed herein. In some embodiments, an isolated cell comprises two isolated nucleic acid sequences as disclosed herein, wherein one nucleic acid encodes polypeptide A and the other nucleic acid encodes polypeptide B. In some embodiments, an isolated cell comprises two expression vectors as disclosed herein, wherein one vector comprises a nucleic acid encoding polypeptide A and the other vector comprises a nucleic acid encoding polypeptide B.

[00425] In certain embodiments, the first vector and the second vector may or may not be introduced simultaneously. In certain embodiments, the first vector and the second vector may be introduced together into the host cell. In certain embodiments, the first vector may be introduced first into the host cell, and then the second vector may be introduced. In certain embodiments, the first vector may be introduced into the host cell which is then established into a stable cell line expressing the first polypeptide, and then the second vector may be introduced into the stable cell line.

[00426] In certain embodiments, the host cells comprise a vector encoding for a first polypeptide and a second polypeptide.

[00427] In certain embodiments, the present disclosure provides methods of expressing the polypeptide provided herein, comprising culturing the host cell containing the vector under conditions in which the inserted polynucleotide in the vector is expressed.

[00428] Suitable conditions for expression of the polynucleotide may include, without limitation, suitable medium, suitable density of host cells in the culture medium, presence of necessary nutrients, presence of supplemental factors, suitable temperatures and humidity, and absence of microorganism contaminants. A person with ordinary skill in the art can select the suitable conditions as appropriate for the purpose of the expression.

[00429] In some embodiments, a method of producing a precursor bispecific antibody construct comprising (a) a first binding domain binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain, comprises steps of culturing a cell or cells comprising a nucleic acid sequence encoding polypeptide A and polypeptide B of the precursor bispecific antibody construct, wherein said precursor bispecific antibody construct polypeptides are expressed and isolated, and wherein said isolated polypeptides A and B form a heterodimer. A disclosed herein in detail, the isolated nucleic acid sequences encoding polypeptides A and B may be comprised within vectors, wherein the same vector or different vectors are used. In some embodiments, each polypeptide may be expressed from a different host cell, wherein dimerization occurs following isolation or purification of the component polypeptides A and B. In some embodiments, polypeptides A and B may be expressed from a same host cell, wherein dimerization occurs in culture or following isolation or purification of the component polypeptides A and B.

[00430] In certain embodiments, the polypeptide expressed in the host cell can form a dimer and thus produce a precursor bispecific antibody construct dimer, for example a heterodimer comprising a polypeptide A and a polypeptide B. In certain embodiments, where the host cells express a first polynucleotide and a second polynucleotide, the first polynucleotide (A) and the second polynucleotide (B) can form a polypeptide complex which is a heterodimer.

[00431] In certain embodiments, the polypeptide complex may be formed inside the host cell. For example, the heterodimer may be formed inside the host cell with the aid of relevant enzymes and/or cofactors. In certain embodiments, the polypeptide complex may be secreted out of the cell. In certain embodiments, the first polypeptide (A) and the second polypeptide (B) may be secreted out of the host cell and form a heterodimer outside of the host cell.

[00432] In certain embodiments, the first polypeptide and the second polypeptide may be separately expressed and allowed to dimerize under suitable conditions. For example, the first polypeptide (A) and the second polypeptide (B) may be combined in a suitable buffer and allow the first protein monomer (A) and the second protein monomer (B) to dimerize through appropriate interactions such as hydrophobic interactions. For another example, the first polypeptide (A) and the second polypeptide (B) may be combined in a suitable buffer containing an enzyme and/or a cofactor which can promote the dimerization of the first polypeptide (A) and the second polypeptide (B). For another example, the first polypeptide (A) and the second polypeptide (A) may be combined in a suitable vehicle and allow them to react with each other in the presence of a suitable reagent and/or catalyst.

[00433] In certain embodiments, the first polypeptide (A) and the second polypeptide (B) may be generated by DNA synthesis and PCR. In certain embodiments, the generated sequences may be subcloned into an expression vector. In certain embodiments, the generated sequences may be subcloned into two expression vectors. In certain embodiments, said expression vector is a plasmid. In certain embodiments, said plasmid is pTT5-based plasmid.

[00434] In certain embodiments, transient expression is performed by co-transfecting the expression vector encoding the first polypeptide (A) and the second polypeptide (B) or by transfecting an expression vector encoding both into a suitable cell. A skilled artisan would appreciate that there are a number of transfection methods and protocols that can be used for this purpose. In certain embodiments, transfection or co-transfection is executed using the PEI method. In certain embodiments, 1L of CHO cells at approximately 2.3xl0 ⁶ /ml in a 3L shake flask is used as the host. Transfection is initiated by adding a mixture of 2mg of total DNA and 4mg PEI in lOOml OptiMEM medium (Invitrogen) to the cells and gentle mixing. Cells are then cultured in an incubator shaker at 120 rpm, 37°C, and 8% C02, for 8-10 days. Feeding with peptone and glucose is carried out 24h later and every 2-3 days thereafter depending on the cell density and viability. The cell culture is terminated on day 8-10 when cell viability reduces to <70%. The conditioned medium is then harvested for protein purification.

[00435] The expressed polypeptides (A) and (B) and/or the polypeptide complex can be collected using any suitable methods. The polypeptides (A) and (B) and/or the polypeptide complex can be expressed intracellularly, in the periplasmic space or be secreted outside of the cell into the medium. If the polypeptides (A) and (B) and/or the polypeptide complex is expressed intracellularly, the host cells containing the polypeptides (A) and (B) and/or the polypeptide complex may be lysed and polypeptide and/or the polypeptide complex may be isolated from the lysate by removing the unwanted debris by centrifugation or ultrafiltration. If the polypeptides (A) and (B) and/or the polypeptide complex is secreted into periplasmic space of E. coli, the cell paste may be thawed in the presence of agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)). If the polypeptides (A) and (B) and/or the polypeptide complex is secreted into the medium, the supernatant of the cell culture may be collected and concentrated using a commercially available protein concentration filter, for example, an Amincon or Millipore Pellicon ultrafiltration unit. A protease inhibitor and/or a antibiotics may be included in the collection and concentration steps to inhibit protein degradation and/or growth of contaminated microorganisms.

[00436] The expressed polypeptides (A) and (B) and/or the polypeptide complex can be further purified by a suitable method, such as without limitation, affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (see, for review, Bonner, P. L., Protein purification, published by Taylor & Francis, 2007 ; Janson, J. C, et al, Protein purification: principles, high resolution methods and applications, published by Wiley- VCH, 1998).

[00437] In certain embodiments, the polypeptides (A) and (B) and/or polypeptide dimer complexes can be purified by affinity chromatography. In certain embodiments, protein A chromatography or protein A/G (fusion protein of protein A and protein G) chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising a component derived from antibody CH2 domain and/or CH3 domain (Landmark et al., J. Immunol. Meth. 62:1- 13 (1983)); Zettlit, K. A., Antibody Engineering, Part V, 531-535, 2010). In certain embodiments, a precursor bispecific antibody construct disclosed herein does not bind to protein A. In certain embodiments, protein G chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising IgGy3 heavy chain (Guss et al., EMBO J. 5: 1567 1575 (1986)). In certain embodiments, protein L chromatography can be useful for purification of polypeptides and/or polypeptide complexes comprising K light chain (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, B. H. K. et al, J. Biol. Chem, 267, 2234- 2239 (1992)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.

[00438] Following any preliminary purification step(s), the mixture comprising the precursor bispecific antibody construct and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 -4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

[00439] In certain embodiments, the polypeptides (A) and (B) and/or polypeptide dimer complexes can be purified by affinity chromatography and size exclusion chromatography (SEC). A skilled artisan would appreciate that there are a number of methods and protocols suitable for this purpose. In certain embodiments, protein purification by affinity chromatography and SEC is performed using an AKTA pure instrument (GE Lifesciences). In certain embodiments, affinity capture of the precursor bispecific antibody is achieved by passing the harvested supernatants over a column of Captures elect™ CH1-XL Affinity Matrix (Thermo Scientific). After washing column with PBS, the protein is eluted with 0.1M Glycine, pH 2.5, and immediately neutralized with 1/6 volume of 1M Tris-HCl, pH 8.0. The affinity purified protein is then concentrated to 5-l0mg/ml using Amicon 30kD concentrator (Merck Millipore) and subjected to SEC purification on a Superdex200 column (GE Lifesciences) equilibrated with PBS. Protein fractions are then collected and analyzed using SDS-PAGE and HPLC-SEC.

Methods of Use of Precursor Bispecific Antibody Constructs

[00440] In some embodiments, described herein are compositions comprising the precursor bispecific antibody construct as described herein and administration of such composition in a variety of therapeutic settings.

[00441] Administration of the precursor bispecific antibody constructs described herein, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions can be prepared by combining a precursor bispecific antibody construct or a precursor bispecific antibody construct-containing composition with an appropriate physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmaceutically active ingredients (including other anti-cancer agents as described elsewhere herein) and/or suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition. Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or topical. In some embodiments, modes of administration depend upon the nature of the condition to be treated or prevented. An amount that, following administration, reduces, inhibits, prevents or delays the progression and/or metastasis of a cancer is considered effective. A skilled artisan would appreciate that the term“physiologically acceptable carrier, diluent or excipient”, may in some embodiments be used interchangeably with the term “pharmaceutically acceptable carrier” having all the same means and qualities.

[00442] In some embodiments, a pharmaceutical composition described herein comprises a nucleotide sequence encoding a precursor bispecific antibody construct. In some embodiments, a nucleotide sequence encoding a precursor construct disclosed herein, comprises a single linear nucleotide sequence. In some embodiments, a nucleotide sequence encoding a precursor construct disclosed herein, comprises two nucleotide sequences. In some embodiments, a nucleotide sequence encoding a precursor construct disclosed herein, comprises two nucleotide sequences present on the same vector. In some embodiments, a nucleotide sequence encoding a precursor construct disclosed herein, comprises two nucleotide sequences present on different vectors.

[00443] In some embodiments, the nucleotide sequence encodes polypeptide A and polypeptide B. In some embodiments, the same nucleotide sequence encodes polypeptide A and polypeptide B. In some embodiments, different nucleotide sequences encode polypeptide A and polypeptide B. In some embodiments, one nucleotide sequence encodes polypeptide A and another nucleotide sequence encodes polypeptide B. In some embodiments, one nucleotide sequence encodes polypeptide A and another nucleotide sequence encodes polypeptide B having a protease cleavage sequence between them, thus allowing polypeptide A and polypeptide B to hetero-dimerize, as described in Duperret EK et al., Cancer Res, Oct. 4 ( doi: K).l l58/0008-5472.CAN-l8-l429)In some embodiments, a method of treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject, or any combination thereof, comprises a step of administering a pharmaceutical composition comprising a precursor bispecific antibody construct comprising (a) a first binding domain binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain; to a subject in need, wherein the method treats, prevents, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in said subject, or reduces the minimal residual disease, increases remission, increases remission duration, reduces tumor relapse rate, prevents metastasis of said tumor or said cancer, or reduces the rate of metastasis of said tumor or said cancer, or any combination thereof, compared with a subject not administered said pharmaceutical composition.

[00444] In some embodiments, a method of treating, preventing, inhibiting the growth of, delaying disease progression, reducing the tumor load, or reducing the incidence of a cancer or a tumor in a subject, or any combination thereof, comprises a step of administering a pharmaceutical composition comprising a nucleotide sequence encoding a precursor bispecific antibody construct comprising (a) a first binding domain binding to a cell surface tumor associated antigen (TAA binding domain); (b) a second binding domain binding to an extracellular epitope of human CD3e (CD3 binding domain); and (c) a regulatory domain; to a subject in need, wherein the method treats, prevents, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in said subject, or reduces the minimal residual disease, increases remission, increases remission duration, reduces tumor relapse rate, prevents metastasis of said tumor or said cancer, or reduces the rate of metastasis of said tumor or said cancer, or any combination thereof, compared with a subject not administered said pharmaceutical composition.

[00445] A skilled artisan would appreciate that the term“treating” and grammatical forms thereof, may in some embodiments encompass both therapeutic treatment and prophylactic or preventative measures with respect to a tumor or cancer as described herein, wherein the object is to prevent or lessen the targeted tumor or cancer as described herein. Thus, in some embodiments of methods disclosed herein, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof; for example when said disease or disorder comprises a cancer or tumor. Thus, in some embodiments, “treating” encompasses preventing, delaying progression, inhibiting the growth of, delaying disease progression, reducing tumor load, reducing the incidence of, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof. In some embodiments, “preventing” encompasses delaying the onset of symptoms, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, or a combination thereof. In some embodiments,“suppressing” or“inhibiting”, encompass reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

[00446] In some embodiments, the size of a cancer or tumor is reduced. In some embodiments, the growth rate of a cancer or tumor is reduced. In some embodiments, the size or the growth rate or a combination thereof, of a cancer or tumor is reduced. In some embodiments, the survival of the subject in need is increased. In some embodiments, the size or the growth rate or a combination thereof, of a cancer or tumor is reduced, or wherein the survival of the subject in need is increased or a combination thereof.

[00447] In some embodiments, the subject in need is a human subject. In some embodiments, the subject in need is a human child. In some embodiments, the subject in need is an adult human. In some embodiments, the subject in need is a human infant.

[00448] In certain embodiments, the amount administered is sufficient to result in tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least a 50% decrease in tumor mass, or by altered (e.g., decreased with statistical significance) scan dimensions. In other embodiments, the amount administered is sufficient to result in clinically relevant reduction in disease symptoms as would be known to the skilled clinician.

[00449] The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.

[00450] The precursor bispecific antibody construct-containing compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. In some embodiments, compositions comprising nucleotide sequences encoding a precursor bispecific antibody construct, may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions may also be administered in combination with antibiotics.

[00451] Typical routes of administering these and related pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques. Pharmaceutical compositions according to certain embodiments as described herein, are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described precursor bispecific antibody construct in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of a precursor bispecific antibody construct of the present disclosure, for treatment of a disease or condition of interest in accordance with teachings herein.

[00452] A pharmaceutical composition may be in the form of a solid or liquid. In one embodiment, the pharmaceutically acceptable carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The pharmaceutically acceptable carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi- solid, semi liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[00453] As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible pharmaceutically acceptable carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid pharmaceutically acceptable carrier such as polyethylene glycol or oil.

[00454] The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

[00455] The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[00456] A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a precursor bispecific antibody construct as herein disclosed such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the precursor bispecific antibody construct in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the precursor bispecific antibody construct. In certain embodiments, pharmaceutical compositions and preparations according to the embodiments described herein, are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the precursor bispecific antibody construct prior to dilution.

[00457] The pharmaceutical composition may be intended for topical administration, in which case the pharmaceutically acceptable carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

[00458] The pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The pharmaceutical composition in solid or liquid form may include an agent that binds to the antibody as disclosed herein, and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include other monoclonal or polyclonal antibodies, one or more proteins or a liposome. The pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.

[00459] The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining a composition that comprises a precursor bispecific antibody construct as described herein and optionally, one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the precursor bispecific antibody construct composition so as to facilitate dissolution or homogeneous suspension of the precursor bispecific antibody construct in the aqueous delivery system.

[00460] The compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound (e.g., precursor bispecific antibody construct) employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. Generally, a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g).

[00461] Compositions comprising the precursor bispecific antibody construct of the present disclosure or comprising a nucleotide sequence encoding the precursor bispecific antibody construct may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound as disclosed herein, and one or more additional active agents, as well as administration of compositions comprising precursor bispecific antibody construct as disclosed herein, and each active agent in its own separate pharmaceutical dosage formulation. For example, a precursor bispecific antibody construct or comprising a nucleotide sequence encoding the precursor bispecific antibody construct, as described herein, and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Similarly, a precursor bispecific antibody construct or comprising a nucleotide sequence encoding the precursor bispecific antibody construct, as described herein, and the other active agent can be administered to the patient together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions comprising precursor bispecific antibody construct or comprising a nucleotide sequence encoding the precursor bispecific antibody construct, and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.

[00462] Thus, in certain embodiments, also contemplated is the administration of precursor bispecific antibody construct compositions of this disclosure or comprising a nucleotide sequence encoding the precursor bispecific antibody construct, in combination with one or more other therapeutic agents. Such therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as cancer, inflammatory disorders, allograft transplantation, type I diabetes, and multiple sclerosis. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutics, radiotherapeutics, or other active and ancillary agents.

[00463] In certain embodiments, the precursor bispecific antibody construct or comprising a nucleotide sequence encoding the precursor bispecific antibody construct, disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimu stine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL.RTM., Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE.RTM., Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-l l; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin.TM. (bexarotene), Panretin.TM. (alitretinoin); ONTAK.TM. (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[00464] A variety of other therapeutic agents may be used in conjunction with the precursor bispecific antibody construct described herein. In one embodiment, the precursor bispecific antibody construct or comprising a nucleotide sequence encoding the precursor bispecific antibody construct, is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.

[00465] The compositions comprising herein described precursor bispecific antibody construct or comprising a nucleotide sequence encoding the precursor bispecific antibody construct may be administered to an individual afflicted with a disease as described herein, including, but not limited to cancer and autoimmune and inflammatory diseases. For in vivo use for the treatment of human disease, the precursor bispecific antibody construct or comprising a nucleotide sequence encoding the precursor bispecific antibody construct described herein are generally incorporated into a pharmaceutical composition prior to administration. A pharmaceutical composition comprises one or more of the precursor bispecific antibody construct or comprising a nucleotide sequence encoding the precursor bispecific antibody construct described herein in combination with a pharmaceutically acceptable carrier or excipient as described elsewhere herein. To prepare a pharmaceutical composition, an effective amount of one or more of the precursor bispecific antibody constructs or comprising a nucleotide sequence encoding the precursor bispecific antibody construct is mixed with any pharmaceutically acceptable carrier(s) or excipient known to those skilled in the art to be suitable for the particular mode of administration.

[00466] A pharmaceutically acceptable carrier may be liquid, semi-liquid or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include, for example, a sterile diluent (such as water), saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methyl parabens, phenols or cresols, mercurials, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride); antioxidants (such as ascorbic acid and sodium bisulfite; methionine, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisol, butylated hydroxytoluene, and/or propyl gallate) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates). If administered intravenously, suitable pharmaceutically acceptable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.

[00467] The compositions comprising precursor bispecific antibody construct as described herein may be prepared with pharmaceutically acceptable carriers that protect the precursor bispecific antibody construct against rapid elimination from the body, such as time release formulations or coatings. Such pharmaceutically acceptable carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art.

[00468] The present precursor bispecific antibody construct are useful for the treatment of a variety of cancers or tumors. In some embodiments, the cancer or tumor comprises a solid tumor. In some embodiments, the cancer or tumor comprises a non-solid tumor. In some embodiments, the cancer or tumor comprises a metastasis of a cancer or tumor.

[00469] For example, some embodiments of a method for the treatment of a cancer are directed to cancers including, but not limited to, melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renal cell carcinoma, uterine cancer, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, testicular cancer, gastric cancer, esophageal cancer, multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL), or other cancers, by administering to a cancer patient a therapeutically effective amount of a herein disclosed precursor bispecific antibody construct or a nucleotide sequence encoding the precursor bispecific antibody construct.

[00470] Solid tumors may be benign (not cancer), or malignant (cancer). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors for which treatment may be provided include sarcomas, carcinomas, and lymphomas. In some embodiments, solid tumors for which treatment may be provided include neoplasms (new growth of cells) or lesions (damage of anatomic structures or disturbance of physiological functions) formed by an abnormal growth of body tissue cells other than blood, bone marrow or lymphatic cells. In some embodiments, a solid tumor for which treatment may be provided consists of an abnormal mass of cells which may stem from different tissue types such as liver, colon, breast, or lung, and which initially grows in the organ of its cellular origin. However, such cancers may spread to other organs through metastatic tumor growth in advanced stages of the disease.

[00471] In some embodiments of a method for treatment of a cancer or tumor, the solid tumor or cancer comprises a sarcoma or a carcinoma, adrenocortical tumor (adenoma and carcinoma), a fibrosarcoma, a myxo-sarcoma, a liposarcoma, a chondrosarcoma, an osteogenic sarcoma, a chordoma, an angiosarcoma, an endothelio sarcoma, a lymphangiosarcoma, a lymphangioendothelio sarcoma, a synovioma, a mesothelioma, an Ewing's tumor, a leiomyosarcoma, a rhabdomyosarcoma, a colon carcinoma, a pancreatic cancer or tumor, a breast cancer or tumor, an ovarian cancer or tumor, a prostate cancer or tumor, a squamous cell carcinoma, a squamous cell carcinoma of the lung, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinomas, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an endocrine tumor, a germ cell tumor, a hepatoblastoma, a hepatocellular carcinoma, a melanoma, a neuroblastoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a soft tissue sarcoma other than rhabdomyosarcoma, a Wilms, Tumor, a cervical cancer or tumor, a uterine cancer or tumor, a testicular cancer or tumor, a lung carcinoma, a small cell lung carcinoma, an anal cancer, a glioblastoma, an epithelial tumor of the head and neck, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodenroglioma, a schwannoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma.

[00472] In some embodiments of a method for treatment of a cancer or tumor, the tumor or cancer comprises a non-solid tumor, that is a non-solid cancer. In some embodiments methods for treatment of a cancer or tumor may be for a diffuse cancer, wherein the cancer is widely spread; not localized or confined. In some embodiments, a diffuse cancer may comprise a non-solid tumor. Examples of diffuse cancers include leukemias. Leukemias comprise a cancer that starts in blood- forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream.

[00473] In some embodiments of a method for treatment of a cancer or tumor, the diffuse cancer comprises a B-cell malignancy. In some embodiments, the diffuse cancer comprises leukemia. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is large B-cell lymphoma.

[00474] In some embodiments of a method for treatment of a cancer or tumor, the diffuse cancer or tumor comprises a hematological tumor. In some embodiments, hematological tumors are cancer types affecting blood, bone marrow, and lymph nodes. Hematological tumors may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages, and masT-cells, whereas the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas (e.g. Hodgkin's Lymphoma), lymphocytic leukemias, and myeloma are derived from the lymphoid line, while acute and chronic myelogenous leukemia (AML, CML), myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

[00475] In some embodiments of a method for treatment of a cancer or tumor, the non-solid (diffuse) cancer or tumor comprises a hematopoietic malignancy, a blood cell cancer, a leukemia, a myelodysplastic syndrome, a lymphoma, a multiple myeloma (a plasma cell myeloma), an acute lymphoblastic leukemia, an acute myelogenous leukemia, a chronic myelogenous leukemia, a Hodgkin lymphoma, a non-Hodgkin lymphoma, or plasma cell leukemia.

[00476] An amount that, following administration, inhibits, prevents reduces the incidence of, reduces the tumor load, or delays the growth, progression and/or metastasis of a cancer in a statistically significant manner (i.e., relative to an appropriate control as will be known to those skilled in the art) is considered effective.

[00477] Another embodiment provides a method for preventing metastasis of a cancer including, but not limited to a solid or non-solid tumor or cancer as disclosed above, by administering to a cancer patient a therapeutically effective amount of a herein disclosed precursor bispecific antibody construct or a nucleotide sequence encoding the precursor bispecific antibody construct (e.g., an amount that, following administration, inhibits, prevents or delays metastasis of a cancer in a statistically significant manner, i.e., relative to an appropriate control as will be known to those skilled in the art).

[00478] Another embodiment provides a method for preventing a cancer including, but not limited to a solid or non-solid tumor or cancer as disclosed above, by administering to a cancer patient a therapeutically effective amount of a herein disclosed precursor bispecific antibody construct or a nucleotide sequence encoding the precursor bispecific antibody construct.

[00479] Another embodiment provides a method for treating, inhibiting the progression of a tumor or cancer including but not limited to a solid or non-solid tumor or cancer as disclosed above, by administering to a patient afflicted by one or more of these diseases a therapeutically effective amount of a herein disclosed precursor bispecific antibody construct or a nucleotide sequence encoding the precursor bispecific antibody construct.

[00480] In one embodiment, the present disclosure provides a method for directing T cell activation, comprising administering to a patient in need thereof an effective amount of a precursor bispecific antibody construct that comprises a CD3 binding domain, as described herein, that is able to specifically binds TCRa, TCRp, CD3y, CD35, CD3e, or a combination thereof, and a TAA first binding domain that specifically binds a TAA target, for instance, a tumor- specific antigen (e.g., EGFR) or other antigens of choice at a site or cell where T -cell activation is desired, and a regulatory domain, as described above.

EXAMPLES

[00481] Examples 1-2 demonstrate basic elements and steps of producing and purifying precursor bispecific antibodies, wherein the representative TAA (anti-TAA first binding site) is hEGFR. Examples 3-9 expand on the functional and therapeutic properties of these constructs. Similar techniques and methods were used to prepare and purify precursor bispecific antibodies used in later Examples, wherein the TAA (anti-TAA first binding site) was 5T4 or ROR1 (Examples 10- 15). In an effort to not repeat technical methods used, reference has been made back to Examples 1-9, wherein based on the disclosure therein, the skilled artisan would surely be able to make and use the bispecific antibodies produced and tested in Examples 10-15.

[00482] A standardized terminology is used throughout the Examples to identify different precursor constructs. This terminology is used for precursors wherein the first binding site recognizes the human EGFR antigen, the human 5T4 antigen, or the ROR1 antigen. If not noted otherwise, the second binding domain in each of these constructs comprised an Fab CD3e binding domain as described in detail herein. Further, the regulatory domain comprised a region (N-terminal to C-Terminal): CAP-linker-HSA-linker, wherein the linkers may be any linkers disclosed, and wherein the linker C-terminal to the HSA may be a protease cleavable, non-cleavable, or multi protease cleavable linker.

[00483] The terminology used is as follows: Micro-Environment Activated T cell Engager construct (MATE); Precursor Tumor Micro-Environment Activated T cell Engager construct (PTMATE); Precursor Tumor Micro-Environment Activated T cell Engager construct with cleavable linker comprising a MMP2/9 cleavage site (PTMATE-C); Precursor Tumor Micro- Environment Activated T cell Engager construct with non-cleavable linker (PTMATE-NC); and Precursor Tumor Micro-Environment Activated T cell Engager construct with multiple cleavage linker having both MMP and Matriptase/uPA/Legumain cleavage sequences in tandem (PTMATE- MC). The first binding site comprised an scFv wherein the target antigen is generally identified following the construct name, for example PTMATE-EGFR-MC or PTMATE-C EGFR, wherein the scFv target TAA is EGFR. A skilled artisan would appreciate that in certain embodiments herein and in the figures, the appearance of the name differs slightly though it is clear which construct was produced and tested, for example but not limited to EGFR -PTMATE-C, which refers to a Precursor Tumor Micro-Environment Activated T cell Engager construct directed to TAA EGFR that has a cleavable MMP2/9 linker. Additional target TAAs include 5T4 and ROR1. The linker in these precursor constructs is between the regulatory arm and a polypeptide of the Fab second binding site. Figures 1A-1C provide examples of MATE and PTMATE structures, and order of domains and elements within these structures.

Example 1: Precursor Bispecific Antibody with Regulatory Arm

[00484] Objective : To produce precursor bispecific antibody constructs with a regulated therapeutic window, wherein the precursor bispecific antibodies have an optimized half-life or a regulated T-cell binding activity, or both.

[00485] Methods: Precursor bispecific antibody constructs (precursor bispecific antibody construct) comprising (1) a single chain fragment variable (scFv) binding domain that binds to an epidermal growth factor receptor (EGFR) epitope, (2) an antibody fragment (Fab) binding domain that binds to a human CD3e epitope, and (3) a regulatory arm, will be manufactured as follows.

[00486] Anti-EGFR x antiCD3e precursor bispecific antibody construct (Figure 1A)

[00487] Figures 4A, 4B, 4G, 4H, 41, and 4J present amino acid and nucleotide sequence information, respectively, of embodiments of precursor bispecific antibody construct shown in Figures 2A and 2B. For example, a heterodimer formed by the combination of polypeptides represented by the sequences shown in Figure 4A (SEQ ID NO: 106) and Figure 41 (SEQ ID NO: 114) represents a precursor bispecific antibody construct with a cleavable peptide (cleavable by an MMP) as shown in Figure 2A. The polypeptide sequences of this heterodimer could be encoded by the nucleotide sequences presented in Figures 4B (SEQ ID NO: 107) and 4J (SEQ ID NO: 115). A similar heterodimer formed by the combination of polypeptides represented by the sequences shown in Figure 4C (SEQ ID NO: 108) and Figure 41 (SEQ ID NO: 114), while appearing to present a similar structure, would not have the same functionality (regulated half-life and regulated T-cell binding activity), as it does not include a cleavable peptide (CP).

[00488] Another example of a precursor bispecific antibody construct heterodimer is that formed by the combination of polypeptides represented by the sequences shown in Figure 4A (SEQ ID NO: 106) and Figure 4G (SEQ ID NO: 112), which also includes a cleavable peptide (cleavable by an MMP) as shown in Figure 2B. The polypeptide sequences of this heterodimer could be encoded by the nucleotide sequences presented in Figures 4B (SEQ ID NO: 107) and 4H (SEQ ID NO: 113). A similar heterodimer formed by the combination of polypeptides represented by the sequences shown in Figure 4C (SEQ ID NO: 108) and Figure 4G (SEQ ID NO: 112), while appearing to present a similar structure, would not have the same functionality (regulated half-life and regulated T-cell binding activity), as it does not include a cleavable peptide (CP).

[00489] Two alternative heterodimers of an active bispecific antibody construct antibody would be formed by the combination of polypeptides presented by the sequence set forth in Figure 4E (SEQ ID NO: 110) in combination with the polypeptide of either Figure 4G (SEQ ID NO: 112) or Figure 41 (SEQ ID NO: 114) A representative activated bispecific antibody construct of a heterodimer of polypeptides from Figure 4E and 41 is shown in Figure 2F. The nucleotide sequence encoding the polypeptide of Figure 4E is presented in Figure 4F (SEQ ID NO: 11 l).As can be recognized from the structures and sequences of Figures 2A and 2B, different variants of precursor bispecific antibody constructs may be created by rearranging the VL and VH regions of the ScFV. Similarly, variants of precursor bispecific antibody constructs could be formed by rearranging components of the regulatory domain, and /or by exchanging places of the regulatory domain with the anti-TAA domain (Figures 1A-1B and Figures 2A-E; not all possible structures are shown)

[00490] Embodiments of an anti-EGFR x antiCD3e precursor bispecific antibody construct variant 1 are presented in Figures 2C); of an anti-EGFR x antiCD3e precursor bispecific antibody construct variant 2 (Figure 2D); and of an anti-EGFR x antiCD3e precursor bispecific antibody construct variant 3 (Figure 2E)

[00491] For each construct, humanized anti-CD3 epsilon framework sequences were used in the anti-CD3e domains. For example, when SP34 anti-CD3epsilon Fab clone sequences are used, the anti-CD3e framework sequences are humanized sequences while the CDR sequences remain“as is” in the SP34 mouse hybridoma clone. The CDR sequences of the mouse SP34 mouse hybridoma, SEQ ID NO: 71-79. The sequences as used to produce an Anti-EGFR x antiCD3e precursor bispecific antibody construct are provided above in Table 3. In certain embodiments, the anti-EGFR domain sequences used include scFv sequences based on the sequence of Panitumumab (International Nonproprietary Name (INN); See United States Patent Nos. 6,235,883 and 8,628,773). In certain embodiments, in the constructs including a cleavable linker, the linker will include a matrix metalloproteinase-2 (MMP2) protease cleavage site. Exemplary amino acid sequences used in a precursor bispecific antibody construct anti-EGFR x anti-CD3e bispecific construct include those presented in Table 3 below.

[00492] Table 3: Amino Acid Sequences comprised in EGFR and CD3a binding domains, and an MMP2 cleavage site

[00493] Analysis of constructs was by methods well known in the art, for example use of SDS- PAGE, SEC-HPLC, and LC-MS, all which are well known and in used in the field of polypeptide and protein analysis.

[00494] Results.

[00495] The anti-EGFR x anti-CD3e precursor bispecific antibody construct is a bivalent antibody precursor that includes a CAP component and a human serum albumin (HS A) component, wherein the CAP and HSA are part of a protease cleavable regulatory arm. The bivalent antibody precursor comprises an scFv EGFR binding domain and a Fab CD3e binding domain.

[00496] The specific structural order N-terminal to C-terminal of the regulatory arm is: CAP- linker-HSA-cleavable linker-C-terminus fused to an N-terminal Variable chain of the Fab. The amino acid sequence of the CAP component is AA 1-27 of CD3e (SEQ ID NO: 4). The amino acid sequence of the HSA component is set forth in SEQ ID NO: 116.

[00497] The anti-EGFR x anti-CD3e precursor bispecific antibody construct (Figure 2A) has a unique structure comprising a specific order of components with two amino acid chains:

Polypeptide 1: N’-VLl-Ll-VHl-L2-VL2-CL-L3- C’; and

Polypeptide 2: N’-CAP-L4-HSA-L5-CP-L6-VH2-CHl-L7- C’

wherein VL1 and VH1 comprise an EGFR binding domain, wherein VL2 and VH2 comprise a CD3e binding domain, wherein Ll, L2, L3, L4, L5, L6, and L7 are linkers, wherein CP is a cleavable peptide including the protease cleavable site, and wherein CH1 and CL are constant regions. In certain embodiments, the amino acid sequences of Ll is set forth in SEQ ID NO: 100, the amino acid sequence of L2 is set forth in SEQ ID NO: 100 or SEQ ID NO: 93, the amino acid sequences of L4 is set forth in SEQ ID NO: 98, the amino acid sequence of L5 is set forth in SEQ ID NO: 99, and the amino acid sequence of L6 is set forth in SEQ ID NO: 99.

[00498] The precursor anti-EGFR x anti-CD3e bispecific construct (anti-EGFR x anti-CD3e precursor bispecific antibody construct) has an increased therapeutic window due to the presence of the HSA, and protease activation of the anti-CD3e domain in order that T-cell activation occurs within the cancer micro-microenvironment, wherein Fab CD3e binding is inhibited prior to the precursor bispecific antibody being in the cancer microenvironment, due to the CAP being specifically bound to or blocking the Fab CD3 binding site.

[00499] The anti-EGFR x anti-CD3e precursor bispecific antibody construct variant 1 construct (Figure 2C) is a bivalent antibody precursor that includes a CAP component and a human serum albumin (HSA) component, wherein the CAP and HSA are part of a protease cleavable regulatory arm. However, unlike the anti-EGFR x anti-CD3e precursor bispecific antibody construct, the positioning of the protease cleave site in the precursor bispecific antibody construct variant 1 construct is between the CAP and the HSA. The amino acid sequences of the bivalent antibody precursor scFv EGFR binding domain and an Fab CD3e binding domain are as above for the anti- EGFR x anti-CD3e precursor bispecific antibody construct.

[00500] The specific structural order N-terminal to C-terminal of the regulatory arm is: CAP- linker-protease cleavage site-linker-HSA- linker-C-terminus fused to an N-terminal Variable chain of the Fab. Thus, the half-life of the anti-EGFR x anti-CD3e precursor bispecific antibody construct variant 1 construct is extended compared with the anti-EGFR x anti-CD3e precursor bispecific antibody construct. The amino acid sequences of the CAP, HSA, and cleavable linker are as above for the anti-EGFR x anti-CD3e precursor bispecific antibody construct.

[00501] The anti-EGFR x anti-CD3e precursor bispecific antibody construct variant 1 construct has a unique structure comprising a specific order of components with two amino acid chains:

Polypeptide 1: N’-VFl-Fl-VHl-F2-VF2-CF-F3- C’; and

Polypeptide 2: N’-CAP-F4-CP-F5-HSA-F6-VH2-CHl-F7- C’

wherein VF1 and VH1 comprise an EGFR binding domain, wherein VF2 and VH2 comprise a CD3e binding domain, wherein El, F2, F3, F4, F5, F6, and F7 are linkers, wherein CP is a cleavable peptide including the protease cleavable site, and wherein CH1 and CF are constant regions.

[00502] The precursor anti-EGFR x anti-CD3e bispecific variant 1 construct (anti-EGFR x anti- CD3e precursor bispecific antibody construct variant 1) has an increased therapeutic window due to the continued presence of the HSA, and protease activation of the anti-CD3e domain in order that T-cell activation occurs within the cancer microenvironment, wherein prior to the bispecific antibody being in the cancer microenvironment, the Fab CD3e binding is inhibited due to the CAP being specifically bound or blocking the Fab CD3 binding site.

[00503] The anti-EGFR x anti-CD3e precursor bispecific antibody construct variant 2 construct (Figure 2D) is a bivalent antibody precursor that includes a human serum albumin (HSA) component, wherein the HSA is part of a protease cleavable regulatory arm. The amino acid sequences of the bivalent antibody precursor scFv EGFR binding domain and an Fab CD3e binding domain are as above for the anti-EGFR x anti-CD3e precursor bispecific antibody construct.

[00504] The specific structural order N-terminal to C-terminal of the regulatory arm is: HSA- linker-protease cleavage site-linker-C-terminus fused to an N-terminal Variable chain of the Fab. Thus, the half-life of the anti-EGFR x anti-CD3e precursor bispecific antibody construct variant 2 construct is extended until the half-life becomes limited upon entry of the precursor bispecific anti- EGFR x anti-CD3e precursor bispecific antibody construct variant 2 construct and cleavage of the regulatory arm. The amino acid sequences of the HSA and cleavable linker are as above for the anti-EGFR x anti-CD3e precursor bispecific antibody construct.

[00505] The anti-EGFR x anti-CD3e precursor bispecific antibody construct variant 2 construct has a unique structure comprising a specific order of components with two amino acid chains:

Polypeptide 1: N’-VLl-Ll-VHl-L2-VL2-CL-L3- C’; and

Polypeptide 2: N’-HSA-L4-CP-L5-VH2-CHl-L7- C’

wherein VL1 and VH1 comprise an EGFR binding domain, wherein VL2 and VH2 comprise a CD3e binding domain, wherein Ll, L2, L3, L4, L5, and L7 are linkers, wherein CP is a cleavable peptide including the protease cleavable site, and wherein CH1 and CL are constant regions.

[00506] The precursor anti-EGFR x anti-CD3e bispecific variant 2 construct (anti-EGFR x anti- CD3e precursor bispecific antibody construct variant 2) has an increased therapeutic window due to the presence of the HSA, wherein following to the bispecific antibody entry into the cancer microenvironment and cleavage of the regulatory arm including the HSA, the bispecific antibody will have a limited half-life of hours.

[00507] The anti-EGFR x anti-CD3e precursor bispecific antibody construct variant 3 construct (Figure 2E) is a bivalent antibody precursor that includes a CAP component as part of a protease cleavable regulatory arm. The amino acid sequences of the bivalent antibody precursor scFv EGFR binding domain and a Fab CD3e binding domain are as above for the anti-EGFR x anti-CD3e precursor bispecific antibody construct.

[00508] The specific structural order N-terminal to C-terminal of the regulatory arm is: CAP- linker-protease cleavage site-linker -C-terminus fused to an N-terminal Variable chain of the Fab. Thus, the binding of the anti-EGFR x anti-CD3e precursor bispecific antibody construct variant 3 construct to a CD3e epitope is inhibited until entry of the pre-cursor antibody into a cancer microenvironment, wherein the CAP component is cleaved. The amino acid sequences of the CAP and the cleavable linker are as above for the anti-EGFR x anti-CD3e precursor bispecific antibody construct.

[00509] The anti-EGFR x anti-CD3e precursor bispecific antibody construct variant 3 construct has a unique structure comprising a specific order of components with two amino acid chains:

Polypeptide 1: N’-VLl-Ll-VHl-L2-VL2-CL-L3- C’; and

Polypeptide 2: N’-CAP-L4-CP-L5-VH2-CHl-L7- C’

wherein VL1 and VH1 comprise an EGFR binding domain, wherein VL2 and VH2 comprise a CD3e binding domain, wherein Ll, L2, L3, L4, L5, and L7 are linkers, wherein CP is a cleavable peptide including the protease cleavable site, and wherein CH1 and CL are constant regions.

[00510] The precursor anti-EGFR x anti-CD3e bispecific variant 3 construct (anti-EGFR x anti- CD3e precursor bispecific antibody construct variant 3) has restricted binding to T-cells and regulated T-cell activation due to the presence of the CAP preventing or inhibiting access to the anti-CD3e domain. Thus, T-cell activation is restricted and occurs within the cancer microenvironment.

[00511] For the precursor constructs (precursor bispecific antibody construct) produced certain linker amino acid sequences have been used, for example, a particular linker (L) is present in the precursor bispecific antibody construct. In other embodiments, the skilled artisan would recognize that a particular linker (L) is not present in the precursor bispecific antibody construct. The linkers between components and between domains are identified by an“L” followed by a numeral, e.g., Ll, L2, L3, L4, L5, L6, and L7. In some embodiments, for a precursor bispecific antibody construct wherein the TAA is EGFR, as is shown in Figures 2A and 2B, the amino acid sequence of L4 is set forth in SEQ ID NO: 98, the amino acid sequence of L5 is set forth in SEQ ID NO: 99, the amino acid sequence of L6 is set forth in SEQ ID NO: 99, and the amino acid sequence of L2 is set forth in SEQ ID NO: 100, but it should be clear to the skilled artisan that other linkers known in the art and/or presented here could be sued.

[00512] Examples 2- 9 present results including analysis of each construct for size (molecular weight), half-life, antigen binding (EGFR), and T-cell engagement (CD3e binding). Further, each construct was analyzed for aggregation formation using SDS-PAGE, CE-SDS, of the size exclusion chromatography (SEC) and analytical HPLC-SEC of the purified product. The constructs were tested for their binding affinities towards the soluble antigens (extracellular CD3epsison and extracellular EGFR) by ELISA binding assay. Cytotoxicity of human peripheral blood mononuclear cells (PBMC) against EGFR expressing cells was analyzed as a test of functionality, where that the presence of the regulatory arm provided a regulatory cytotoxicity effect. Furthermore, constructs were tested for its anticancer activity in xenograft mouse model, both in A NSG mouse model.

Example 2: Expression and Purification of Bispecific Antibody Constructs

[00513] Objective : To express and purify a cleaved precursor bispecific antibody, a non-cleaved precursor bispecific antibody, and a bispecific antibody construct.

[00514] Methods: Gene synthesis and plasmid construction. The coding sequences for the heavy chain (HC) and light chain (LC) of the precursor bispecific antibody were generated by DNA synthesis and PCR, subsequently subcloned into pTT5-based plasmid (NRC Biotechnology Research Institute) for protein expression in mammalian cell system. Finally, the gene sequences in the expression vectors were confirmed by DNA sequencing.

[00515] Expression of precursor bispecific antibody construct. Transient expression of the precursor bispecific antibodies was performed by co-transfection of paired HC and LC constructs into CHO cells using PEI method. Briefly, 1L of CHO cells at approximately 2.3xl06/ml in a 3L shake flask was used as the host, Transfection was initiated by adding a mixture of 2mg of total DNA and 4mg PEI in lOOml OptiMEM medium (Invitrogen) to the cells and gentle mixing. Cells were then cultured in an incubator shaker at 120 rpm, 37°C, and 8% C02, for 8-10 days. Feeding with peptone and glucose was carried out 24h later and every 2-3 days thereafter depending on the cell density and viability. The cell culture was terminated on day 8-10 when cell viability reduced to <70%. The conditioned medium was harvested for protein purification.

[00516] Purification of precursor bispecific antibody construct. Protein purification by affinity chromatography and SEC was performed using an AKTA pure instrument (GE Lifesciences). Affinity capture of the precursor bispecific antibody was achieved by passing the harvested supernatants over a column of CaptureSelect™ CH1-XL Affinity Matrix (Thermo Scientific). After washing column with PBS, the protein was eluted with 0.1M Glycine, pH 2.5, and immediately neutralized with 1/6 volume of 1M Tris-HCl, pH 8.0. The affinity purified protein was then concentrated to 5-l0mg/ml using Amicon 30kD concentrator (Merck Millipore) and subjected to SEC purification on a Superdex200 column (GE Lifesciences) equilibrated with PBS. Protein fractions were collected and analyzed using SDS-PAGE and HPLC-SEC.

[00517] SEC-HPLC analysis of precursor bispecific antibody construct. Analytical SEC-HPLC was performed using a TSK G3000SWXL column (Tosoh Instruments), Shimadzu LC-10 HPLC instrument (Shimadzu Corp.), and common conditions for IgG, i.e. mobile phase buffer, PBS; flow rate, lml/min; running cycle, 30min; protein sample concentration, lmg/ml diluted in PBS; and 20pl/injection/run.

[00518] Results. The expressed HC and LC constructs attached to form a single molecule, as indicated by the single -75 kDA band observed in the SDS-PAGE, and by a single major peak at RT of -9 min in SEC-HPLC (Figures 5A and 5B, respectively). Similar results were observed for the non-cleaved bispecific antibody construct, which produced a -100 kDA band in SDS-PAGE, and a single major peak at RT of -7.5 min in SEC-HPLC (Figures 5C and 5D, respectively), and for the purified cleaved bispecific antibody construct, which produced a -100 kDA band in SDS- PAGE, and a major peak at RT of -7.5 min in SEC-HPLC (Figures 5E and 5F, respectively).

[00519] Conclusion: A cleaved precursor bispecific antibody, a non-cleaved precursor bispecific antibody, and a bispecific antibody construct can be successfully expressed and purified.

Example 3: Binding of Bispecific Antibody Constructs to EGFR and CD3s

[00520] Objective : To study the binding efficacy of a variety of precursor bispecific antibody constructs that recognize EGFR (scFv first binding domain) and CD3e (Fab second binding domain), including precursor constructs comprising a cleavable linker, precursor constructs comprising a non-cleavable linker, precursor constructs lacking a CAP element, precursor constructs lacking a half-life extending element, and an activated bispecific antibody construct (control).

Methods:

Antibody expression and purification:

All antibodies were produced under serum-free conditions (FreeStyle medium) by cotransfecting relevant heavy and light chain expression vectors in CHO cells, using PEI.

Antibodies were purified by CH1 affinity chromatography (CaptureSelect™ CH1-XL Affinity Matrix; ThermoFisher) and further purified by superdex 200 column (GE Healthcare) to remove soluble aggregates. Purified antibodies were filter-sterilized over 0.2-mM dead-end filters. Concentration of purified antibodies was determined by absorbance at 280 nm (specific extinction coefficients were calculated for each protein). Purified proteins were analyzed by SDS/PAGE, SEC-HPLC and mass spectrometry.

Protease cleavage:

[00521] Conversion of the Precursor Tumor Micro-Environment Activated T cell Engager constructs (PTMATE) variants to the active Micro-Environment Activated T cell Engager (MATE) was performed by recombinant Human MMP-9 (R&D Systems). Briefly, the activated MMP9 is diluted to lOOug/ml in the TCBN buffer (50 mM Tris, 10 mM CaQ2, 150 mM NaCl, 0.05% Brij35(w/v), pH 7.5). The PTMATE variants were buffer-exchanged to PBS by diluting and concentrating then mixed with MMP9 protease with mass ratio 100:1. The proteolytic reaction was performed at room temperature for l6h. The cleavage products were detected by SDS-PAGE. ELISA of bispecific antibody constructs to antigens EGFR and CD3epsilon:

[00522] Method 1: EGFR antigen proteins, hEGFR-Fc (Cat # 344-ER-050, Bio-techne) and rhesus EGFR-Fc (Cat # EGR-C5252, Aero Biosystems)) were diluted to 0.05 ug/ml in PBS and CD3epsilon (hCD3epsilon-His (Cat # 10977-H08S, Sino Biological) and cyno CD3epsilon (Cat # CDE-C5226, Aero Biosystems)) to O.Olug/ml in PBS and coat 100 ul/well on ELISA plate (Cat # 9018, Coming), respectively, and incubated over-night at 4°C. The plates were blocked with 250ul 1% BSA in PBST for lhr at 37°C, and washed four times with PBST. All washes were done using Biotek (Elx 405). All the bispecific antibody constructs were diluted to 15 ug/ml and prepared with 3-fold serial dilutions (12 points, including 0 ug/ml). lOOul/well of diluted antibody construct solution were added to the plate and incubated for lhr at 37°C. Plates were washed four times with PBST, and then 100 ul/well of anti-human kappa light chain-HRP antibody (1: 10000) was added for 0.5 hr at 37°C. Plates were washed 4 times with PBST, then 100 ul/well of TMB substrate was added at RT for 5 min. 100 ul/well of 1.0 N HC1 was added to terminate the reaction. Plates were read using an ELISA plate reader at 450 nm wavelength (SpectraMax M5e). Data Analysis was performed using Graphpad Prism 5 software by using nonlinear regression (curve fit): log (agonist) vs. response, agonist is antibody concentration (nM) and response is OD value.

[00523] Method 2: MATE, PTMATE-C (construct with cleavable“C” linker), and PTMATE- NC (construct with non-cleavable“NC” linker) variants (anti-EGFR [scFv]/anti-CD3 antibody [Fab]) on recombinant human antigens. (Figure 1C (MATE), Figure ID (PTMATE), Figure IE (PTMATE A-CAP also termed“HSA-MATE”-C), Figure IF (PTMATE A-HSA also termed “CAP-MATE-C”))

[00524] As an example, coating recombinant human CD3e antigen at optimized concentration (O.Olug/ml) on 96-well plate (Coming#90l8) and incubating for overnight at 4°C. After blocking with 5% BSA and washed 3 times with lxPBST (lxPBS+0.05%Tween-20), diluted PTMATE variants were added to the plate and incubated for lhr at 37 °C. Bound PTMATE variants were detected with anti-human lambda light chain-HRP (1:10000) (SouthemBiotech, cat#2072-05). Optical density 450nm was read after adding TMB substrate and stop solution 1N HC1. Data was analyzed with Graphpad prism software by using nonlinear regression (curve fit): log (agonist) vs. response.

Results:

Initial Results: [00525] The expressed bispecific constructs were analyzed for its binding to CD3e and to EGFR in the absence of protease. The EC50 to human CD3e (hCD3e) was of 65.83 nM for the non-cleaved construct (squares), 440 nM for the cleaved construct (triangles), and 0.1063 nM for the active bispecific antibody construct (circles) (Figure 6A). The EC50 to cynomolgus CD3e (cyno CD3e) was of 10.1 nM for the non-cleaved construct (squares), 829 nM for the cleaved construct (triangles), and 0.0945 nM for the active bispecific antibody construct (circles) (Figure 6B). The EC50 to human EGFR (hEGFR) was of 0.2136 nM for the non-cleaved construct (squares), 0.173 nM for the cleaved construct (triangles), and 0.067 nM for the active bispecific antibody construct (circles) (Figure 6C). The EC50 to rhesus monkey EGFR (rEGFR) was of 0.2669 nM for the non- cleaved construct (squares) and 0.009133 nM for the active bispecific antibody construct (circles) (Figure 6D).

[00526] Follow-up Results: A broader range of variant constructs were expressed and analyzed for binding to human EGFR and human CD3e in the absence of protease. Figure 7A shows that the active MATE construct (circles) bound human EGFR at much lower concentrations of antibody than any of the PTMATE constructs (PTMATE-C [squares]; CAP-MATE-C [triangles]; HSA- MATE-C [inverse triangles]; PTMATE -NC [diamonds]). Similarly, Figure 7B shows that the active MATE construct (circles) bound human CD3e at much lower concentrations of antibody than any of the PTMATE constructs (PTMATE-C [squares]; CAP-MATE-C [triangles]; HSA-MATE- C [inverse triangles]; PTMATE-NC [diamonds]). The HSA-MATE construct, which lacks a CAP element, showed binding to CD3e between that of the MATE and other PTMATE constructs. Effect of Protease Cleavage.

Figures 8A-8B demonstrate similar binding to human EGFR antigen by MATE and PTMATE constructs (Figure 8A) while binding to human CD3e is influence by the presence or absence of a CAP element (Figure 8B). Absence of a CAP in the MATE construct results in increased binding affinity compared to the PTMATE constructs. The results shown in Figure 8C demonstrate regulatory binding of constructs to human CD3e, wherein the incubation of PTMATE-C constructs with protease leads to increased affinity of the resultant structure with the CD3e antigen (blue inverted triangles). A similar increase in affinity was not observed in the construct having a non- cleavable linker (PTMATE-NC) and incubated with protease (purple squares). Note the similar binding affinity of the PTMATE-C without protease and PTMATE-NC with or without protease. The double headed arrow points out the maximal and minimal binding affinities of the different constructs, wherein it is clear binding affinity may be regulated in PTMATE by cleavage of the regulatory domain by a protease.

[00527] Conclusion: The cleaved (active) construct (MATE) and the non-cleaved construct (PTMATE) have a relatively similar binding affinity to EGFR. All PTMATE variants similarly bind human EGFR, regardless of the presence or absence of protease. Binding to human CD3e was influenced by the presence of a CAP element. Constructs lacking a CAP element showed increased binding. Binding affinity to CD3e of PTMATE constructs could be regulated by incubation with protease, wherein incubation of a cleavable PTMATE construct in the presence of protease resulted in a significant increased binding affinity of the construct. Orders of magnitude increased EC50s of CD3e binding between masked precursor PTMATE (no protease or non-cleavable linker) and cleaved PTMATE forms. Protease-cleaved PTMATE has restored CD3e binding.

Example 4: Binding of Bispecific Antibody Constructs to Cancer cells

[00528] Objective : To study the binding efficacy of cleaved and non-cleaved variants of precursor bispecific antibody constructs, to cells. Specifically, to study the binding efficacy of MATE, PTMATE-C and PTMATE-NC variants to Jurkat T-lymphocytes (CD3e) and 293F-human EGFR cells (EGFR).

[00529] Methods:

[00530] Antibody constructs were produced as described in Examples 1- 3.

[00531] FACS analysis of bispecific antibody constructs to cancer cells:

[00532] PTMATE variants were tested by FACS using Jurkat cells or 293F-human EGFR cells to assess binding affinity of variants to cell-surface expressed CD3e or EGFR.

[00533] Briefly, the cells were harvested and stained with a titration of each PTMATE variants for lhr at 4°c. After staining, cells were washed 2 times with cold FACS buffer (1XPBS, 2%FBS). Bound antibodies were detected with FITC conjugated Mouse anti-Human Lambda Light Chain Secondary Antibody (Thermo Fisher, MA1-10395). The fluorescence intensity of the staining was measured using flow cytometer (BD, FACSVerse). The geometric mean fluorescence intensity (GMFI; median fluorescence intensity (MFI)) of MATE set antibodies staining was calculated (BD FACSuite software). Dose-response curves were generated and EC50s for PTMATE variants’ binding were calculated using GraphPad Prism software.

[00534] Results: The expressed precursor and/or cleaved bispecific constructs were analyzed for their ability to bind to tumor cells expressing CD3 epsilon (Jurkat T cells) and cell expressing hEGFR (293F). Both cleaved and non-cleaved antibody constructs bound to EGFR expressing cells with relatively similar affinity (Figure 9A). The bispecific antibody (MATE) bound CD3e expressing Jurkat T-cells. The precursor bispecific antibody constructs (PTMATE-C, PTMATE- NC )showed reduced binding to CD3e expressing Jurkat T-cells (Figure 9B arrow). [00535] Conclusion: Only when cleaved would the precursor antibody recognizes CD3e- expressing T cells. However, both cleaved and non-cleaved regulatory antibodies bind EGFR expressing tumor cells.

Example 5: In Vitro Evaluation of Precursor Bispecific EGFR Binding Constructs

Objective: To evaluate in vitro, dose dependent T-cell mediated cytotoxicity of PTMATE-EGFR variant constructs of breast cancer (MDA-MB-231 cells) and colorectal cancer cells (HCT-116 cells).

Methods:

[00536] Antibody constructs were produced as described in Examples 1, 2 and 3.

LDH Cytotoxicity Assay: MATE, PTMATE-C and PTMATE-NC variants (anti-EGFR/anti-CD3 antibody) in MDA-MB-231 cells. Similar assays were performed with colorectal cancer cell line HCT-116.

PTMATE variants were analyzed for their potential to induce T cell-mediated cytotoxicity in EGFR high-expressing MDA-MB-231 cells. HCT-116 similarly express EGFR on their cell surface. MDA-MB-231 cells were harvested and resuspended in RPMI 1640 medium supplemented with 5% fetal bovine serum. Approximately, 10,000 cells per well were plated in a round-bottom 96- well plate in duplicates; Human CD3+ T cells isolated from PBMCs (effector cells) were added into the wells to obtain different E:T ratios. Negative control groups were represented by effector or target cells only. For normalization, maximal lysis of MDA-MB-231 target cells (= 100%) was determined by incubation of the target cells with lysis solution in kit, inducing cell death. Minimal lysis (= 0%) was represented by target cells co-incubated with effector cells only, i.e. without any T cell bispecific antibody. After 24h incubation at 37°C, 5% C0 ₂ , LDH release from MDA-MB- 231 cells into the supernatant was then measured with the LDH detection kit (Promega, G1780), following the manufacturer's instructions. The percentage of specific cell lysis was calculated as [Sample release - Effector spontaneous release-Target spontaneous release]/[Maximum release - Target spontaneous release] xlOO. The percentage of LDH release was plotted against the concentrations of anti-EGFR/anti-CD3 bispecific antibodies in concentration-response curves. The EC50 values were measured using GraphPad Prism software.

Results:

[00537] Using a 24-hour lactate dehydrogenase assay, PTMATE-EGFR constructs having masked anti-CD3e binding domains (PTMATE-MC and PRMATE-NC) showed significantly reduced killing of breast cancer and colorectal cancer cell line cells (Figures 10A and 10B) compared with the MATE-EGFR constructs. MATE-EGFR showed dose-dependent T-cell mediated cytotoxicity of MDA-MB-231 (breast cancer) and HCT116 (colorectal cancer) cell lines. In the case of MDA-MB-231 cells the EC50 was as low as 0.6pM.

[00538] Conclusion: A pronounced difference in cell killing can be observed between MATE and anti-CD3e masked PTMATE-MC and PTMATE-NC constructs.

Example 6: In-Vitro Cell-Based Assays of PT MATE-EGFR:

T-cell Activation & Cytokine Release

Objective: To examine T-cell activation and cytokine release in the presence of variant precursor PTMATE constructs.

Methods:

[00539] Antibody constructs were produced as described in Examples 1- 3.

[00540] T-Cell Activation and Cytokine Release

[00541] MDA-MB-231 cells were re-suspended in X-VIV015 medium containing 2 mM Glutamax and dispensed to 96-well round bottom plate at a density of lxlO ⁴ cells per well. Test precursor antibodies were added and then plates were incubated for 30 minutes. PBMCs were isolated from fresh human blood and dispensed to plate at a density of 5xl0 ⁴ cells or lxlO ⁵ per well. Plate was incubated for 48 hours. Cytokine contents of supernatant culture media were measured by Luminex kit for IFN-g and TNF-a according to the manufacturer’s instruction (R&D). Meanwhile, the cell pellets were harvested and treated with the following antibodies: anti-CD45- FITC (clone: HI30), anti-CD4-APC (clone: RPA-T4), anti-CD8-PE (clone: PRA-T8) and anti- CD69-BV605 (clone: FN50). Flow cytometry was performed on a Thermo Attune NxT and analyzed with Flow Jo V10 software, CD69+ cells percentage among CD4+ and CD8+ T cell population were monitored.

PTMATE constructs tested included MATE EGFR, PTMATE-C EGFR, and PTMATE-NC EGFR.

[00542] Results: The results of T-cell activation and cytokine release are shown in Figures 11A- 111 for cytokines CD69 (Figures 11A-11C), IFNy (Figures 11D-11F), and TNFa (Figures 11G- 111). CD3/CD28 activation represents the positive control, whereby once both receptors are bound, it activates T-cells. Not shown is the data for cytokines CD45, CD4, and CD8, which followed the trends shown for CD69, INFy, and TNFa. The results indicate that T-cells secrete cytokines in the presence of MATE, PTMATE-C, and to much lower extent, to PTMATE-NC, due to T-cell activation.

[00543] CD8+-CD69+ T-cell activation we detected when using PTMATE-C variants at 48 hours (data not shown), wherein activation was observed using concentrations as low as single and sub-pM range. Marked differences in T-cell activation and cytokine release were observed between MATE and the precursor PTMATE-C construct in all cases. Cytokine release is reduced when using the PTMATE-C.

[00544] Conclusion: As the CD3 is capped in PTMATE-C and there is no apparent cleavage in the conditioned medium of the assay, the results of PTMATE-C and PTMATE-NC are relatively similar.

Example 7: Pharmacokinetics of PTMATE-EGFR in Mice

[00545] Objective: To examine if the half-life of precursor constructs administered to mice is extended compared with the half-life of the MATE construct lacking the HSA domain.

[00546] Methods:

[00547] Antibody constructs were produced as described in Examples 1- 3.

[00548] Pharmacokinetics:

[00549] Male C57BL/6 mice were dosed via tail vein injection of antibodies. Approx. 80 pL of blood were taken from the animals via retro orbital puncture for semi- serial bleeding or cardiac puncture (under anesthesia with isoflurane) for terminal bleeding into tubes at pre-dose, and 10 min, 1, 2, 4, 8, 24 hr and 2, 4, 7, 10, 14 d post dosing. The blood samples were placed at room temperature for 0.5 hr and centrifuged (2000 g*5 min, 4°C) to obtain the serum samples. All serum samples were diluted 20-fold in assay diluent (lxPBS/l% BSA/0.05%Tween-20/0.05% proclin 300) first and then additionally diluted in 5% pooled mouse serum. The diluted samples were loaded at 50 pL/well into the recombinant Human EGFR Fc chimera-coated plates. Following loading, plates were sealed and incubated for one hour at 37°C. The reaction on the plates could be stopped and read at 450/630 nm wavelength following reaction with HRP-conjugated detection antibody working solution and incubation with premixed TMB substrate. Pharmacokinetic (PK) parameters were estimated by non-compartmental model using WinNonlin 6.4 software.

[00550] Constructs analyzed included MATE (VL-VH) EGFR, PTMATE-C (VL-VH) EGFR, and PTMATE-NC (VL-VH) EGFR.

[00551] Dosing was by intravenous injection at 0.5 mg/kg or 2 mg/kg.

[00552] Half-life was observed over the course of 10 days.

[00553] Results:

[00554] Marked differences in serum Ti _/2 between MATE and the pro-form reduced-activity PTMATE format precursor constructs was observed and is shown by comparing Figures 12A, 12B, and 12C. There was an extended“Long” serum Ti/2 in its PTMATE-C EGFR and PRMATE-NC EGFR constructs (Figures 12B and 12C). Compared with this extended presence of the PTMATE antibody“drug”, administration of MATE EGFR tumor activated constructs resulted in a much shorter Ti/2 (Figure 12A). The data show that the concentration of the MATE EGFR construct was reduced to less than 100 ng/ml by day 1, wherein the concentration of the PTMATE EGFR remained above 100 ng/ml for 6-10 days.

[00555] Once the PTMATE-C construct would be localized to a tumor micro-environment, cleavage of the regulatory arm would be expected wherein the smaller-sized MATE may provide improve tumor penetration.

[00556] It is well established that human semm albumin has lower recycling in mouse due to sequence differences. Therefore, the T observed in mice is mostly governed by mass, and less to serum albumin recycling. Unlike in mice, the sequence identity between serum albumin of humans and non-human primates allows serum albumin receptor mediated recycling. Therefore, it is expected that the Tl/2 will be much longer in humans and non-human primates. Thus, longer Tm is expected in future tests in primates/humans (serum albumin recycling).

[00557] Conclusion: The regulatory arm comprising an HSA domain provides for an extended half-life of the precursor construct.

Example 8: In vivo Xenograft of PTMATE EGFR in NSG Mouse Model

[00558] Objective: To examine the effects of variant PTMATE constructs on morbidity, mortality,“tumor take rate”, and tumor growth, in a mouse model.

[00559] Methods :

[00560] Antibody constructs were produced as described in Examples 1- 3.

In-vivo xenograft assay:

[00561] NOD/S CID/IL2RY ^nu11 (NSG) Mice (Charles River Laboratory) were used in accordance with a protocol reviewed and approved by the Institutional Animal Care and User Ethical Committee. Mice were housed in sterile conditions using high-efficiency particulate arrestance filtered micro-isolators and fed with irradiated food and acidified water. Xenograft tumors were generated by SC injection of 3xl0e ⁶ MDA-MB-231 cells (in 200 pl of PBS) into 6-8 weeks old mice. When tumors of -50 mm ³ were formed, PBMC cells derived from volunteers’ peripheral blood were IV injected into tail vain at E:T ratio of 3 : 1. When tumors of -80- 120 mm3 were formed, PTMATE variants were daily tail-injected (0.5-l5ug/kg/day). After tumor inoculation, the animals were checked daily for morbidity and mortality. At the time of routine monitoring, the animals were checked for any effects of tumor growth and treatments on normal behavior such as mobility, food and water consumption, body weight gain/loss (body weights were measured twice weekly), eye/hair, matting and any other abnormal effect. The major endpoint was the tumor take rate and the tumor growth curve. Tumor sizes were measured twice weekly in two dimensions using a caliper, and the volume expressed in mm ³ using the formula: V = 0.5 a x b 2 where a and b are the long and short diameters of the tumor, respectively. Body weights were measured twice weekly.

[00562] Constructs tested included MATE-EGFR, PTMATE-C EGFR, PTMATE-NC EGFR, and control (no construct; Two controls were used in the assay, cancer cells alone (not treated) and cancer cells+ PBMC (treated with PBS as control)).

[00563] Results:

[00564] A pilot Xenograft trial with the NSG mice was run using the following parameters: NSG mice: MDA-MB-231 cancer cells for inoculation (tumor generation); 10 animals each (2 donors, 5+5); Initiation of treatment -lOOmm ² (administration of antibody constructs); E:T ration 3:1; and Dose: 2ug/kg/day. Figure 13A provides a schematic showing the flow of events. Figure 13B presents the fold change in tumor size for the following samples administered: MATE with hPBMC 2ug/kg /day (green); PTMATE-C with hPBMC 2ug/kg/day (red); PTMATE-NC with hPBMC 2ug/kg/day (yellow); and Control (Cancer cells with hPBMC alone).

[00565] Administration of MATE and PTMATE-C showed similar results, with reduced fold change in tumor size compared with the PTMATE-NC and control samples. (Figure 13B) The results indicate that in the xenograft model, the tumors have secreted MMPs that have cleaved the CAP and the HAS regulatory arm, thus modifying the construct, which liberates the CD3e for T- cell binding and activation.

[00566] Thus, both the EGFR first binding site and CD3e second binding site of the PTMATE- C construct provide therapeutic functionality, wherein they are available for binding to the tumor (anti-EGFR binding) and to therapeutic T-cells (anti-CD3e).

[00567] Summary: The results demonstrate that the PTMATE-C precursor construct, once activated in-vivo at the tumor microenvironment by cancer tissue protease, provides an effective tool against tumor growth.

Example 9: PTMATE-MC Constructs Cleavage and Binding Characteristics

[00568] Objective: To analyze the cleavage products and binding characteristics of antibody variants comprising single and multi-cleavage site linkers.

[00569] Methods:

[00570] Antibody constructs were produced as described in Examples 1- 3. Cleavage product analysis and ELISA assays were performed essentially as described in Examples 2 and 3. Briefly, for cleavage analysis 2 ug per sample was used, wherein the mass ratio of antibody construct to protease was as follows: protein to MMP9= 100:1; and protein to Matriptase=25:l. The reaction was run at room temperature overnight. Samples were then run on an SDS-PAGE (4-20%) under non-reducing conditions.

[00571] For ELISA binding analysis the following variant constructs were analyzed: MATE EGFR (VL-VH), PTMATE-C EGFR(VL-VH), and PTMATE-MC EGFR (VL-VH).

[00572] Results. Figure 14 shows that cleavage of the PTMATE-MC EGFR with either MMP9 or Matiptase resulted in identical patterns of the cleaved PTMATE-MC EGFR construct products (MATE EGFR + Regulatory arm), wherein in the absence of protease a single“band” is observed. The distribution of the cleaved and uncleaved products agreed well with those observed using MMP9 protease to cleave the PTMATE-C EGFR construct.

[00573] The binding affinities of the different constructs to CD3e are shown in Figure 15A, and to EGFR in Figure 15B. Binding to human CD3e is influenced by the presence or absence of a CAP element within the regulatory domain of the PTMATE constructs (Figure 15A), wherein the MATE construct shows the highest binding affinity followed by the PTMATE-MC construct cleaved using either MMP9 or Matriptase and the PTMATE-C construct cleaved using MMP9. Products retaining their regulatory arm had greatly reduce binding affinity. Figure 15B demonstrates similar binding to human EGFR antigen by MATE and PTMATE constructs.

[00574] Conclusion: All PTMATE variants similarly bind human EGFR, regardless of the presence or absence of protease. Binding to human CD3e was influenced by the presence of a CAP element, wherein effective cleavage of the regulatory arm was observed using MMP9 or matriptase in the multi-cleavage linker. Thus, as shown above in Example 3, binding affinity to CD3e of PTMATE constructs may be regulated by incubation in the presence of proteases found in tumor environments.

Example 10: Expression and Purification of Variant 5T4 PTMATE Constructs

[00575] Objective: To express and purify 5T4 PTMATE-C, 5T4 PTMATE-NC, and 5T4 MATE bispecific antibody constructs. 5T4, also known as trophoblast glycoprotein, is an antagonist of the Wnt/p-catenin signaling pathway and is found on a number of carcinomas.

[00576] Methods: Methods for producing and purifying constructs, and methods for SDS and SEC analysis, are provided in Examples 1- 3, and 9. The 5T4 bispecific constructs are similar to each of PTMATE-C EGFR, PTMATE-NC EGFR, and MATE EGFR, respectively, wherein the EGFR scFv region was replaced by a 5T4 scFv region (i.e., the TAA in this Example is 5T4), for example in the case of the VL-VH scFv 5T4 constructs, the amino acid sequence of the scFv was the sequence set forth in SEQ ID NO: 137 (encoded by SEQ ID NO: 147). In the case of a VH-VL scFv 5T4 constructs, the amino acid sequence of the scFv was the sequence set forth in SEQ ID NO: 140 (encoded by SEQ ID NO: 148.

[00577] Protease cleavage was by MMP9 at a mass ratio protein to MMP9 of 100: 1. Buffers were changed to PBS via diluting and concentrating. The reaction was carried out at room temperature overnight.

[00578] Results. The expressed and purified 5T4 bispecific antibody constructs appear as single, intact molecules in SDS-PAGE run under non-reducing conditions, as indicated in Figures 16A, 16C, 16E, 17A and 17B, compared with the two polypeptide bands observed under reducing conditions (scFv-kC polypeptide and Fd polypeptide (for MATE) or Fd-fusion polypeptide (for PTMATE constructs), wherein the Fd-fusion includes the regulatory arm domain). A single major peak was observed following SEC-HPLC (Figures 16B, 16D, 16F, 17C, and 17D), wherein details of each scan are provided below the scans.

[00579] In order to show successful cleavage of the regulatory arm in the presence of a tumor microenvironment protease PTMATE -C and PTMATE-NC constructs were incubated in vitro in the presence of MMP9. Figure 18 clearly shows the control (VL-VH) and (VH-VL) MATE products compared with PTMATE-C and PTMATE-NC constructs incubated in the presence and absence of MMP9 protease. Inclusion of MMP9 with the PTMATE-C 5T4 constructs resulted in two bands - the released MATE portion and the regulatory arm, wherein incubation with MMP9 had no effect on the PTMATE-NC construct.

[00580] Conclusion: 5T4 PTMATE-C, 5T4 PTMATE-NC, and 5T4 MATE (VL-VH) and (VH- VL) bispecific antibody constructs were successfully expressed, purified, and could have their regulatory arm cleaved.

Example 11: Binding Affinities of 5T 4 Bispecific Constructs for h5T4 and hCD3s

[00581] Objective : To analyze the ELISA binding characteristics of 5T4 PTMATE antibody variants comprising single cleavage site linkers.

[00582] Methods: Antibody constructs were produced and isolated, and ELISA binding affinity tested as described at least in Examples 1- 3, and 9.

[00583] For ELISA binding analysis the following variant constructs were produced and analyzed: MATE 5T4 (VL-VH), MATE 5T4 (VH-VL), PTMATE-C 5T4 (VL-VH), PTMATE-C 5T4 (VH-VL), and PTMATE-NC 5T4 (VL-VH).

[00584] Results: Figure 19 shows that ELISA binding of the variant 5T4 constructs was not significantly influenced by the presence or absence of a regulatory arm. Further (VL-VH) and (VH- VL) constructs had very similar binding kinetics, as can be seen by the EC50 values presented. Figure 21 presents the ELISA binding affinity curves of the different 5T4 constructs to CD3e in the presence and absence of the protease MMP9. Binding to human CD3e is influenced by the presence or absence of a CAP element within the regulatory domain of the PTMATE 5T4 constructs, wherein the MATE constructs shows the highest binding affinities (pink and purple circles) followed by the PTMATE-C constmcts cleaved using MMP9 (blue and green triangles) Products retaining their regulatory arm (PTMATE-NC or PTMATE-C in the absence of protease) had greatly reduce binding affinities.

[00585] Conclusion: All PTMATE 5T4 variants similarly bind human 5T4, regardless of the presence or absence of protease or a regulatory arm. In contrast, binding to human CD3e was influenced by the presence of a CAP element, wherein effective cleavage of the regulatory arm was observed using MMP9. Thus, as shown above in Examples 3 and 9, binding affinity to CD3e of PTMATE constructs may be regulated by the presence of proteases found in tumor microenvironments .

Example 12: Binding Affinities of Bispecific 5T4 Antibody Constructs to Cancer Cells

[00586] Objective: To study the binding efficacy of cleaved and non-cleaved 5T4 variant precursor bispecific antibody constructs to cells. Specifically, to study the binding efficacy of MATE 5T4, PTMATE-C 5T4, and PTMATE-NC 5T4 variants to CHO-K1 cells and to Jurkat T- lymphocytes (CD3e) and 293F-human 5T4 cells (5T4).

[00587] Methods:

[00588] Antibody constructs were produced and purified as described in Examples 1- 3. FACS analysis methods were as described above in Example 4.

[00589] Constructs tested included MATE (VL-VH) 5T4, PTMATE-C (VL-VH) 5T4, PTMATE-C (VH-VL) 5T4, and PTMATE-NC (VL-VH) 5T4,

[00590] Tissue culture cell lines used included CHO-Kl-h5T4 cells, CHO-Kl-blank (empty cassette), and Jurkat T-cells. PTMATE variants were tested by FACS using the CHO-K1 and Jurkat cell lines assess binding affinity of variants to cell-surface expressed CD3 or 5T4.

[00591] Results:

[00592] The expressed precursor and/or cleaved bispecific constructs were analyzed for their ability to bind CHO-K1 cells expressing 5T4 or control CHO-K1 cells that do not express 5T4. Figure 20A shows that all of the constructs (MATE 5T4 (VL-VH), MATE 5T4 (VH-VL) PTMATE-C 5T4 (VL-VH), PTMATE-C 5T4 (VH-VL), and PTMATE-NC 5T4) bound to 5T4 expressing CHO-K1 cells with relatively similar affinities. There was no binding by any of the variant constructs to CHO-K1 cells not expressing 5T4 (Figure 20B). As expected, the bispecific antibody MATE variants (VL-VH and VH-VL) bound CD3e expressing Jurkat T-cells with the highest affinity. In the absence of protease (MMP9) the precursor bispecific antibody constructs (PTMATE-C, PTMATE-NC) showed reduced binding affinity to CD3e expressing Jurkat T-cells (Figure 22). As well, PTMATE-NC plus MMP9 also had a reduced binding affinity o CD3e expressing Jurkat T-cells (Figure 22). MMP9 cleaved PTMATE-C 5T4 (VL-VH) and (VH-VL) constructs showed increased binding affinity compared to the uncleaved constructs, indicating availability of the anti-CD3e binding site (Figure 22).

[00593] Conclusion: Only when cleaved would the precursor PTMATE-C 5T4 antibodies recognizes CD3e-expressing Jurkat lymphoma T cells. However, both cleaved and non-cleaved regulatory 5T4 antibodies bound 5T4 expressing CHO-K1 cells.

Example 13: Expression and Purification of Variant ROR1 PTMATE Constructs

[00594] Objective : To express and purify PTMATE-C ROR1, PTMATE-NC ROR1, and MATE ROR1 bispecific antibody constructs. ROR1 is receptor tyrosine kinase-like orphan receptor that modulates neurite growth in the central nervous system. The gene encoding ROR1 is highly expressed during early embryonic development but expressed at very low levels in adult tissues. Increased expression of this gene is associated with B-cell chronic lymphocytic leukemia.

[00595] Methods : Methods for expressing and purifying constructs, and methods for SDS and SEC analysis, are provided in Examples 1- 3, and 9. The ROR1 bispecific constructs are similar to each of PTMATE-C ROR1, PTMATE-NC ROR1, and MATE ROR1, respectively, wherein the EGFR scFv region was replaced by a ROR1 scFv region (i.e., the TAA in this Example is ROR1), for example in the case of the VL-VH scFv ROR1 constructs, the amino acid sequence of the scFv was the sequence set forth in SEQ ID NO: 141 (encoded by SEQ ID NO: 149). In the case of a VH- VL scFv ROR1 constructs, the amino acid sequence of the scFv was the sequence set forth in SEQ ID NO: 144 (encoded by SEQ ID NO: 150).

[00596] Protease cleavage was by MMP9 at a mass ratio of protein to MMP9 of 100: 1. Buffers were changed to PBS via diluting and concentrating. The reaction was carried out at room temperature overnight.

[00597] Results. The expressed and purified ROR1 bispecific antibody constructs appear as single, intact molecules in SDS-PAGE run under non-reducing conditions, as indicated in Figures 23A, 23C, and 23E, compared with the two polypeptide bands observed under reducing conditions (scFv-kC polypeptide and Fd polypeptide (for MATE) or Fd-fusion polypeptide (for PTMATE constructs), wherein the Fd-fusion includes the regulatory arm domain). A single major peak was observed following SEC-HPLC (Figures 23B, 23D, and 23F), wherein details of each scan are provided below the scans.

[00598] In order to show successful cleavage of the regulatory arm in the presence of a tumor microenvironment protease PTMATE -C and PTMATE-NC constructs were incubated in vitro in the presence of MMP9. Figure 26 clearly shows the control (VL-VH) MATE ROR1 products compared with PTMATE-C and PTMATE-NC constructs incubated in the presence and absence of MMP9 protease. Inclusion of MMP9 with the PTMATE-C ROR1 construct resulted in two bands - the released MATE portion and the regulatory arm, wherein incubation with MMP9 had no effect on the PTMATE-NC construct.

[00599] Conclusion: PTMATE-C ROR1, PTMATE-NC ROR1, and MATE ROR1 (VL-VH) bispecific antibody constructs were successfully expressed, purified, and could have their regulatory arm cleaved by an MMP9 protease.

Example 14: Binding Affinities ofRORl Bispecific Constructs for ROR1 Antigen

[00600] Objective : To analyze using ELISA binding assays, the ROR1 binding characteristics of ROR1 PTMATE antibody variants.

[00601] Methods : Antibody constructs were produced and isolated, and ELISA binding affinity assayed as described at least in Examples 1- 3 and 9.

[00602] For ELISA binding analysis the following variant constructs were produced and analyzed: MATE ROR1 (VL-VH), PTMATE-C ROR1 (VL-VH), PTMATE-MC ROR1 (VH-VL), and PTMATE-NC ROR1 (VL-VH).

[00603] Results. Figure 24 shows that binding of the variant ROR1 constructs to the human hRORl antigen was relatively the same for constructs comprising (PTMATE) or not comprising (MATE) a regulatory arm domain. The EC50 values provided below the graph support the observation that MATE (VL-VH) ROR1, PTMATE-C (VL-VH) ROR1, and PTMATE-NC (VL- VH) ROR1 bind human ROR1 antigen with similar affinities. Figure 25 presents ELISA binding affinity curves of the different ROR1 constructs to hRORl, including a construct comprising a multiple cleavage site linker. Again, the variant constructs bound hRORl with similar affinities.

[00604] The binding curves of Figure 28 show that binding to human CD3e is influenced by the presence or absence of a CAP element of the regulatory domain of the PTMATE ROR1 constructs, wherein the MATE constructs show the highest binding affinities (pink circles and orange squares, (MATE ROR1 +/- MMP9 respectively), followed by the PTMATE-C cleaved using MMP9 (green inverted triangles-see arrow). Products retaining their regulatory arm (PTMATE-NC and PTMATE-C in the absence of protease) had greatly reduce binding affinities (black tringles, blue diamonds, and purple circles).

[00605] Conclusion: All PTMATE ROR1 variants similarly bind human ROR1, regardless of the presence or absence of protease or a regulatory arm domain. In contrast, binding to human CD3e was influenced by the presence of a CAP element, wherein effective cleavage of the regulatory arm was observed using MMP9. Thus, as shown above in Examples 3 and 9, binding affinity to CD3e of PTMATE constructs may be regulated by the presence of a protease found in tumor microenvironments .

Example 15: Binding Affinities of Bispecific ROR1 Antibody Constructs to Jurkat Cancer

Cells

[00606] Objective: To study the binding efficacy of cleaved and non-cleaved ROR1 variant precursor bispecific antibody constructs to Jurkat T lymphoma cells. Specifically, to study the binding efficacy of MATE ROR1, PTMATE-C ROR1, and PTMATE-NC ROR1 variants to Jurkat T-lymphocytes (CD3e) in the presence and absence of MMP9 protease.

[00607] Methods:

[00608] Antibody constructs were produced as described in Examples 1- 3. FACS analysis methods were as described above in Example 4.

[00609] Constructs tested included MATE ROR1, PTMATE-C ROR1, PTMATE-C ROR1, and PTMATE-NC ROR1,

[00610] Tissue culture cell lines used included Jurkat T lymphoma cells. Binding of PTMATE variants were tested by FACS using the Jurkat cell lines to assess binding affinity of variants to cell- surface expressed CD3e.

[00611] Results: Figure 27 shows that the bispecific antibody MATE variant bound CD3e expressing Jurkat T-cells with the highest affinity, independent of the presence or absence of protease (pink circles and orange squares, respectively). In the absence of protease (MMP9) the precursor bispecific antibody constructs (PTMATE-C, PTMATE-NC) showed reduced binding affinity to CD3e expressing Jurkat T-cells (olive triangles and blue diamonds, respectively, see arrow, note curves superimposed at the bottom of the graph). As well, PTMATE-NC plus MMP9 also had a reduced binding affinity to CD3e expressing Jurkat T-cells (purple circles, see arrow). MMP9 cleaved PTMATE-C ROR1 construct showed increased binding affinity compared to the uncleaved constructs, indicating availability of the anti-CD3e binding site (green inverted triangles, see arrow).

Conclusion: Only when cleaved did the precursor PTMATE-C ROR1 antibodies recognizes CD3e- expressing Jurkat lymphoma T cells.

[00612] While certain features of precursor bi-specific antibodies have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the embodiments of a precursor bispecific antibody construct, as disclosed herein.