DOSING REGIMEN FOR CD3 BINDING PROTEINS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/571,755 filed October 12, 2017, and 62/571,767 filed October 12, 2017, both of which are incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] CD3 denotes an antigen that is expressed on human T cells as part of the multimolecular T cell receptor complex, consisting of five chains: 2 CD3-epsilon, a CD3-gamma, a CD3-delta, and a CD3 zeta. Clustering of CD3 on T cells e.g. by anti-CD3 antibodies leads to T cell activation similar to the binding of an antigen but independent from the clonal specificity of the T cell subset.

[0003] CD33 is a transmembrane cell surface glycoprotein receptor that is specific for myeloid cells. The CD33 antigen is expressed on approximately 90% of AML myeloblasts, including leukemic stem cells and on cells of other myeloproliferative disorders. Studies with gemtuzumab ozogamicin have validated CD33 as a target for antibody-based therapeutics; however, many patients fail to derive benefit from antibody-based drug conjugates, and thus other approaches that utilize CD33 as a target but employ different killing mechanisms such as T-cell redirection could be more efficacious.

SUMMARY OF THE INVENTION

[0004] Provided herein, in one aspect is a method for the treatment of cancer in a subject comprising administering to a subject in need thereof, a protein that binds to human CD3 and a target antigen at a dose and frequency of administration sufficient to achieve a maximum concentration (C _max ) in blood in 1 to 28 days. In another aspect, provided herein is a method for the treatment of cancer in a subject comprising administering to a subject in need thereof, a protein that binds to human CD33 and CD3 at a dose and frequency of administration sufficient to achieve a maximum concentration (C _ma x) in blood in 1 to 28 days. In some embodiments, the dose and frequency of administration is sufficient to achieve a steady state concentration (C _ss ) in 1 to 28 days.

[0005] In some embodiments, the protein is administered as a continuous dose, an intermittent dose, a single dose, multiple doses, or a combination thereof. In other embodiments, the protein is administered as a continuous dose of about 0.5 μg to about 3000 μg per day. In yet other embodiments, the administration is over a period of time of at least 1 hour, 1 day, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, or at least 12 weeks.

[0006] In some embodiments, the administration provides a C _max of about 20 pg/mL to about 20000 pg/mL. In other embodiments, the administration provides a C _ss of about 20 pg/mL to about 20000 pg/mL. In other embodiments, the administration provides an AUC of about 200 day*pg/mL to about 600000 day*pg/mL.

[0007] In some embodiments, the administration is intravenous, intramuscular, intralesional, topical or subcutaneous. In some embodiments, the administration is by bolus or continuous infusion.

[0008] In some embodiments, the administration provides for gradual T-cell or monocyte activation over 1 to 28 days. In other embodiments, the administration provides for gradual cytokine release over 1 to 28 days. In certain instances, the cytokine is T Fa, IL-2, IL-4, IL-6, IL-8, IL-10, TGF-β, or ΠΤΝΓγ.

[0009] In further embodiments, the administration reduces C-reactive protein levels. In certain instances, the administration increases the levels of monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes, or platelets. In other instances, the administration increases neutrophil levels. In other instances, the administration increases erythrocyte levels.

[0010] In further embodiments, the administration reduces myeloblasts. In further embodiments, the administration reduces myeloid-derived suppressor cells (MDSCs).

[0011] In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the target antigen is EGFR, HER2, HER3, EGFRviii, PSMA, BCMA, CD19, MSLN, CD123, CD33, EpCAM, c-MET, CEA, cd79b, CD66, CD30, EphA2, CSPG4, DLL3, or VEGF.

[0012] In some embodiments, the cancer has cells that express CD33. In other embodiments, the tumor microenvironment has cells that express CD33. In some instances, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), precursor B-cell lymphoblastic leukemia, myeloid sarcoma, multiple myeloma, acute lymphoma, acute lymphoblastic lymphoma, myelodysplastic syndrome (MDS),

myeloproliferative neoplasms, chronic myelomonocytic leukemia (CMML), pancreatic cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, liver cancer, thyroid cancer, colorectal cancer, prostate cancer, ovarian cancer, cervical cancer, head and neck cancer, stomach cancer, bladder cancer, intestinal cancer, testicular cancer, uterine cancer, oral cancer, nasopharynx cancer, skin cancer, renal cancer, glioblastoma, astrocytoma, epithelial carcinoma, bone cancer, mesothelioma and melanoma. In some instances, the cancer is a solid tumor cancer. In some instances, the cancer is AML or MDS. In further instances the AML is relapsed or refractory. In further instances the MDS is relapsed or refractory.

[0013] Provided herein, in another aspect is a method for the treatment of myelodysplastic syndrome in a subject comprising to a subject in need thereof, a protein that binds to human CD3 and a target antigen such as CD33 at a dose and frequency of administration sufficient to achieve a maximum concentration (C _max ) in blood in 1 to 28 days.

[0014] Provided herein, in another aspect is a method for the treatment of a myeloproliferative disease in a subject comprising to a subject in need thereof, a protein that binds to human CD3 and a target antigen such as CD33 at a dose and frequency of administration sufficient to achieve a maximum concentration (C _max ) in blood in 1 to 28 days.

[0015] Provided herein, in another aspect is a method for the treatment of immune suppression or inflammation by myeloid derived suppressor cells (MDSCs) in a subject comprising to a subject in need thereof, a protein that binds to human CD3 and a target antigen such as CD33 at a dose and frequency of administration sufficient to achieve a maximum concentration (C _max ) in blood in 1 to 28 days.

[0016] Provided herein, in another aspect is a method for reducing MDSCs in a subject comprising administering to a subject in need thereof, a protein that binds to human CD3 and a target antigen such as CD33 at a dose and frequency of administration sufficient to achieve a maximum blood concentration (C _max ) in 1 to 28 days.

[0017] Provided herein, in another aspect is a method for the treatment of cancer in a subject comprising administering to a subject in need thereof, a protein that binds to human CD3 and a target antigen such as CD33 at a dose and frequency of administration sufficient to reduce MDSCs in the subject.

[0018] In any of the above aspects, the protein is an antibody or antibody derivative. In any of the above aspects, the protein comprises Fab, Fab', or F(ab') ₂ fragments. In any of the above aspects, the protein comprises a single-chain Fv, tandem single-chain Fv, bi-specific T-cell engager, dual affinity retargeting antibody, diabody, single domain antibody, a bispecific antibody, a bivalent, bispecific (2x2) T-cell engager or a tandem diabody.

[0019] In any of the above aspects, the protein is a bivalent, bispecific (2x2) T-cell engager. In some embodiments, the protein is a bivalent, bispecific (2x2) T-cell engager that comprises a first polypeptide and a second polypeptide, each polypeptide having at least four variable chain domains linked one after another, wherein each polypeptide comprise

(i) a variable heavy chain (VH) domain specific to the target antigen;

(ii) a variable light chain (VL) domain specific to the target antigen;

(iii) a VH domain specific for human CD3, and (iv) a VL domain specific for human CD3.

[0020] In some instances, in each polypeptide, the four variable chain domains are linked with one after another by peptide linkers LI, L2 and L3 in the order of:

VL(CD3)-Ll-VH(target antigen)-L2-VL(target antigen)-L3-VH(CD3);

VH(CD3)-Ll-VL(target antigen)-L2-VH(target antigen)-L3-VL(CD3);

VL(target antigen)-Ll-VH(CD3)-L2-VL(CD3)-L3-VH(target antigen); or

VH(target antigen)-Ll-VL(CD3)-L2-VH(CD3)-L3-VL(target antigen).

[0021] In some embodiments, the bivalent, bispecific (2x2) T-cell engager comprises a first polypeptide and a second polypeptide, each polypeptide having at least four variable chain domains linked one after another, wherein each polypeptide comprise

(v) a variable heavy chain (VH) domain specific to human CD33;

(vi) a variable light chain (VL) domain specific to human CD33;

(vii) a VH domain specific for human CD3, and

(viii) a VL domain specific for human CD3.

[0022] In some instances, in each polypeptide, the four variable chain domains are linked with one after another by peptide linkers LI, L2 and L3 in the order of:

VL(CD3 )-L 1 - VH(CD33 )-L2- VL(CD33 )-L3 - VH(CD3 );

VH(CD3 )-L 1 - VL(CD33 )-L2- VH(CD33 )-L3 - VL(CD3 );

VL(CD33 )-L 1 - VH(CD3 )-L2- VL(CD3 )-L3 - VH(CD33 ); or

VH(CD33 )-L 1 - VL(CD3 )-L2- VH(CD3 )-L3 - VL(CD33 ) .

[0023] In some instances, the VL domain specific to human CD33 comprises a CDRl consisting of the sequence selected from the group consisting of SEQ ID NOs:21-27, a CDR2 consisting of the sequence selected from the group consisting of SEQ ID NOs:28-34 and a CDR3 consisting of the sequence of the group consisting of SEQ ID NOs:35-41.

[0024] In some instances, the VH domain specific to human CD33 comprises a CDRl consisting of the sequence selected from the group consisting of SEQ ID NOs:42-48, a CDR2 consisting of the sequence selected from the group consisting of SEQ ID NOs:49-55 and a CDR3 consisting of a sequences selected from the group consisting of SEQ ID NOs:56-63.

[0025] In some instances, the CDRl, CDR2 and CDR3 of the VL domain specific to human CD33 are sequences selected from the group consisting of:

(i) SEQ ID NOs:21, 28 and 35;

(ii) SEQ ID NOs:22, 29 and 36;

(iii) SEQ ID NOs:23, 30 and 37;

(iv) SEQ ID NOs:24, 31 and 38;

(v) SEQ ID NOs:25, 32 and 39; (vi) SEQ ID NOs:26, 33 and 40; and

(vii) SEQ ID NOs:27, 34 and 41.

[0026] In some instances, the CDRl, CDR2 and CDR3 of the VH domain specific to CD33 are sequences selected from the group consisting of:

(i) SEQ ID NOs :42, 49 and 56;

(ϋ) SEQ ID NOs A3, 50 and 57;

(iii) SEQ ID NOs: A3, 50 and 58;

(iv) SEQ ID NOs A3, 50 and 59;

(v) SEQ ID NOs A3, 50 and 60;

(vi) SEQ ID NOs :44, 51 and 61;

(νϋ) SEQ ID NOs :45, 52 and 62;

(viii) SEQ ID NOs :46, 53 and 63;

(ix) SEQ ID NOs :47, 54 and 63;. and

(x) SEQ ID NOs :48, 55 and 63.

[0027] In some instances, the VL and VH domains specific to CD33 are sequences selected from the group consisting of:

(i) SEQ ID NO 1 and SEQ ID NO: 1 1 ;

(ϋ) SEQ ID NO 2 and SEQ ID NO: 12;

(iii) SEQ ID NO 3 and SEQ ID NO: 13;

(iv) SEQ ID NO A and SEQ ID NO: 14;

(v) SEQ ID NO 5 and SEQ ID NO: 15;

(vi) SEQ ID NO 6 and SEQ ID NO: 16;

(νϋ) SEQ ID NO :7 and SEQ ID NO: 17;

(viii) SEQ ID NO :8 and SEQ ID NO: 18;

(ix) SEQ ID NO :9 and SEQ ID NO: 19; and

(x) SEQ ID NO : 10 and SEQ ID NO:20.

[0028] In some instances, the VH domain specific for human CD3 comprises a CDRl sequence of STYAMN (SEQ ID NO:72), a CDR2 sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO:73) and a CDR3 sequence of HGNFGNS YVSWFAY (SEQ ID NO:74) or

HGNFGNSYVSYFAY (SEQ ID NO:75).

[0029] In some instances, the VL domain specific for human CD3 comprises a CDRl sequence of RSSTGAVTTSNYAN (SEQ ID NO:90), a CDR2 sequence of GTNKRAP (SEQ ID NO:91), and a CDR3 sequence of ALWYSNL (SEQ ID NO:92).

[0030] In some instances, the VL and VH domains specific to CD3 are sequences selected from the group consisting of: (i) SEQ ID NO:64 and SEQ ID NO:68;

(ii) SEQ ID NO:65 and SEQ ID NO:69;

(iii) SEQ ID NO:66 and SEQ ID NO:70; and

(iv) SEQ ID NO:67 and SEQ ID N0:71.

[0031] In some instances, each polypeptide comprises four variable chain domains selected from the group consisting of:

(i) SEQ ID NOs:2, 12, 65 and 69;

(ϋ) SEQ ID NOs:3, 13, 65 and 69;

(iii) SEQ ID NOs:4, 14, 65 and 69;

(iv) SEQ ID NOs:5, 15, 65 and 69;

(v) SEQ ID NOs: l, 11, 64 and 68;

(vi) SEQ ID NOs:2, 12, 64 and 68;

(νϋ) SEQ ID NOs:2, 12, 66 and 70;

(viii) SEQ ID NOs:4, 14, 66 and 70;

(ix) SEQ ID NOs:5, 15, 66 and 70;

(x) SEQ ID NOs:3, 13, 64 and 68;

(xi) SEQ ID NOs:3, 13, 67 and 71;

(xii) SEQ ID NOs:4, 14, 64 and 68;

(xiii) SEQ ID NOs:5, 15, 64 and 68;

(xiv) SEQ ID NOs:7, 17, 64 and 68;

(XV) SEQ ID NOs:6, 16, 64 and 68;

(xvi) SEQ ID NOs:6, 16, 67 and 71;

(xvii) SEQ ID NOs:8, 18, 64 and 68;

(xviii) SEQ ID NOs:9, 19, 64 and 68;

(xix) SEQ ID NOs:9, 19, 67 and 71; and

(XX) SEQ ID NOs: 10, 20, 64 and 68.

[0032] In some instances, the bivalent, bispecific (2x2) T-cell engager comprises a sequence selected from the group consisting of SEQ ID NOs:98-121. In some instances, the bivalent, bispecific (2x2) T-cell engager comprises a sequence selected from the group consisting of SEQ ID Nos 123-146.

INCORPORATION BY REFERENCE

[0033] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0035] Figure 1 Schematic representation of the gene organization and a domain order of a CD3/CD33 bivalent, bispecific (2x2) T-cell engager. Exemplary 2x2 T-cell engagers are expressed as a single polypeptide comprised of four variable domains connected via short peptide linkers LI, L2 and L3. Following expression, two monomeric polypeptides associate non-covalently head-to-tail to form the functional homodimeric molecule. LI, L2, L3 : Linker; V _H : Heavy chain variable domain; V _L : Light chain variable domain.

[0036] Figure 2 CD3 engaging 2x2 T-cell engager and its mode of action. 2x2 T-cell engagers are bispecific proteins with two binding sites for each antigen that leads to activation and proliferation of T-cells. A CD33/CD3 2x2 T-cell engager binds to a CD33 ⁺ tumor cell with two of four binding domains and to CD3 with the other two binding domains. This T-cell/target cell binding forms an immunological synapse that promotes activation of the T-cell and promotes the subsequent destruction of the tumor cell via apoptosis.

[0037] Figure 3 Domain order variants of CD33/CD3 2x2 T-cell engagers. Variations of domain order of variable heavy (VH) and variable light (VL) chains within gene sequences encoding 2x2 T-cell engagers allows production of antibodies with CD33 and CD3 specificities located on the inside or outside of the molecule. Domain specificities, location of signal sequences (ss) and linkers (LI, L2, L3) and affinity tags (His) as well as 5 ^' - and 3 ^' -ends are indicated.

[0038] Figure 4 Comparison of positively enriched vs. negatively selected healthy donor T-cells. KG-la cells were incubated with 10 pM (approx. 1 ng/mL) and 25 pM ( approx. 2.5 ng/mL) of one of 10 selected 2x2 T-cell engagers and either negatively selected healthy donor T-cells or positively selected healthy donor T-cells at an E:T cell ratio of 1 : 1 or 3 : 1, as indicated. After 48 hours, cell counts were determined and cytotoxicity was assessed with DAPI staining. Results are shown as mean±SEM for the percentage of dead cells (upper panels) and the percentage of specific cytotoxicity (lower panels) from 3 independent experiments performed in duplicate wells.

[0039] Figure 5 Analysis strategy. Scatter and histogram plots from one healthy donor T- cell aliquot and 1 representative AML cell line (HL-60) and primary AML specimen (AMP002) each illustrating the strategy pursued to determine 2x2 T-cell engager-induced cytotoxicity. FSC, forward scatter; SSC, side scatter.

[0040] Figure 6 Screening cytotoxicity assays in CD33+ AML cell lines. Parental HL-60 (A,B) and KG-la (C,D) cells were incubated with 10 pM (approx. 1 ng/mL) and 25 pM (approx. 2.5 ng/mL) of one of 22 CD33/CD3 2x2 T-cell engager molecules or a non-binding control 2x2 T-cell engager (00) and healthy donor T-cells at an E:T cell ratio of either 1 : 1 (A,C) or 5: 1 (B,D) as indicated. After 48 hours, cell counts were determined and cytotoxicity was assessed with DAPI staining to quantify drug-specific cytotoxicity. Results are shown as mean±SEM for the percentage of DAPI ⁺ cells from 3 independent experiments performed in duplicate wells.

Qualitatively similar results were obtained when cytotoxicity was expressed as the percentage of specific cytotoxicity.

[0041] Figure 7 Selection of primary AML specimens for study. Frozen aliquots from a total of primary human AML specimens were obtained for analysis. The percentage of AML blasts upon thaw was determined by flow cytometry based on CD45/side-scatter properties. Viability of the specimens was determined upon thaw as well after 48 hours in cytokine- containing liquid culture (without addition of 2x2 T-cell engager molecules or healthy donor T- cells) via flow cytometry using DAPI as live/dead cell marker. Results for viability after thawing as well as after 48 hours are depicted for all specimens, which had >58% AML blasts. Square: Primary AML specimens that showed a viability of >50% at thaw as well as >50% after 48 hours in cytokine-containing liquid culture which were included in the final analyses.

[0042] Figure 8 2x2 T-cell engager-induced cytotoxicity in primary AML specimens. Primary AML specimens were incubated with 2.5 pM (approx. 250 pg/mL), 10 pM (approx. 1 ng/mL), and 25 pM (approx.2.5 ng/mL) of one of 9 2x2 T-cell engager molecules without healthy donor T-cells added (A) or with healthy donor T-cells at an E:T cell ratio of either 1 :3 (B) or 1 : 1 (C) as indicated. After 48 hours, cell counts were determined and cytotoxicity was assessed with DAPI staining to quantify drug-specific cytotoxicity. Results are shown as mean±SEM for the percentage of specific cytotoxicity from experiments performed in duplicate wells.

[0043] Figure 9 Amino acid sequence of extracellular domain of human CD33 (aa 18 -

259) (SEQ ID NO: 93);

[0044] Figure 10 Amino acid sequences

(A) complete sequence of 2x2 T-cell engager 1 with C-terminal hexa-histidine (6xHis)- tag

(SEQ ID NO: 98); (B) complete sequence of 2x2 T-cell engager 2 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 99);

(C) complete sequence of 2x2 T-cell engager 3 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 100);

(D) complete sequence of 2x2 T-cell engager 4 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 101);

(E) complete sequence of 2x2 T-cell engager 5 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 102);

(F) complete sequence of 2x2 T-cell engager 6 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 103);

(G) complete sequence of 2x2 T-cell engager 7 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 104);

(H) complete sequence of 2x2 T-cell engager 8 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 105);

(I) complete sequence of 2x2 T-cell engager 9 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 106);

(J) complete sequence of 2x2 T-cell engager 10 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 107);

(K) complete sequence of 2x2 T-cell engager 11 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 108);

(L) complete sequence of 2x2 T-cell engager 12 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 109);

(M) complete sequence of 2x2 T-cell engager 13 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 10);

(N) complete sequence of 2x2 T-cell engager 14 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 1 1);

(O) complete sequence of 2x2 T-cell engager 15 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 12);

(P) complete sequence of 2x2 T-cell engager 16 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 13);

(Q) complete sequence of 2x2 T-cell engager 17 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 14);

(R) complete sequence of 2x2 T-cell engager 18 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 15); (S) complete sequence of 2x2 T-cell engager 19 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 16);

(T) complete sequence of 2x2 T-cell engager 20 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 17);

(U) complete sequence of 2x2 T-cell engager 21 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 18);

(V) complete sequence of 2x2 T-cell engager 22 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 1 19);

(W) complete sequence of 2x2 T-cell engager 23 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 120); and

(X) complete sequence of 2x2 T-cell engager 24 with C-terminal hexa-histidine (6xHis)- tag (SEQ ID NO: 121). Underlined sequences represent linkers LI , L2 and L3.

[0045] Figure 11 Amino acid sequences

(A) complete sequence of 2x2 T-cell engager 1 (SEQ ID NO: 123);

(B) complete sequence of 2x2 T-cell engager 2 (SEQ ID NO: 124);

(C) complete sequence of 2x2 T-cell engager 3 (SEQ ID NO: 125);

(D) complete sequence of 2x2 T-cell engager 4 (SEQ ID NO: 126);

(E) complete sequence of 2x2 T-cell engager 5 (SEQ ID NO: 127);

(F) complete sequence of 2x2 T-cell engager 6 (SEQ ID NO: 128);

(G) complete sequence of 2x2 T-cell engager 7 (SEQ ID NO: 129);

(H) complete sequence of 2x2 T-cell engager 8 (SEQ ID NO: 130);

(I) complete sequence of 2x2 T-cell engager 9 (SEQ ID NO: 131);

(J) complete sequence of 2x2 T-cell engager 10 (SEQ ID NO: 132);

(K) complete sequence of 2x2 T-cell engager 1 1 (SEQ ID NO: 133);

(L) complete sequence of 2x2 T-cell engager 12 (SEQ ID NO: 134);

(M) complete sequence of 2x2 T-cell engager 13 (SEQ ID NO: 135);

(N) complete sequence of 2x2 T-cell engager 14 (SEQ ID NO: 136);

(O) complete sequence of 2x2 T-cell engager 15 (SEQ ID NO: 137);

(P) complete sequence of 2x2 T-cell engager 16 (SEQ ID NO: 138);

(Q) complete sequence of 2x2 T-cell engager 17 (SEQ ID NO: 139);

(R) complete sequence of 2x2 T-cell engager 18 (SEQ ID NO: 140);

(S) complete sequence of 2x2 T-cell engager 19 (SEQ ID NO: 141);

(T) complete sequence of 2x2 T-cell engager 20 (SEQ ID NO: 142);

(U) complete sequence of 2x2 T-cell engager 21 (SEQ ID NO: 143);

(V) complete sequence of 2x2 T-cell engager 22 (SEQ ID NO: 144); (W) complete sequence of 2 2 T-cell engager 23 (SEQ ID NO: 145); and (X) complete sequence of 2x2 T-cell engager 24 (SEQ ID NO: 146). Underlined sequences represent linkers LI, L2 and L3.

[0046] Figure 12 Effect of 2x2 T-cell engagers 16 and 12 on the growth of HL-60 cells in NOD/scid mice. Eight experimental groups of immunodeficient NOD/scid mice were xenotransplanted by subcutaneous injection with a suspension of 4xl0 ⁶ HL-60 cells on day 0. Prior to injection HL-60 cells were mixed with 3xl0 ⁶ purified T-cells from healthy donors. All animals of the experimental groups transplanted with tumor cells and T-cells received an intravenous bolus on days 0, 1, 2, 3 and 4 of either vehicle (control) or 2x2 T-cell engagers 16 or 12 at three different dose levels as indicated (0.1 μg, 1 μg, and 10 μg). One group without effector cells and vehicle treatment served as an additional negative control.

[0047] Figure 13 Anti-tumor activity of 2x2 T-cell engager 16 in an AML Xenograft Model. NOD/scid mice were sublethally irradiated (2 Gy) and subcutaneously inoculated with 4xl0 ⁶ HL-60 cells. On day 9 the animals received a single bolus injection of anti-asialo GM1 rabbit Ab. When tumors reached a volume between 50-150 mm ³ (mean 73 ± 11 mm ³ ) on day 10 animals were allocated to 3 treatment groups. Groups 2 and 3 (n = 8) were intraperitoneally injected with 1.5xl0 ⁷ expanded and activated human T-cells. From day 13 to day 21 (qdxd9) animals received either 2x2 T-cell engager 16 (Group 3) or vehicle into the lateral tail vein (Group 1 and Group 2).

[0048] Figure 14 Relative amount (A) and absolute counts (B) of human AML blasts in the bone marrow (BM) and spleen of NSG mice at day 38 after treatment with 5 μg (0.25 mg/kg) or 50 μg (2.5 mg/kg) CD33/CD3 2x2 T-cell engager 12 and 16.

[0049] Figure 15 Kinetics of CD33/CD3 2x2 T-cell engager 16-mediated target cell lysis. lxlO ⁴ calcein-labeled HL-60 target cells were incubated with primary human T-cells as effector cells at an E:T ratio of 25 : 1 in the presence of serial dilutions of 2x2 T-cell engager 16 or without antibody (w/o) for 30 min, 1 h, 2 h, 3 h, 4 h, or 5 h. At each time point, the fluorescent calcein released from lysed target cells was used to calculated specific lysis. Mean and SD of three replicates are plotted.

[0050] Figure 16 Kinetics of EC ₅₀ and specific lysis values for CD33/CD3 2x2 T-cell engager 16. EC ₅₀ values (black solid circles) and 2x2 T-cell engager 16-mediated target cell lysis (open squares) were determined in calcein-release cytotoxicity assays at the indicated incubation times by non-linear regression/sigmoidal dose-response and plotted.

[0051] Figure 17 Cytotoxic activity in newly diagnosed, relapsed and refractory AML patient samples. [0052] Figure 18 Serum concentration of CD33/CD3 2 2 T-cell engager 16 (AMV564) in subjects 02-001, -002, and -003 at a dose of 0^g/day for 14 days.

[0053] Figure 19 Serum concentration of CD33/CD3 2 2 T-cell engager 16 for 14 days at denoted dose levels to patients with to patients with relapsed/refractory acute myeloid leukemia.

[0054] Figure 20 Exemplary levels of myeloblasts (upper left panel), absolute neutrophil counts (upper right panel), hemoglobin (lower left panel) and C-reactive protein (CRP) (lower right panel) in a subject dosed with CD33/CD3 2x2 T-cell engager 16 a dose of 0.5 μg/day for 14 days according to Example 14.

[0055] Figure 21 Exemplary levels of blood counts, i.e., red blood cells (upper left panel) and white blood cells (upper right panel), interleukin-6 (lower left panel) and CRP (lower right panel) in a subject dosed with CD33/CD3 2x2 T-cell engager 16 a dose of 1.5 μg/day for 14 days according to Example 14.

[0056] Figure 22 Improved hemoglobin, neutrophils, platelet, and monocyte counts improvement following administration of AMV564 at a 1.5 meg dose level for 14 days in a subject (upper panels). Improved hemoglobin, neutrophils, and platelet counts as well as decreased CRP levels following administration of AMV564 at a 1.5 meg dose level for 14 days in a subject (lower panels).

[0057] Figure 23 Best relative change in percent bone marrow leukemic blasts from baseline following administration of AMV564 for 14 days to patients with relapsed/refractory acute myeloid leukemia.

[0058] Figure 24 Exemplary dosing regimens for AMV564. Intermittent dosing every other day with titration from 5 μg — » 15 μg— » 100 μg (upper panel). Intermittent dosing with 15 μg continuous infusion x 3 days followed by every other day with titration from 100 μg— » 200 μ _§ .

DETAILED DESCRIPTION OF THE INVENTION

[0059] Described herein are pharmaceutical means and methods for immunological, medical interventions based on administering therapeutic proteins, in particular bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3. The bispecific antibodies described herein are useful for the prevention, treatment or

amelioration of a cancer or a non-solid or solid tumor, wherein said bispecific antibodies are administered to certain dosing schedules and/or treatment regimens.

[0060] Antibody-based therapeutics are widely used in the treatment of human diseases. A common phenomenon observed with certain antibody therapy is the occurrence of side effects, such as cytokine release syndrome ("CRS"). CRS is an immediate complication occurring in response to infusions of antibodies binding to T cells. CRS has been associated with T cell/monocyte activation and, secondarily, with activation of the complement cascade. These processes are mediated through the Fc part of antibodies which are capable of cross-linking T cells and mononuclear cells and activating complement. For example, the pathogenesis of CRS has been attributed to the synthesis of tumor necrosis factor (T F) alpha, IL-2 and IL-6 and gamma-interferon in response to stimulation of T lymphocytes by OKT3, an anti-CD3 antibody. In CRS, the cytokines released by the activated T cells (such as TNF alpha, interleukins (IL-2, IL-6) and interferons (IFN gamma) produce a type of systemic inflammatory response similar to that found in severe infection, and characterized by hypotension, pyrexia and rigors. Other adverse side effects described to be associated with CRS are fatigue, vomiting, tachycardia, hypertension, headache and back pain.

[0061] CRS has been observed when administering various monoclonal antibodies. For example, the anti-tumor activity of the anti-CD20 antibody rituximab is harnessed for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Dose escalation, i.e. increasing doses, achieved by thrice-weekly dosing is necessary for Rituximab to effect significant clinical activity as a single agent. However, this administration scheme triggers release of the cytokines TNF-alpha and IL-6, the serum levels of which peak 90 minutes after starting a respective infusion. This rise in cytokines is accompanied by fever, chills, hypotension and nausea.

Infusion toxicity could be reduced with appropriate pre-treatment and a stepped-up

administration scheme (Lin, 2003, Seminar Oncol. 30, 483-492).

[0062] CRS has also been observed when applying other antibody formats, such as bispecific antibodies. Single infusions of bispecific antibody MDX-2H12 (anti-Fc gamma receptor Ixanti- Her-2/neu) in combination with GCSF (granulocyte colony-stimulating factor) in breast carcinoma patients led to maximal levels of TNF-alpha and IL-6 at 2 and 4 hours, respectively. Peak levels of TNF-alpha and IL-6 did not correlate to the dose of bispecific antibody applied (Repp, 2003, Br. J. Cancer, 89, 2234-43). In WO 99/54440, CRS has been observed in an internal clinical study performed with anti-CD19xanti-CD3 bispecific single chain antibody applied in repeated infusions to a patient with B-cell derived chronic lymphatic leukemia (B- CLL). As shown in FIGS. 19 and 20 of WO 99/54440, release of TNF, IL-6 and IL-8 has been found in response to each of the two administered 20 minute-infusions (3 μg and 10 μg of the bispecific single chain antibody, respectively), with cytokine release after each administration. Maximal cytokine release was observed after administration of 10 μg of bispecific single chain antibody.

[0063] Accordingly, it is therefore an aim of the present embodiments described herein to provide provide pharmaceutical means and methods for antibody-based medical therapies with increased patient tolerability. Specifically, it is an aim that such means and methods allow for maximal retention of bioactivity of the antibody administered, while undesired and adverse side effects due to this administration are minimized.

Bispecific Antibodies to CD33 and CD3

[0064] According to a first aspect, described herein are bispecific antibodies having specificity to an antigen expressed on a target cell and an antigen expressed on a T-cell. In some embodiments, the bispecific antibodies have specificity for at least CD33, preferably human CD33. In some embodiments, the CD33 binding domains of the bispecific antibodies described herein have specificity for human and cynomolgus CD33, i.e. are cross-reactive. In some embodiments, these cross-reactive binding domains bind to human and cynomolgous CD33 with similar affinity.

[0065] CD33 is a transmembrane cell surface glycoprotein receptor that is specific for myeloid cells. The CD33 antigen is expressed on approximately 90% of acute myeloid leukemia (AML) myeloblasts and cells of other myeloproliferative disorders, including leukemic stem cells and myeloid derived suppressor cells (MDSCs). CD33 is expressed on monocytes, dendritic cells, neutrophils, resident macrophages, basophils and eosinophils. Two alternatively spliced isoforms have been identified that may have implications for downstream signaling.

[0066] For the isolation of antibody domains specific for CD33, such as human CD33, antibody libraries may be screened. For example IgM phage display libraries can be screened by employing, for example, a recombinant CD33-Fc fusion protein containing amino acids 1-243 of the extracellular domain of human CD33 (Figure 9, SEQ ID NO:93).

[0067] In some embodiments the CD33 binding domain has at least one CD33 binding site comprising a light chain variable domain and a heavy chain variable domain. The light chain variable domain comprises the light chain CDRl, CDR2 and CDR3 and the heavy chain variable domain comprises the heavy chain CDRl, CDR2 and CDR3. In some embodiments these light chain CDRs (CDRl, CDR2 and CDR3) are selected from the human CDR sequences shown in Table 1 (SEQ ID NOs:21-41). In certain instances, the light chain CDRl is selected from SEQ ID NOs:21-27. In certain instances, the light chain CDR2 is selected from SEQ ID NOs:28-34. In certain instances, the light chain CDR3 is selected from SEQ ID NOs:35-41.

[0068] In some embodiments these heavy chain CDRs (heavy chain CDRl, CDR2 and CDR3) are selected from the human CDR sequences shown in Table 2 (SEQ ID NOs:42-63). In certain instances, the heavy chain CDRl is selected from SEQ ID NOs:42-48. In certain instances, the heavy chain CDR2 is selected from SEQ ID NOs:49-55. In certain instances, the heavy chain CDR3 is selected from SEQ ID NOs: 56-63. [0069] In some embodiments, the light and heavy CDRs are selected without the surrounding framework sequences of the respective variable domains, which include framework sequences from other immunoglobulins or consensus framework regions, optionally are further mutated and/or replaced by other suitable framework sequences. Therefore provided herein in some embodiments, is a CD33 binding domain comprising a light chain variable domain, wherein the light chain CDRl is SEQ ID NO:21; the light chain CDR2 is SEQ ID NO:28 and the light chain CDR3 is SEQ ID NO:35. In some embodiments, a CD33 binding domain comprises a light chain variable domain, wherein the light chain CDRl is SEQ ID NO:22; the light chain CDR2 is SEQ ID NO:29 and the light chain CDR3 is SEQ ID NO:36. In some embodiments, a CD33 binding domain comprises a light chain variable domain, wherein the light chain CDRl is SEQ ID NO:23; the light chain CDR2 is SEQ ID NO:30 and the light chain CDR3 is SEQ ID NO:37. In some embodiments, a CD33 binding domain comprises a light chain variable domain, wherein the light chain CDRl is SEQ ID NO:24; the light chain CDR2 is SEQ ID NO:31 and the light chain CDR3 is SEQ ID NO:38. In some embodiments, a CD33 binding domain comprises a light chain variable domain, wherein the light chain CDRl is SEQ ID NO:25; the light chain CDR2 is SEQ ID NO:32 and the light chain CDR3 is SEQ ID NO:39. In some embodiments, a CD33 binding domain comprises a light chain variable domain, wherein the light chain CDRl is SEQ ID NO:26; the light chain CDR2 is SEQ ID NO:33 and the light chain CDR3 is SEQ ID NO:40. In some embodiments, a CD33 binding domain comprises a light chain variable domain, wherein the light chain CDRl is SEQ ID NO:27; the light chain CDR2 is SEQ ID NO:34 and the light chain CDR3 is SEQ ID NO:41.

[0070] Also provided herein in some embodiments, is a CD33 binding domain comprising a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO:42; the heavy chain CDR2 is SEQ ID NO:49 and the heavy chain CDR3 is SEQ ID NO:56. In some embodiments, a CD33 binding domain comprises a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO:43; the heavy chain CDR2 is SEQ ID NO:50 and the heavy chain CDR3 is SEQ ID NO:57. In some embodiments, a CD33 binding domain comprises a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO:43; the heavy chain CDR2 is SEQ ID NO:50 and the heavy chain CDR3 is SEQ ID NO:58. In some embodiments, a CD33 binding domain comprises a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO: 43; the heavy chain CDR2 is SEQ ID NO: 50 and the heavy chain CDR3 is SEQ ID NO: 59. In some embodiments, a CD33 binding domain comprises a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO: 43; the heavy chain CDR2 is SEQ ID NO: 50 and the heavy chain CDR3 is SEQ ID NO:60. In some embodiments, a CD33 binding domain comprises a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO:44; the heavy chain CDR2 is SEQ ID NO:51 and the heavy chain CDR3 is SEQ ID N0:61. In some embodiments, a CD33 binding domain comprises a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO:45; the heavy chain CDR2 is SEQ ID NO:52 and the heavy chain CDR3 is SEQ ID NO:62. In some embodiments, a CD33 binding domain comprises a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO:46; the heavy chain CDR2 is SEQ ID NO:53 and the heavy chain CDR3 is SEQ ID NO:63. In some embodiments, a CD33 binding domain comprises a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO:47; the heavy chain CDR2 is SEQ ID NO:54 and the heavy chain CDR3 is SEQ ID NO:63. In some embodiments, a CD33 binding domain comprises a heavy chain variable domain, wherein the heavy chain CDRl is SEQ ID NO:48; the heavy chain CDR2 is SEQ ID NO: 55 and the heavy chain CDR3 is SEQ ID NO: 63.

[0071] In further embodiments, a CD33 binding domain comprises a variable light chain domain selected from amino acid sequences SEQ ID NOs. : l-10 shown in Table 3. In further

embodiments, a CD33 binding domain comprises a variable heavy chain domain selected from amino acid sequences SEQ ID NO: 11-20 shown in Table 4. In yet further embodiments, a CD33 binding domain comprises a variable light chain domain selected from amino acid sequences SEQ ID NOs.: 1-10 shown in Table 3 and a variable heavy chain domain selected from amino acid sequences SEQ ID NO: 11-20 shown in Table 4.

[0072] The term "binding domain" refers to an immunoglobulin derivative with antigen binding properties, i.e. immunoglobulin polypeptides or fragments thereof that contain an antigen binding site. The binding domain comprises variable domains of an antibody or fragments thereof. Each antigen-binding domain is formed by an antibody, i.e. immunoglobulin, variable heavy chain domain (VH) and an antibody variable light chain domain (VL) binding to the same epitope, whereas the variable heavy chain domain (VH) comprises three heavy chain

complementarity determining regions (CDR): CDRl, CDR2 and CDR3; and the variable light chain domain (VL) comprises three light chain complementarity determining regions (CDR): CDRl, CDR2 and CDR3. In some instances, the binding domain according to some

embodiments herein is devoid of immunoglobulin constant domains. In some instances, the variable light and heavy chain domains forming the antigen binding site is covalently linked with one another, e.g. by a peptide linker, or in other instances, the variable light and heavy chain domains non-covalently associate with one another to form the antigen binding site. The term "binding domain" refers also to antibody fragments or antibody derivatives including, for example, Fab, Fab ^' , F(ab ^' ) ₂ , Fv fragments, single-chain Fv, tandem single-chain Fv ((scFv) ₂ , Bi- specific T-cell engagers (BiTE®), dual affinity retargeting antibodies (DART™), diabody, tandem diabody (TandAb®), DuoBody® IgG molecules, single domain antibodies (e.g., VHH), 2x2 T-cell engagers, TriTacs, and the like. Furthermore, in certain instances, the binding domain is multivalent, i.e. has two, three or more binding sites for the target antigen, e.g., CD33, or CD3.

Table 1: Amino acid sequences of anti-CD33 variable light chain CDRl, CDR2 and CDR3

SEQ ID NO:41 AAWDDSLSA

Table 2: Amino acid sequences of anti-CD33 variable heavy chain CDRl, CDR2 and CDR3

SEQ ID NO:62 HKRGSDAFDI

SEQ ID NO:63 EYYYDSSEWAFDI

Table 3: Amino acid sequences of all anti-CD33 variable light chain domains (amino acid sequences of variable light chain CDRl, CDR2 and CDR3 are in bold and underlined) anti-

Sequence

CD33 Variable light chain (VL) domain Sequence

identifier

clone

SYELTQPPSVSVAPGQTAMITCGGNNIGSTTVHWYQQKPGQAPVLVV

01 SEQ ID NO: 1 YDDNERPSGIPERFSGSNSGSTATLTINRVEAGDEADYYCOVWDSGSD

HVVFGGGTKLTVL

OSVLTOPPSASGTPGORVTISCSGSRSNIGSNTVNWYOOLPGTAPKLLI

02 SEQ ID NO:2 YGNNORPSGVPDRFSGSKSGTSASLAISGLOSEDEADYYCATWDDSLI

GWVFGGGTKLTVL

OSVLTOPPSASGTPGORVTISCSGSRSNIGSNTVNWYOOLPGTAPKLLI

03 SEQ ID NO:3 YGNNORPSGVPDRFSGSKSGTSASLAISGLOSEDEADYYCATWDDSLI

GWVFGGGTKLTVL

OSVLTOPPSASGTPGORVTISCSGSRSNIGSNTVNWYOOLPGTAPKLLI

04 SEQ ID NO:4 YGNNORPSGVPDRFSGSKSGTSASLAISGLOSEDEADYYCATWDDSLI

GWVFGGGTKLTVL

OSVLTOPPSASGTPGORVTISCSGSRSNIGSNTVNWYOOLPGTAPKLLI

05 SEQ ID NO:5 YGNNORPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSLI

GWVFGGGTKLTVL

OSVLTOPPSASGTPGORVTISCSGSSSNIGSNTVNWYOOLPGTAPKLLI

06 SEQ ID NO:6 YSDNORPSGVPDRFSGSKSGSSASLAISGLOSDDEADYYCATWDDSLN

GAVFGGGTKLTVL

OSVLTOPPSVSGAPGORVTISCTGSSSNIGAGYDVHWYOOLPGTAPKL

07 SEQ ID NO:7 LIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCOSYDSSL

SDVVFGGGTKLTVL

OSVLTOPPSASGTPGORVTISCSGSSSNIGSNIVNWYOOLPGTAPKLLIY

08 SEQ ID NO:8 SNNORPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLKG

YVFGGGTKLTVL

OSVLTOPPSASGTPGORVTISCSGSSSNIGSNTVKWYOOLPGTAPKLLI

09 SEQ ID NO:9 YSNNORSSGVPDRFSGSKSGSSASLAISGLOSEDEADYYCAAWDDSLN

GYVFGGGTKLTVL

OSVLTOPPSASGTPGORVTISCSGSSSNIGDNWNWYOOLPGTAPKLLI

10 SEQ ID NO: 10 YSTNKRPSGVPDRFSGSKSGSSASLAISGLOSEDEADYYCAAWDDSLS

AYVFGGGTKLTVL

Table 4: Amino acid sequence of anti-CD33 variable heavy chain domain (amino acid sequences of variable heavy chain CDRl, CDR2 and CDR3 are in bold and underlined) anti-

Sequence

CD33 Variable heavy chain (VH) domain Sequence

identifier

clone

OVOLOESGGGVVOPGRSLRLSCAASGFSFSNYGIHWVROAPGKGLEWVA

01 SEQ ID NO: 11 LISYDGNKKFYADSVKGRFAISRDTSKNTVDLOMTSLRPEDTAVYYCAK

DRLESAAFDYWGQGTLVTVSS

OVOLVOSGAEVKKPGASVKVSCKASGYTFTSYDINWVROAPGOGLEWM

02 SEQ ID NO: 12 GWMNPNSGNTGFAQKFOGRVTMTRDTSTSTVYMELS SLRSEDT AVYYC

ARDRANTDFSYGMDVWGQGTLVTVSS

OVOLVOSGAEVKKPGASVKVSCKASGYTFTSYDINWVROAPGOGLEWM

03 SEQ ID NO: 13 GWMNPNSGNTGFAOKFQGRVTMTRDTSTSTVYMELS SLRSEDT AVYYC

ARDRAVTDYYYGMDVWGQGTLVTVSS OVOLVOSGAEVKKPGASVKVSCKASGYTFTSYDINWVROAPGOGLEWM

04 SEQ ID NO: 14 GWMNPNSGNTGFAOKFOGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC

ARDRANTDYSFGMDVWGOGTLVTVSS

OVOLVOSGAEVKKPGASVKVSCKASGYTFTSYDINWVROAPGOGLEWM

05 SEQ ID NO: 15 GWMNPNSGNTGFAOKFOGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC

ARDRANTD YSLGMD VWGOGTL VT VS S

OVOLVOSGAEVKKPGASVKVSCKASGYTFTSYYMHWVROAPGOGLEW

06 SEQ ID NO: 16 MGIINPSGGSTSYAOKFOGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC

ARD WPAAID YYGMD VWGOGTT VT VS S

OVOLVOSGAEVKKPGESLKISCKGSGYSFTSYWIGWVROMPGKGLEWM

07 SEQ ID NO: 17 GIIYPGDSDTRYSPSFOGOVTISADKSISTAYLOWSSLKASDTAMYYCAR

HKRGSD AFDIWGOGTT VT VS S

OVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVROAPGOGLEWMG

08 SEQ ID NO: 18 GIYPIFGSANYAOKFOGRVTITADESTSTAYMELSSLRSEDTAVYYCARE

YYYDSSEWAFDIWGOGTLVTVSS

OVOLVOSGAEVKKPGSSVKVSCKASGGTFSSYGISWVROAPGOGLEWM

09 SEQ ID NO: 19 GGIIPIFGSAHYAOKFOGRVTITADESTSTAYMELSSLRSEDTAVYYCAR

EYYYDSSEWAFDIWGOGTLVTVSS

OVOLVOSGAEVKKPGSSVKVSCKASGGTFDSYAISWVROAPGOGLEWM

10 SEQ ID NO:20 GGIIPIFGSAHYSOKFOGRVTITADESTSTAYMELSSLRSEDTAVYYCARE

YYYD S SE W AFDIWGQGTL VT VS S

[0073] In some embodiments, a binding domain conferring specificity to CD33 is selected from one of the following combinations of a variable heavy chain domain and a variable light chain domain forming the human CD33 binding site shown in Table 3 and in Table 4. Non-limiting examples include (i) SEQ ID NO: 1 and SEQ ID NO: 11, (ii) SEQ ID NO: 2 and SEQ ID NO: 12, (iii) SEQ ID NO:3 and SEQ ID NO: 13, (iv) SEQ ID NO:4 and SEQ ID NO: 14, (v) SEQ ID NO:5 and SEQ ID NO: 15, (vi) SEQ ID NO:6 and SEQ ID NO: 16, (vii) SEQ ID NO:7 and SEQ ID NO: 17, (viii) SEQ ID NO:8 and SEQ ID NO: 18, (ix) SEQ ID NO:9 and SEQ ID NO: 19, and (x) SEQ ID NO: 10 and SEQ ID NO:20.

[0074] The bispecific antibodies described herein have binding domains that not only have specificity for CD33, but also have at least one further functional domain. In a further embodiment at least one further functional domain is an effector domain. An "effector domain" comprises a binding site of an antibody specific for an effector cell, which can stimulate or trigger cytotoxicity, phagocytosis, antigen presentation, cytokine release. Such effector cells are, for example, but not limited to, T-cells. In particular, the effector domain comprises at least one antibody variable heavy chain domain and at least one variable light chain domain forming an antigen binding site for an antigen on T-cells, such as, for example, human CD3.

[0075] Thus, in some embodiments, the bispecific antibody described herein is multifunctional. The term multifunctional as used herein means that a binding protein exhibits two or more different biological functions. For example, the different biological functions are different specificities for different antigens. In certain instances, the multifunctional CD33 binding protein is multispecific, i.e. has binding specificity to CD33 and one or more further antigens. In certain instances, the binding protein is bispecific with specificities for CD33 and CD3 and may be masked or unmasked with other proteins, protein fragments or chemical structures.

Such bispecific binding proteins include, for example, bispecific monoclonal antibodies of the classes IgA, IgD, IgE, IgG or IgM, diabodies, single-chain diabodies (scDb), single chain antibodies, nanobodies, tandem single chain Fv (scFv)2, for example Bi-specific T-cell engagers (BiTE®), dual affinity retargeting antibodies (DART™), tandem diabodies (TandAb®),2x2 T- cell engagers and flexibodies.

[0076] CD3, as used herein, denotes an antigen that is expressed on human T cells as part of the multimolecular T cell receptor complex, consisting of five chains: 2 CD3-epsilon, a CD3- gamma, a CD3-delta, and a CD3 zeta. Clustering of CD3 on T cells e.g. by anti-CD3 antibodies leads to T cell activation similar to the binding of an antigen but independent from the clonal specificity of the T cell subset, as described above. Thus, a bispecific antibody specifically binding with one of its specificities the human CD3 antigen relates to a CD3-specific construct capable of binding to the human CD3 complex expressed on human T cells and capable of inducing elimination/lysis of target cells, wherein such target cells carry/display an antigen which is bound by the other, non-CD3 -binding portion of the bispecific single chain antibody. Binding of the CD3 complex by CD3 -specific binders (e.g. a bispecific single chain antibody as administered according to the pharmaceutical means and methods of the invention) leads to activation of T cells.

[0077] In certain embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has specificity for human CD3 and, in some instances, cynomolgus CD3. Examples of such a binding site are polypeptides comprising the VH domain CDR1, CDR2 and CDR3 from the sequences shown in Table 5 (SEQ ID NOs:64-67) and VL domain CDR1, CDR2 and CDR3 from the sequence shown in Table 6 (SEQ ID NOs: 68-71). In certain instances, a CD3 binding site is the combination of the variable heavy chain domain of SEQ ID NO:64 and the variable light chain domain of SEQ ID NO:68. In certain instances, a CD3 binding site is the

combination of the variable heavy chain domain of SEQ ID NO: 65 and the variable light chain domain of SEQ ID NO:69. In certain instances, a CD3 binding site is the combination of the variable heavy chain domain of SEQ ID NO: 66 and the variable light chain domain of SEQ ID NO:70. In certain instances, a CD3 binding site is the combination of the variable heavy chain domain of SEQ ID NO:67 and the variable light chain domain of SEQ ID NO:71.

Table 5: Amino acid sequence of an anti-CD3 variable heavy chain domain (amino acid sequences of variable heavy chain CDR1, CDR2 and CDR3 are in bold and underlined)

anti-CD3 VH domain Sequence

SEQ ID NO:64 EVOLVESGGGLVOPGGSLRLSCAASGFTFSTYAMNWVROAPGKGLEWVGRIRSKYNNY CD3-01 ATYYADSVKDRFTISRDDSKNSLYLOMNSLKTEDTAVYYCARHGNFGNSYVSYFAYWG

QGTLVTVSS

SEQ ID NO:65 EVOLVESGGGLVOPGGSLRLSCAASGFTFSTYAMNWVROAPGKGLEWVGRIRSKYNNY CD3-02 ATYYADSVKDRFTISRDDSKNSLYLOMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWG QGTLVTVSS

SEQ ID NO:66 EVOLVESGGGLVOPGGSLRLSCAASGFTFSTYAMNWVROAPGKGLEWVGRIRSKYNNY CD3-03 ATYYADSVKDRFTISRDDSKNSLYLOMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWG

QGTLVTVSS

SEQ ID NO:67 EVOLVESGGGLVOPGGSLRLSCAASGFTFSTYAMNWVROAPGKGLEWVGRIRSKYNNY CD3-04 ATYYADSVKDRFTISRDDSKNSLYLOMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWG

QGTLVTVSS

Table 6: Amino acid sequence of an anti-CD3 variable light chain domain (amino acid sequences of variable light chain CDR1, CDR2 and CDR3 are in bold and underlined)

anti-CD3 VL domain Sequence

SEQ ID NO:68 DIOMTOSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVOOKPGKAPKALIGGTNKRAP CD3-01 GVPSRFSGSLIGDKATLTISSLOPEDFATYYCALWYSNLWVFGOGTKVEIK

SEQ ID NO:69 DIOMTOSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVOOKPGKAPKGLIGGTNKRAP CD3-02 GVPARFSGSGSGTDFTLTISSLOPEDFATYYCALWYSNLWVFGOGTKVEIK

SEQ ID NO:70 DIOMTOSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVOOKPGKAPKGLIGGTNKRAP CD3-03 GVPSRFSGSLIGDKATLTISSLOPEDFATYYCALWYSNLWVFGOGTKVEIK

SEQ ID NO:71 DIOMTOSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVOOKPGKAPKGLIGGTNKRAP CD3-04 GVPSRFSGSLIGTDFTLTISSLQPEDFATYYCALWYSNLWVFGOGTKVEIK

[0078] In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable heavy chain domain comprising a CDR1 sequence of STYAMN (SEQ ID

NO:72). In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable heavy chain domain comprising a CDR2 sequence of

RIRSK YNNYAT YYAD S VKD (SEQ ID NO:73). In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable heavy chain domain comprising a CDR3 sequence of HGNFGNSYVSWFAY (SEQ ID NO:74). In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable heavy chain domain comprising a CDR3 sequence of HGNFGNS YVS YF AY (SEQ ID NO:75). In yet further embodiments, the CD3 binding site has a variable heavy chain domain comprising a CDR1, CDR2 and CDR3 sequence of SEQ ID NOs:72-74 respectively. In yet further embodiments, the CD3 binding site has a variable heavy chain domain comprising a CDR1, CDR2 and CDR3 sequence of SEQ ID NOs: 72, 73 and 75 respectively.

[0079] In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable heavy chain domain comprising a CDR1 sequence selected from the group consisting of NTYAMN (SEQ ID NO:76), NTYAMH (SEQ ID NO:77) and NKYAMN (SEQ ID NO:78). In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable heavy chain domain comprising a CDR2 sequence selected from the group consisting of RIRNKYNNYATYYADSVKD (SEQ ID NO:79), RIRNKYNNYATEYADSVKD (SEQ ID NO: 80), RIRSKYNNYATEYAASVKD (SEQ ID NO: 81),

RIRNKYNNYATEYAASVKD (SEQ ID NO: 82), RIRSK YNNYAT YYAD SVKG (SEQ ID NO:83) and RIRSKYNNYATEYADSVKS (SEQ ID NO:84). In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable heavy chain domain comprising a CDR3 sequence selected from the group consisting of HG FGDSYVSWFAY (SEQ ID NO:85), HG FGNTYVSWFAY (SEQ ID NO:86), HGNFGC S YVS WF AY (SEQ ID NO:87), HGNFGNSYISYWAY (SEQ ID NO:88) and HGNFGNSYVSFFAY (SEQ ID NO:89).

[0080] ). In yet further embodiments, the CD3 binding site has a variable heavy chain domain comprising a CDR1, CDR2 and CDR3 sequence of SEQ ID NOs:76, 73 and 74 respectively, SEQ ID NOs:76, 79 and 74 respectively, SEQ ID NOs:76, 80 and 74 respectively, SEQ ID NOs:76, 81 and 74 respectively, SEQ ID NOs:76, 82 and 74 respectively, SEQ ID NOs:76, 83 and 74 respectively, SEQ ID NOs:72, 83 and 74 respectively, SEQ ID NOs:72, 83 and 85 respectively, SEQ ID NOs:76, 83 and 86 respectively, SEQ ID NOs:77, 83 and 74 respectively, SEQ ID NOs:72, 83 and 87 respectively, SEQ ID NOs:78, 73 and 88 respectively or SEQ ID NOs:78, 84 and 89 respectively.

[0081] In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable light chain domain comprising a CDR1 sequence of RSSTGAVTTSNYAN (SEQ ID NO:90). In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable light chain domain comprising a CDR2 sequence of GTNKRAP (SEQ ID NO:91). In further embodiments, the CD3 binding site of a bispecific antibody to CD33 and CD3 has a variable light chain domain comprising a CDR3 sequence of ALWYSNL (SEQ ID NO:92). In yet further embodiments, the CD3 binding site has a variable light chain domain comprising a CDR1, CD2 and CD3 sequence of SEQ ID NOs:90-92 respectively.

[0082] In certain instances, the CD3 binding site has a high affinity to CD3. Alternatively, in other instances, the CDR1, CDR2, CDR3 from the heavy-chain domain as well as the light- chain domain or, optionally, the variable light-chain domains and variable heavy-chain domains is derived from other CD3 antibodies, such as, for example UCHT1, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), and the like.

[0083] In another aspect, described herein are bispecific antibodies to CD33 and CD3 that are humanized or fully human, i.e. of human origin. In further embodiments, described herein are bispecific antibodies to CD33 and CD3 that are camelid or llama.

[0084] In some embodiments, a bispecific antibody to CD33 and CD3 has one of the following combinations providing CD33 and CD3 specificity by variable light and heavy chain domains for CD33 and CD3 : include, but are not limited to, (i) SEQ ID NOs:2, 12, 65 and 69, (ii) SEQ ID NOs:3, 13, 65 and 69, (iii) SEQ ID NOs:4, 14, 65 and 69, (iv) SEQ ID NOs:5, 15, 65 and 69, (v) SEQ ID NOs: 1, 11, 64 and 68, (vi) SEQ ID NOs:2, 12, 64 and 68, (vii) SEQ ID NOs:2, 12, 66 and 70, (viii) SEQ ID NOs:4, 14, 66 and 70, (ix) SEQ ID NOs:5, 15, 66 and 70, and (x) SEQ ID NOs:3, 13, 64 and 68, (xi) SEQ ID NOs:3, 13, 67 and 71, (xii) SEQ ID NOs:4, 14, 64 and 68, (xiii) SEQ ID NOs:5, 15, 64 and 68, (xiv) SEQ ID NOs:7, 17, 64 and 68, (xv) SEQ ID NOs:6, 16, 64 and 68, (xvi) SEQ ID NOs:6, 16, 67 and 71, (xvii) SEQ ID NOs:8, 18, 64 and 68, (xviii) SEQ ID NOs:9, 19, 64 and 68; (xix) SEQ ID NOs:9, 19, 67 and 71, and (xx) SEQ ID NOs: 10, 20, 64 and 68.

Conserved Variants of CDR Sequences and Heavy and Light Chain Domains

[0085] In alternative embodiments, the heavy and light chain domains incorporate

immunologically active homologues or variants of the CDR sequences described herein.

Accordingly in some embodiments, a CDR sequence in a heavy or light chain domain that binds to CD33 or CD3 is similar to, but not identical to, the amino acid sequence depicted in SEQ ID NOs: 21-63 or 72-92. In certain instances, a CDR variant sequence has a sequence identity of 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% compared to the sequence of SEQ ID NOs: 21-63 or 72-90 and which is immunologically active.

[0086] In further instances, a CDR variant sequence incorporates 1, 2, 3, 4, or 5 conserved amino acid substitutions. Conservative substitutions include amino acid substitutions that substitute a given amino acid with another amino acid of similar characteristics and further include, among the aliphatic amino acids interchange of alanine, valine, leucine, and isoleucine; interchange of the hydroxyl residues serine and threonine, exchange of the acidic residues aspartate and glutamate, substitution between the amide residues asparagine and glutamine, exchange of the basic residues lysine and arginine, and replacements among the aromatic residues phenylalanine and tyrosine.

[0087] In yet further instances, a CDR variant sequence incorporates substitutions that enhance properties of the CDR such as increase in stability, resistance to proteases and/or binding affinities to CD33 or CD3.

[0088] In other instances, a CDR variant sequence is modified to change non-critical residues or residues in non-critical regions. Amino acids that are not critical can be identified by known methods, such as affinity maturation, CDR walking, site-directed mutagenesis, crystallization, nuclear magnetic resonance, photoaffinity labeling, or alanine-scanning mutagenesis.

[0089] In further alternative embodiments, the bispecific antibodies to CD33 and CD3 comprise heavy and light chain domains that are immunologically active homologues or variants of heavy and light chain domain sequences provided herein. Accordingly, in some embodiments, a CD33 and CD3 binding protein comprises a heavy or light chain domain sequence that is similar to, but not identical to, the amino acid sequence depicted in SEQ ID NOs: 1-20 or 64-71. In certain instances, a variant heavy or light chain domain sequence has a sequence identity of 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% compared to the sequence of SEQ ID NOs: 1-20 or 64-71 and which is

immunologically active.

[0090] In further instances, a variant heavy or light chain domain sequence incorporates 1, 2, 3, 4, or 5 conserved amino acid substitutions. Conservative substitutions include amino acid substitutions that substitute a given amino acid with another amino acid of similar characteristics and further include, among the aliphatic amino acids interchange of alanine, valine, leucine, and isoleucine; interchange of the hydroxyl residues serine and threonine, exchange of the acidic residues aspartate and glutamate, substitution between the amide residues asparagine and glutamine, exchange of the basic residues lysine and arginine, and replacements among the aromatic residues phenylalanine and tyrosine.

[0091] In yet further instances, a variant heavy or light chain domain sequence incorporates substitutions that enhance properties of the CDR such as increase in stability, resistance to proteases and/or binding affinities to CD33 or CD3.

[0092] In other instances, a variant heavy or light chain domain sequence is modified to change non-critical residues or residues in non-critical regions. Amino acids that are not critical can be identified by known methods, such as affinity maturation, CDR walking, site-directed mutagenesis, crystallization, nuclear magnetic resonance, photoaffinity labeling, or alanine- scanning mutagenesis.

CD33 and CD3 2x2 T-cell Engagers

[0093] In another aspect, a bispecific antibody to CD33 and CD3 is a dimer, i.e. comprises two polypeptides with antigen binding sites for CD33 and CD3.

[0094] Also provided herein in another aspect, is a dimeric and bispecific antibody to CD33 and CD3 in the format of a 2x2 T-cell engager. Such 2x2 T-cell engagers are constructed by linking four antibody variable binding domains (two heavy-chain variable domains (VH) and two light- chain variable domains (VL) in a single gene construct (Figure 1) enabling homo-dimerization. In such 2x2 T-cell engagers the linker length is such that it prevents intramolecular pairing of the variable domains so that the molecule cannot fold back upon itself to form a single-chain diabody, but rather is forced to pair with the complementary domains of another chain. The domains are also arranged such that the corresponding VH and VL domains pair during this dimerization. Following expression from a single gene construct, two identical polypeptide chains fold head -to-tail forming a functional non-covalent homodimer of approximately 105 kDa (Figure 1). Despite the absence of intermolecular covalent bonds, the homodimer is highly stable once formed, remains intact and does not revert back to the monomelic form. [0095] 2x2 T-cell engagers have a number of properties that provide advantages over traditional monoclonal antibodies and other smaller bispecific molecules. 2x2 T-cell engagers contain only antibody variable domains and therefore are contemplated to lack side effects or non-specific interactions that may be associated with an Fc moiety. For example, Fc receptors which can bind to Fc domains are found on numerous cell types such as white blood cells (e.g., basophils, B-cells, eosinophils, natural killer cells, neutrophils and the like) or Kuppfer cells. Because 2x2 T-cell engagers allow for bivalent binding to each of CD33 and CD3, the molecules have avidity that is similar to that of an IgG antibody against a single target. The size of a 2x2 T-cell engager, at approximately 105 kDa, is smaller than that of an IgG, which may allow for enhanced tumor penetration. However, this size is well above the renal threshold for first-pass clearance, offering a pharmacokinetic advantage compared with smaller bispecific formats based on antibody-binding domains or non-antibody scaffolds. Moreover 2x2 T-cell engagers are advantageous over other bispecific binding proteins such as BiTE or DART molecules based on these pharmacokinetic and avidity properties resulting in longer intrinsic half-lives and rapid cytotoxicity. 2x2 T-cell engagers are well expressed in host cells, for example, mammalian CHO cells. It is contemplated that robust upstream and downstream manufacturing processes are available for 2x2 T-cell engagers.

[0096] The CD33 and CD3 bispecific 2x2 T-cell engagers described herein are designed to allow specific targeting of tumor cells and cells in the tumor microenvironment, such as MDSCs, that express CD33 by recruiting cytotoxic T-cells. In contrast, by engaging CD3 molecules expressed specifically on these cells, the 2x2 T-cell engager can bind cytotoxic T- cells and CD33 expressing cells in a highly specific fashion, thereby significantly increasing the cytotoxic potential of such molecules. This mechanism is outlined in Figure 2. It is reported that T-cells can play a role in controlling tumor growth. For example, the presence of cytotoxic T-cells in colorectal tumors as well as lymph nodes from NHL patients was shown to correlate with a better clinical outcome. Furthermore, the potential of therapies designed to induce T-cell responses has been demonstrated for melanoma vaccines, as well as the antibody directed against CTLA-4, a negative regulator of T-cell activation. The 2x2 T-cell engagers described herein engage cytotoxic T-cells via binding to the surface-expressed CD3, which forms part of the T-cell receptor. Simultaneous binding of this 2x2 T-cell engager to CD3 and to CD33 expressed on the surface of particular tumor cells causes T-cell activation and mediates the subsequent lysis of the malignant cell (Figure 2).

[0097] Therefore, in a further aspect is a multispecific, 2x2 T-cell engager. In some

embodiments, a multispecific 2x2 T-cell engager has specificities to two, three or more different epitopes, wherein two or more epitopes can be of the same antigen target or of different antigen targets. In certain embodiments the multispecific, 2x2 T-cell engager is bispecific and tetravalent, i.e. comprises four antigen-binding sites. Such a bispecific 2x2 T-cell engager binds with at least one antigen-binding site, to human CD3 and to human CD33, wherein in certain instances, the 2x2 T-cell engager binds with two antigen-binding sites to human CD3 and with two other antigen-binding sites to human CD33, i.e. the 2x2 T-cell engager binds bivalently to each antigen.

[0098] In some embodiments, a bispecific, antigen-binding 2x2 T-cell engager is specific to human CD33 and human CD3, wherein said 2x2 T-cell engager comprises a first polypeptide and a second polypeptide, each polypeptide having at least four variable chain domains linked one after another, wherein each polypeptide comprises

(i) a variable heavy chain (VH) domain specific to human CD33;

(ii) a variable light chain (VL) domain specific to human CD33;

(iii) a VH domain specific for human CD3, and

(iv) a VL domain specific for human CD3.

[0099] In particular embodiments, a bispecific 2x2 T-cell engager specifically binds to an epitope of human CD33 which is within ₆₂ DQEVQEETQ ₇₀ (SEQ ID NO:94) (amino acid residues 62-70 of SEQ ID NO:93) of human CD33. In particular instances, such a 2x2 T-cell engager comprises a first polypeptide and a second polypeptide, each polypeptide having at least four variable chain domains linked one after another, wherein each polypeptide comprises

(i) a variable heavy chain domain specific to an epitope of human CD33 which is within ₆₂ DQEVQEETQ ₇₀ (SEQ ID NO: 94) (amino acid residues 62-70 of SEQ ID NO:93) of human CD33;

(ii) a variable light chain domain specific to an epitope of human CD33 which is within ₆₂ DQEVQEETQ ₇₀ (SEQ ID NO: 94) (amino acid residues 62-70 of SEQ ID NO:93) of human CD33;

(iii) a variable heavy chain domain specific for human CD3, and

(iv) a variable light chain domain specific for human CD3.

[0100] In other embodiments, described herein are CD33/CD3 2x2 T-cell engagers that have an affinity to CD33 on CD33 ⁺ cells with a K _D of 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. The CD33 ⁺ cells can be selected from tumor cells such as, for example, HL-60 or KG-1.

[0101] In a further embodiment a CD33/CD3 2x2 T-cell engager described herein binds CD3 and in certain instances, the epsilon chain of CD3 on CD3 ⁺ cells, particularly T-cells, with a K _D of 10 nM or less, 5 nM or less or 2 nM or less. [0102] In some embodiments, each polypeptide of a bispecific 2x2 T-cell engager comprises one of the following combinations of the four variable chain domains: (i) SEQ ID NOs:2, 12, 65 and 69, (ii) SEQ ID NOs:3, 13, 65 and 69, (iii) SEQ ID NOs:4, 14, 65 and 69, (iv) SEQ ID NOs:5, 15, 65 and 69, (v) SEQ ID NOs: l, 11, 64 and 68, (vi) SEQ ID NOs:2, 12, 64 and 68, (vii) SEQ ID NOs:2, 12, 66 and 70, (viii) SEQ ID NOs:4, 14, 66 and 70, (ix) SEQ ID NOs:5, 15, 66 and 70, and (x) SEQ ID NOs:3, 13, 64 and 68, (xi) SEQ ID NOs:3, 13, 67 and 71, (xii) SEQ ID NOs:4, 14, 64 and 68, (xiii) SEQ ID NOs:5, 15, 64 and 68, (xiv) SEQ ID NOs:7, 17, 64 and 68, (xv) SEQ ID NOs:6, 16, 64 and 68, (xvi) SEQ ID NOs:6, 16, 67 and 71, (xvii) SEQ ID NOs:8, 18, 64 and 68, (xviii) SEQ ID NOs:9, 19, 64 and 68; (xix) SEQ ID NOs:9, 19, 67 and 71, and (xx) SEQ ID NOs: 10, 20, 64 and 68.

[0103] As used herein, "dimer" refers to a complex of two polypeptides. In certain

embodiments, the two polypeptides are non-covalently associated with each other, in particular with the proviso that there is no covalent bond between the two polypeptides. In certain instances, the two polypeptides have covalent associations such as disulfide bonds that form to aid in stabilization of the dimer. In certain embodiments, the dimer is homodimeric, i.e.

comprises two identical polypeptides. The term "polypeptide" refers to a polymer of amino acid residues linked by amide bonds. The polypeptide is, in certain instances, a single chain fusion protein, which is not branched. In the polypeptide the variable antibody domains are linked one after another. The polypeptide, in other instances, may have contiguous amino acid residues in addition to the variable domain N-terminal and/or C-terminal residues. For example, such contiguous amino acid residues may comprise a Tag sequence, in some instances at the C- terminus, which is contemplated to be useful for the purification and detection of the

polypeptide.

[0104] In one aspect, each polypeptide of the bispecific 2x2 T-cell engager comprises four variable domains, a variable light chain (VL) and a variable heavy chain (VH) of a CD3 binding protein as well as a variable light chain (VL) and a variable heavy chain (VH) of a CD33 binding protein. In certain embodiments, four variable domains are linked by peptide linkers LI, L2 and L3 and in some instances arranged from the N- to the C-terminus as follows:

Domain Order:

(1) VL(CD3)-L1-VH(CD33)-L2-VL(CD33)-L3-VH(CD3); or

(2) VH(CD3)-L1-VL(CD33)-L2-VH(CD33)-L3-VL(CD3); or

(3) VL(CD33)-L1-VH(CD3)-L2-VL(CD3)-L3-VH(CD33); or

(4) VH(CD33 )-L 1 - VL(CD3 )-L2- VH(CD3 )-L3 - VL(CD33 ).

[0105] The length of the linkers influences the flexibility of the antigen-binding 2x2 T-cell engager according to reported studies. Accordingly, in some embodiments, the length of the peptide linkers LI, L2 and L3 is such that the domains of one polypeptide can associate intermolecularly with the domains of another polypeptide to form the dimeric antigen-binding 2x2 T-cell engager. In certain embodiments, such linkers are "short", i.e. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, the linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the linker is a peptide bond. Such short linkers favor the intermolecular dimerization of the two polypeptides by binding and forming correct antigen-binding sites between antibody variable light chain domains and antibody variable heavy chain domains of different polypeptides. Shortening the linker to about 12 or less amino acid residues generally prevents adjacent domains of the same polypeptide chain from intramolecular interaction with each other. In some embodiments, these linkers consist of about 3 to about 10, for example 4, 5 or 6 contiguous amino acid residues.

[0106] Regarding the amino acid composition of the linkers, peptides are selected that do not interfere with the dimerization of the two polypeptides. For example, linkers comprising glycine and serine residues generally provide protease resistance. The amino acid sequence of the linkers can be optimized, for example, by phage-display methods to improve the antigen binding and production yield of the antigen-binding polypeptide dimer. Examples of peptide linkers suitable for a 2 2 T-cell engager in some embodiments are GGSGGS (SEQ ID NO:95), GGSG (SEQ ID NO:96), or GGSGG (SEQ ID NO:97).

[0107] Non-limiting examples of 2x2 T-cell engagers as described herein are 2x2 T-cell engagers having an anti-CD33 VL and VH domain, an anti-CD3 VL and VH domain, domain order and linker according to Table 7.

Table 7: Exemplary CD33/CD3 2x2 T-cell engagers

2x2 T- Anti-CD33 domain Anti-CD3 domain Linker

Domain

cell

VL VH VH VL Order L1/L3 L2 engager

01 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:2 NO: 12 NO:65 NO:69 ¹

02 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:3 NO: 13 NO:65 NO:69 ¹

03 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:4 NO: 14 NO:65 NO:69 ¹

04 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:5 NO: 15 NO:65 NO:69 ¹

05 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:4 NO: 14 NO:65 NO:69 ¹ G

06 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:5 NO: 15 NO:65 NO:69 ¹ G

07 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO: l NO: 11 NO:64 NO:68 ¹ GS

08 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:2 NO: 12 NO:64 NO:68 GS

09 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:2 NO: 12 NO:66 NO:70 ¹

10 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:4 NO: 14 NO:66 NO:70 ¹ 11 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG NO:5 NO: 15 NO:66 NO:70 ¹

12 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO:3 NO: 13 NO:64 NO:68 ¹

13 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:3 NO: 13 NO:67 NO:71 ¹

14 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:2 NO: 12 NO:64 NO:68 ¹

15 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:4 NO: 14 NO:64 NO:68 ¹

16 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:5 NO: 15 NO:64 NO:68 ¹

17 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:7 NO: 17 NO:64 NO:68 ¹

18 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:7 NO: 17 NO:64 NO:68

19 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:6 NO: 16 NO:64 NO:68 ¹

20 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:6 NO: 16 NO:67 NO:71 ¹

21 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:8 NO: 18 NO:64 NO:68 ¹

22 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:9 NO: 19 NO:64 NO:68 ¹

23 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

N0:9 NO: 19 NO:67 NO:71 ¹

24 SEQ ID SEQ ID SEQ ID SEQ ID GGSGGS GGSG

NO: 10 NO:20 NO:64 NO:68 ¹

[0108] In some embodiments, a 2x2 T-cell engager is attached to a C-terminal hexa-histidine (6xHis)-tag. In some embodiments, a 2x2 T-cell engager with a C-terminal hexa-histidine (6xHis)-tag is 2x2 T-cell engager 01 (SEQ ID NO:98), 02 (SEQ ID NO:99), 03 (SEQ ID NO: 100), 04 (SEQ ID NO: 101), 05 (SEQ ID NO: 102), 06 (SEQ ID NO: 103), 07 (SEQ ID NO: 104), 08 (SEQ ID NO: 105), 09 (SEQ ID NO: 106), 10 (SEQ ID NO: 107), 11 (SEQ ID NO: 108), 12 (SEQ ID NO: 109), 13 (SEQ ID NO: 110), 14 (SEQ ID NO: 111), 15 (SEQ ID NO: 112), 16 (SEQ ID NO: 113), 17 (SEQ ID NO: 114), 18 (SEQ ID NO: 115), 19 (SEQ ID NO: 116), 20 (SEQ ID NO: 117), 21 (SEQ ID NO: 118), 22 (SEQ ID NO: 119), 23 (SEQ ID NO: 120), or 24 (SEQ ID NO: 121) as depicted in Figure 10A to 10X.

[0109] In some embodiments, a 2x2 T-cell engager is 2x2 T-cell engager 01 (SEQ ID NO: 123), 02 (SEQ ID NO: 124), 03 (SEQ ID NO: 125), 04 (SEQ ID NO: 126), 05 (SEQ ID NO: 127), 06 (SEQ ID NO: 128), 07 (SEQ ID NO: 129), 08 (SEQ ID NO: 130), 09 (SEQ ID NO: 131), 10 (SEQ ID NO: 132), 11 (SEQ ID NO: 133), 12 (SEQ ID NO: 134), 13 (SEQ ID NO: 135), 14 (SEQ ID NO: 136), 15 (SEQ ID NO: 137), 16 (SEQ ID NO: 138), 17 (SEQ ID NO: 139), 18 (SEQ ID NO: 140), 19 (SEQ ID NO: 141), 20 (SEQ ID NO: 142), 21 (SEQ ID NO: 143), 22 (SEQ ID NO: 144), 23 (SEQ ID NO: 145), or 24 (SEQ ID NO: 146) as depicted in Figure 11 A to 1 IX.

[0110] The bispecific antibody to CD33 and CD3 (e.g., CD33/CD3 bispecific 2x2 T-cell engager) described herein is produced, in some embodiments, by expressing polynucleotides encoding the polypeptide of the 2x2 T-cell engager which associates with another identical polypeptide to form the antigen-binding 2x2 T-cell engager. Therefore, another aspect is a polynucleotide, e.g. DNA or RNA, encoding the polypeptide of an antigen-binding 2x2 T-cell engager as described herein.

[0111] The polynucleotide is constructed by known methods such as by combining the genes encoding at least four antibody variable domains either separated by peptide linkers or, in other embodiments, directly linked by a peptide bond, into a single genetic construct operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate expression system such as, for example CHO cells. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. The promoter is selected such that it drives the expression of the polynucleotide in the respective host cell.

[0112] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. This recombinant vector can be constructed according to known methods.

[0113] A variety of expression vector/host systems may be utilized to contain and express the polynucleotide encoding the polypeptide of the described antigen-binding 2x2 T-cell engager. Examples of expression vectors for expression in E.coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0114] Thus, the antigen-binding 2x2 T-cell engager as described herein, in some embodiments, is produced by introducing a vector encoding the polypeptide as described above into a host cell and culturing said host cell under conditions whereby the polypeptide chains are expressed, may be isolated and, optionally, further purified.

[0115] In other aspects, the bispecific antibody to CD33 and CD3 (e.g., CD33/CD3 bispecific 2x2 T-cell engager) described herein has a modification. Typical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of

phosphatidylinositol, drug conjugation, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of

pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. In further embodiments, the bispecific antibody to CD33 and CD3 is modified with additional amino acids, such as a leader or secretory sequence or a sequence for purification of the polypeptide.

[0116] In further aspects, the bispecific antibody to CD33 and CD3 (e.g., CD33/CD3 bispecific 2x2 T-cell engager) described herein comprises a half-life extension domain that extends half- life of the bispecific antibody. Such domains are contemplated to include but are not limited to HSA binding domains, pegylation, small molecules, and other half-life extension domains known in the art. Human serum albumin (HSA) (molecular mass -67 kDa) is the most abundant protein in plasma, present at about 50 mg/ml (600 μΜ), and has a half-life of around 20 days in humans. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma. Noncovalent association with albumin extends the elimination half-time of proteins. In some embodiments, the half-life extension domain is a domain that binds to HSA including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a single chain variable fragments (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived single domain antibody, peptide, ligand or small molecule entity specific for HSA.

[0117] In other aspects, provided herein are pharmaceutical compositions comprising the bispecific antibody to CD33 and CD3, a vector comprising the polynucleotide encoding the polypeptide of the bispecific antibody to CD33 and CD3 or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents. In further aspects, the pharmaceutical compositions comprise excipients for sustained release, e.g. PLGA nanoparticles and the like. In further aspects, the pharmaceutical compositions are coated on a device for insertion into the body for sustained release at a particular site.

[0118] Bispecific antibodies to CD33 and CD3 with high-affinity binding to CD33 and CD3 are highly active in a large number of primary AML specimens, suggesting that these molecules could be active against human AML across the entire cytogenetic/molecular disease spectrum, even in cases of minimal CD33 expression. Of note, drug-specific cytotoxicity is also observed in the presence of residual autologous T-cells and is significantly augmented by the addition of controlled amounts of healthy donor T-cells (see Example 6).

[0119] The bispecific antibodies to CD33 and CD3, in particular 2x2 T-cell engagers, can induce potent cytolysis of CD33 ⁺ leukemic cells in vitro. The data indicate that high-affinity binding to both CD33 and CD3 maximizes bispecific protein-induced T-cell activation and anti- AML efficacy. High-affinity CD33/CD3-directed bispecific binding proteins, such as the 2x2 T-cell engagers described herein display cytolytic activity in primary AML in vitro. Thus, these bispecific antibodies to CD33 and CD3, in particular 2x2 T-cell engagers are suitable for a therapeutic approach for the treatment of acute myeloid leukemia (AML) or other hematologic malignancies, for example, myelodysplastic syndrome (MDS) or myeloproliferative disease (MPD).

[0120] Therefore, provided herein are methods wherein a bispecific antibody to CD33 and CD3 as described herein above is administered in an effective dose to a subject, e.g., a patient, for the treatment of a CD33 ⁺ cancer (e.g. acute myeloid leukemia (AML)), disease or condition. CD33 ⁺ cancers include, but are not limited to, acute leukemias such as acute myeloid leukemia, acute lymphoblastic leukemia (ALL) including precursor B-cell lymphoblastic leukemia, myeloid sarcoma, multiple myeloma, acute lymphomas such as acute lymphoblastic lymphoma, chronic myelomonocytic leukemia and the like.

[0121] Also provided herein are methods wherein a bispecific antibody to CD33 and CD3 as described herein above is administered in an effective dose to a subject, e.g., a patient, for the treatment of a CD33 ⁺ disease and condition. CD33 ⁺ diseases and conditions include immune suppressive states or environments attributed by myeloid derived suppressor cells (MDSCs) in certain cancers and chronic inflammation.

[0122] In some embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of acute myeloid leukemia (AML). In certain embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of an acute myeloid leukemia subtype.

[0123] The French-American-British classification system divides AML into eight subtypes: AML-M0 (minimally differentiated), AML-M1 (without maturation), AML-M2 (with granulocytic maturation), AML-M3 (promyelocytic or acute promyelocytic leukemia), AML-4 (acute myelomonocytic leukemia), AML-M5 (acute monoblastic or monocytic leukemia), AML- M6 (acute erythroid leukemia), and AML-M7 (acute megakaryoblastic leukemia). In certain instances, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of AML-MO, AML-M1, AML-M2, AML-M3, AML-M4, AML-M5, AML-M6, or AML-M7.

[0124] The WHO AML classification scheme organizes AML according to the following subtypes: AML with Recurrent Genetic Abnormalities, AML with myelodysplasia-related changes, Therapy-related myeloid neoplasms, Myeloid sarcoma, Myeloid proliferations related to Down syndrome, Blastic plasmacytoid dendritic cell neoplasm, and AML not otherwise categorized. In certain other instances, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of AML with Recurrent Genetic Abnormalities, AML with myelodysplasia-related changes, Therapy-related myeloid neoplasms, Myeloid sarcoma, Myeloid proliferations related to Down syndrome, Blastic plasmacytoid dendritic cell neoplasm, or AML not otherwise categorized.

[0125] In some other embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of a newly diagnosed, recurrent or refractory AML.

[0126] In further embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of a preleukemia blood disorder such as myelodysplastic syndrome (MDS) or myeloproliferative disease (MPD). In certain instances, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of MDS. In certain instances, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of MPD.

[0127] In other embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of multiple myeloma. In further embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered for the treatment of chronic myelomonocytic leukemia (CMML).

[0128] In other embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered for inhibiting or eliminating myeloid derived suppressor cells (MDSCs). MDSCs highly overexpress CD33 in certain isolated diseased tissues and possess strong

immunosuppressive activities. In certain human cancers (CD33 ⁺ as well as non-CD33 ), MDSCs proliferate and are activated to suppress tumor-specific CD4 ⁺ T-cell responses and induce T _reg cells, allowing the tumor or cancer to flourish in a microenvironment. In chronic inflammation, MDSCs are reportedly expanded and found at inflammation sites to suppress T cell immune function. In other embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered for treating a condition associated with MDSCs. In yet other embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered to treat immune suppression. In yet other embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered to treat inflammation or immune suppression suppressed by MDSCs. In yet other embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered to treat a decreased immune response caused by MDSCs. In yet other embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered to treat angiogenesis, tumor invasion, or metastasis of cancers that are promoted by MDSCs. In yet other embodiments, the bispecific antibody to CD33 and CD3 as described herein is administered to treat a cancer or tumor that is enhanced, augmented, aggravated or increased by MDSCs. Such cancers include blood cancers as well as solid tumors including but not limited to pancreatic cancer, gastric cancer, esophageal cancer, and melanoma.

Dosing and Administration

[0129] The bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein is contemplated for use as a medicament. Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, intralesional, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient ^' s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently.

[0130] An "effective dose" refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology. For example, an "effective dose" useful for treating and/or preventing a CD33 ⁺ cancer such as AML may be determined using known methods. Maximum tolerated doses (MTD) and maximum response doses (MRD) can be determined via established animal and human experimental protocols as well as in the examples described herein.

[0131] In some embodiments, a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein is provided in a dose per day, i.e., 'continuous dose', from about 0.01 μg to about 1000 μg, from about 0.05 μg to about 500 μg, from about 0.1 μg to about 500 μg, or about 0.5 μg to about 300 μg. In certain embodiments, a bispecific antibody to CD33 and CD3 described herein is provided in a daily dose or continuous dose of about 0.01 μg, about 0.02 μg, about 0.05 μg, about 0.07 μg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1.5 μg, about 2 μg, about 2.5 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 12 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μ¾ about 45 μg, about 50 μ¾ about 55 μg, about 60 μg, about 65 μ , about 70 μ , about 75 μ , about 80 μg, about 85 μg, about 90 μ , about 95 μg, about 100 μ , about 125 μg, about 150 μg, about 175 μ , about 200 μg, about 250 μ¾ about 275 μg, about 300 μg, about 350 μ , about 400 μg, about 450 μ¾ about 500 μg, about 600 μg, about 700 μ¾ about 800 μg, about 900 μ§, about 1000 μg, about 2000 μg, about 3000 μ§, or more, or any range derivable therein. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 0.5 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 1.5 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 5 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 15 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 50 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 100 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 150 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 200 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 250 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 300 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 500 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 1000 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 2000 μg. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 3000 μg.

[0132] In further embodiments, a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein is provided in a daily dose from about 0.0001 μg/kg to about 10 μg/kg per body weight. In certain instances, a bispecific described herein is provided in a daily dose of about 0.001 μg/kg, about 0.005 μg/kg, about 0.01 μg/kg, about 0.03 μg/kg, about 0.05 μg/kg, about 0.07 μg/kg, about 0.1 μg/kg, about 0.2 μg/kg, about 0.3 μg/kg, about 0.4 μg/kg, about 0.5 μg/kg, about 0.6 μg/kg, about 0.7 μg/kg, about 0.8 μg/kg, about 0.9 μg/kg, about 1 μg/kg, about 2 μg/kg, about 3 μg/kg, about 4 μg/kg, about 5 μg/kg, about 6 μg/kg, about 7 μg/kg, about 8 μg/kg, about 9 μg/kg, or about 10 μg/kg, about 20 μg/kg, or about 30 μg/kg, or more, or any range derivable therein.

[0133] In further embodiments, a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein is provided in a dose per day from about 0.005 μg/m ² to about 500 μg/m ² patient body surface area, from about 0.025 μg/m 2 to about 250 μg/m 2 , from about 0.05 μg/m 2 to about 250 μg/m 2 , or about 0.25 μg/m 2 to about 150 μg/m ² . In certain embodiments, a bispecific antibody described herein is provided in a daily dose of about 0.005 μ§/ιη ² , about 0.01 μg/m ² , about 0.05 μg/m ² , about 0.1 μ§/ιη ² , about

2 2 2 2 2

0.2 μg/m , about 0.3 μg/m , about 0.4 μg/m , about 0.5 μ§/ιη , about 0.6 μg/m , about 0.7

2 2 2 2 2 2 μg/m , about 0.8 μ§/ιη , about 0.9 μ§/ιη , about 1 μg/m , about 1.5 μg/m , about 2 μg/m , about

2 2 2 2 2 2

2.5 μg/m , about 3 μ§/ιη , about 4 μ§/ιη , about 5 μ§/ιη , about 6 μ§/ιη , about 7 μg/m , about 8

2 2 2 2 2 2 μ§/ιη , about 9 μ§/ιη , about 10 μg/m , about 12 μg/m , about 15 μ§/ιη , about 20 μ§/ιη , about

2 2 2 2 2 2

25 μ§/ιη , about 30 μg/m , about 35 μ§/ιη , about 40 μ§/ιη , about 45 μg/m , about 50 μg/m ,

2 2 2 2 2 about 60 μg/m , about 70 μ§/ιη , about 80 μg/m , about 90 μg/m , about 100 μ§/ιη , about 125

2 2 2 2 2 2 μg/m , about 150 μ§/ιη , about 175 μg/m , about 200 μg/m , about 250 μ§/ιη , about 275 μg/m ,

2 2 2 2 2 about 300 μg/m , about 350 μ§/ιη , about 400 μ ^ , about 450 μ§/ιη , about 500 μ§/ιη , about 600 μ^ ² , about 700 μ νη ² , about 800 μ^ ² , about 1000 μ^ ² , about 1200 μ νη ² , or about 1500 μ§/ιη ² , or more, or any range derivable therein.

[0134] The dose per day described herein can be administered by bolus infusion or continuous infusion. Bolus infusion as used herein refers to an infusion which is interrupted earlier than 6 hours, whereas the term "continuous infusion" refers to an infusion which is allowed to proceed permanently for at least 6 hours without interruption. "Continuous infusion" refers to a permanently administered infusion. Accordingly, in the context of the invention, the terms "permanent" and "continuous" are used as synonyms. In some embodiments, a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein is administered as a continuous infusion over 6 h, 7 h, 8 h, 9 h, 10 h, 1 1 h, 12 h, 14 h, 16, 18, 20, 22, or 24 h per day. In some embodiments, a bispecific antibody is administered as a 12 h continuous infusion. In some embodiments, a bispecific antibody is administered as a 24 h continuous infusion. The dose per day described herein can be given once per day or multiple times per day in the form of sub-doses given b.i.d., t.i.d., q.i.d., or the like where the number of sub-doses equal the dose per day. It is further

contemplated that the dose per day described herein and/or its sub-doses can be administered at the same location site on a patient or different sites.

[0135] The dose per day described herein, in additional embodiments, can be administered at a certain infusion rates. In certain embodiments, a bispecific antibody to CD33 and CD3 described herein is infused at a rate of about 0.01 μg/h, about 0.02 μg/h, about 0.05 μg/h, about 0.07 μg/h, about 0.1 μg/h, about 0.2 μg/h, about 0.3 μg/h, about 0.4 μg/h, about 0.5 μg/h, about 0.6 μg/h, about 0.7 μg/h, about 0.8 μg/h, about 0.9 μg/h, about 1 μg/h, about 1.5 μg/h, about 2 μg/h, about 2.5 μg/h, about 3 μg/h, about 4 μg/h, about 5 μg/h, about 6 μg/h, about 7 μg/h, about 8 μg/h, about 9 μg/h, about 10 μg/h, about 12 μg/h, about 15 μg/h, about 20 μg/h, about 25 μg/h, about 30 μg/h, about 35 μg/h, about 40 μg/h, about 45 μg/h, about 50 μg/h, about 55 μg/h, about 60 μ§/η, about 65 μg/h, about 70 μ§/η, about 75 μg/h, about 80 μ§/η, about 85 μg/h, about 90 μ /1ι, about 95 μ§/η, about 100 μ /1ι μ¾ about 125 μ§/η, about 150 μ§/η, about 175 μ§/1ι, about 200 μ§/η, or more, or any range derivable therein. In certain instances, a bispecific antibody described herein is infused at a rate of about 0.25 μg/h. In certain instances, a bispecific antibody described herein is infused at a rate of about 0.5 μg/h. In certain instances, a bispecific antibody described herein is infused at a rate of about 0.75 μ^. In certain instances, a bispecific antibody described herein is infused at a rate of about 1 μ^. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 1.5 μg/h. In certain instances, a bispecific antibody described herein is infused at a rate of about 2 μg/h. In certain instances, a bispecific antibody described herein is infused at a rate of about 2.5 μg/h. In certain instances, a bispecific antibody described herein is infused at a rate of about 3 μ^. In certain instances, a bispecific antibody described herein is infused at a rate of about 4 μ^. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 5 μg. In certain instances, a bispecific antibody described herein is infused at a rate of about 6 μ^. In certain instances, a bispecific antibody described herein is infused at a rate of about 7 μ^. In certain instances, a bispecific antibody described herein is infused at a rate of about 7.5 μg/h. In certain instances, a bispecific antibody described herein is infused at a rate of about 8 μ^. In certain instances, a bispecific antibody described herein is infused at a rate of about 9 μ^. In certain instances, a bispecific antibody described herein is infused at a rate of about 10 μ^. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 15 μ§/1ι. In certain instances, a bispecific antibody described herein is infused at a rate of about 20 μ§/1ι. In certain instances, a bispecific antibody described herein is infused at a rate of about 25 μ§/1ι. In certain instances, a bispecific antibody described herein is infused at a rate of about 30 μ§/1ι. In certain instances, a bispecific antibody described herein is infused at a rate of about 40 μ§/1ι. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 50 μg/h. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 60 μg/h. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 70 μg/h. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 80 μg/h. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 90 μg/h. In certain instances, a bispecific antibody described herein is provided in a daily dose of about 100 μ^.

[0136] It is further contemplated that the infusion rates can be variable to reduce the risk of side effects, such as cytokine release syndrome, or allows the subject to acclimate to the bispecific antibody. In some instances, an infusion rate can begin at a rate for a certain period of time, i.e., a lead-in dose, and then 'stepped-up' to a high rate. In some instances, an infusion rate can include two or more 'stepped-up' higher rates. In some instances, an infusion rate can begin at a certain rate, and then 'stepped-down' to a lower rate. In some instances, an infusion rate can include two or more 'stepped-down' lower rates. In further instances, an infusion rate can include both a 'stepped-up' and 'stepped-down' rates. In certain embodiments, a bispecific antibody to CD33 and CD3 described herein is provided in a lead-in dose of about 0.01 μg, about 0.02 μg, about 0.05 μg, about 0.07 μg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1.5 μg, about 2 μ¾ about 2.5 μg, about 3 μg, about 4 μ¾ about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 12 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about 75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg, about 250 μg, about 275 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, or about 1000 μg. In certain embodiments, the lead-in dose is 5 μg. In certain embodiments, the lead-in dose is 10 μg. In certain embodiments, the lead-in dose is 15 μg. In certain embodiments, the lead-in dose is 20 μg. In certain embodiments, the lead-in dose is 25 μg. In certain embodiments, the lead-in dose is 30 μg. In certain embodiments, the lead-in dose is 35 μg. In certain embodiments, the lead-in dose is 40 μg. In certain embodiments, the lead-in dose is 45 μg. In certain embodiments, the lead-in dose is 50 μg. In certain embodiments, the lead-in dose is 55 μg. In certain embodiments, the lead-in dose is 60 μg. In certain embodiments, the lead-in dose is 65 μg. In certain embodiments, the lead-in dose is 70 μg. In certain embodiments, the lead-in dose is 75 μg. In certain embodiments, the lead-in dose is 80 μg. In certain embodiments, the lead-in dose is 85 μg. In certain embodiments, the lead-in dose is 90 μg. In certain embodiments, the lead-in dose is 95 μg. In certain embodiments, the lead-in dose is 100 μg.

[0137] In further embodiments, administration of a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3 is at doses described herein or at other dose levels determined and contemplated by a medical practitioner. In certain therapeutic applications, a bispecific antibody is administered to a patient already suffering from a cancer, in an amount sufficient to cure or at least partially arrest the symptoms of the cancer. Amounts effective for this use depend on the severity and course of the cancer, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, a dose escalation clinical trial, such as described in the following example. [0138] In some embodiments, a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein is administered continuously or chronically, i.e., daily dosing for a particular amount of time or cycle. In some embodiments, a bispecific antibody described herein is administered at least 1 week (7 days), at least 2 weeks (14 days), at least 3 weeks (21 days), at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, least about 16 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 56 weeks, at least about 60 weeks, at least about 64 weeks, at least about 68 weeks, at least about 72 weeks, at least about 90 weeks, at least about 100 weeks, at least about 110 weeks, and at least about 120 weeks.

[0139] Administration periods can be further defined as treatment cycles where a given number of days or weeks equates one treatment cycle. In some embodiments, one treatment cycle is an administration period of about 1 week, about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks or about 16 weeks. In certain embodiments, one treatment cycle is 14 days or 2 weeks. Treatment cycles for administration of a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein also include, but are not limited to 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 11 cycles, 12 cycles, 13 cycles, 14 cycles, 15 cycles, 16 cycles, 17 cycles, 18 cycles, 19 cycles, 20 cycles, 25 cycles, 30 cycles, 40 cycles, or more.

[0140] Dosages for a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein can, in some embodiments, be the same for each treatment cycle or the dosages may vary per cycle or the dosages may vary within a cycle. In some embodiments, a higher initial dose of a bispecific antibody described herein is administered for the first cycle and a lower dose is administered for all subsequent cycles. In other embodiments, the dosages are decreased gradually per administration for each cycle. In yet other embodiments, the dosages are increased gradually per administration for each cycle.

[0141] In some embodiments, administration for a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein is withheld or given a "drug holiday" in one or more treatment cycles. For example, a bispecific antibody described herein is administered for one treatment cycle and subsequently withheld for the next treatment cycle. In other embodiments, a bispecific antibody described herein is withheld from a subject every other treatment cycle, every two treatment cycles, every three treatment cycles, every four treatment cycles, or every five treatment cycles. [0142] Administration of a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein can, in other embodiments, also be provided in an intermittent dosing schedule. Intermittent dosing schedules include administering a bispecific antibody described herein for a number of days, withholding administration for a certain period of time, subsequently administering the bispecific antibody again with another subsequent withholding. Intermittent dosing can be used to stay within the safety profile as well as maximize efficacy potential of the bispecific antibody. In a non- limiting example, for a 14-day treatment cycle, a bispecific antibody can be administered for days 1-4 and 8-12. Another intermittent dosing schedule is administration of a bispecific antibody every other day. Other intermittent dosing schedules are contemplated that include administration of a bispecific antibody daily for one, two, three, four, five, six, seven, eight, nine or ten days, a withholding period of one, two, three, four, five, six, seven, eight, nine or ten days and an optional daily and withholding period similar or different from the previous

administration within a treatment cycle. In a non-limiting example, a bispecific antibody described herein is administered daily for three days at a certain dose and then subsequently every other day at the same or different dose of a particular treatment period or cycle (See e.g., Figure 24, bottom).

[0143] In further embodiments, administration of a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, described herein is provided once a week, twice a week, three times a week, four times a week, five times a week or six times a week. In certain instances, administration of a bispecific antibody is provided once a week. In certain instances, administration of a bispecific antibody is provided twice a week. In certain instances, administration of a bispecific antibody is provided three times a week.

[0144] In certain embodiments, intermittent dosing is combined with dose titration. Dose titration refers to administration of a bispecific antibody at certain dosage and then increasing the dosage after the intermittent period. The dose can be titrated one, two, three, four, or five times. For example, an every other day intermittent dosing can have a dose titration of 5— » 15 — »100 μg where the 100 μg dose is reached on the fifth and subsequent days (Figure 24, top).

Pharmacokinetics and Pharmacodynamics

[0145] The dosing and administration regimens for a bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, provided herein further provide unique pharmacokinetic profiles not seen with other therapeutic proteins or bispecific antibodies that bind to and engage T cells. [0146] Many therapeutic proteins have rapid clearance and short half-lives. Examples of such proteins which are commercially marketed include interferon alfa-2a (Roferon-A®, half-life: 3.7-8.5 h, MW 19kDa), filgrastim (Neupogen®, half-life: 3.5 h, MW 18 kDa), and imiglucerase (Cerezyme®, half-life: 4-10 min, MW 60 kDa). Many bispecific antibodies also have rapid clearance and short half-life. For example, blinatumomab, an anti-CD19><CD3 bispecific BiTE® antibody (MW 54 kDa) has a half-life of around 1-2 h.

[0147] The rapid clearance and short half-life of certain proteins and antibodies can require more frequent or longer dosing regimens. Current methods that increase half-life include the addition of Fc domains to encourage FcRn recycling of the protein, modifications such as glycosylation or pegylation, or linkage or binding to serum proteins such as albumin. In contrast, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell described herein, e.g., CD33 and CD3, have long half-lives of

approximately one to two days when administered to a human subject. In some embodiments, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell described herein have a half-life of greater than 2 h, about 3 h, about 4 h, about 6 h, about 8, about h, about 10 h, about 12 h, about 14 h, about 16 h, about 18 h, about 20 h, about 22 h, about 24 h, about 30 h, about 36 h, about 40 h, about 44 h, about 48 h, or greater than 48 h. This is remarkable in that the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell described herein, e.g., CD33 and CD3, are not designed with half- life extension methods such as the ones previously described. The long half-lives of the bispecific antibodies described herein present therapeutic benefits such as less frequent dosing, lower dosing and having a prolonged effective concentration during or after a treatment cycle. In some embodiments, the bispecific antibodies described herein have a half-life of about 48 h or 2 days.

[0148] In another aspect, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a consistent increase in drug concentration with a maximum concentration (C _max ) in the blood achieved in about 1 to 28 days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ma x in 1 day, in 2 days, in in 3 days, in 4 days, in 5 days, in 6 days, in 7 days, in 8 days, in 9 days, in 10 days, in 11 days, in 12 days, in 13 days, in 14 days, in 15 days, in 16 days, in 17 days, in 18 days, in 19 days, in 20 days, or in 21 or more days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _max in 1 day. [0149] In another aspect, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a consistent increase in drug concentration with a steady state concentration (C _ss ) in the blood achieved in about 1 to 21 days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ss in 1 day, in 2 days, in in 3 days, in 4 days, in 5 days, in 6 days, in 7 days, in 8 days, in 9 days, in 10 days, in 11 days, in 12 days, in 13 days, in 14 days, in 15 days, in 16 days, in 17 days, in 18 days, in 19 days, in 20 days, or in 21 or more days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ss in 1 day. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ss in 1 to 3 days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ss in 3 to 7 days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ss in 1 to 7 days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ss in 7 to 14 days.

[0150] In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ss in 3 to 14 days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ss in 14 to 21 days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ss in 7 to 21 days.

[0151] In yet another aspect, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a consistent increase in drug concentration with a C _ma x and a steady state concentration (C _ss ) in the blood achieved in about 1 to 28 days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _max and a C _ss 1 day, in 2 days, in in 3 days, in 4 days, in 5 days, in 6 days, in 7 days, in 8 days, in 9 days, in 10 days, in 11 days, in 12 days, in 13 days, in 14 days, in 15 days, in 16 days, in 17 days, in 18 days, in 19 days, in 20 days, or in 21 or more days. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, further provide a C _ma x and a C _ss in 1 day.

[0152] In some embodiments, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, are administered at a dose and frequency to provide a C _ma x of about 10 pg/mL, about 20 pg/mL, about 30 pg/mL, about 40 pg/mL, about 50 pg/mL, about 60 pg/mL, about 70 pg/mL, about 80 pg/mL, about 90 pg/mL, about 100 pg/mL, about 150 pg/mL, about 200 pg/mL, about 250 pg/mL, about 300 pg/mL, about 350 pg/mL, about 400 pg/mL, about 500 pg/mL, about 600 pg/mL, about 00 pg/mL, about 800 pg/mL, about 900 pg/mL, about 1000 pg/mL, about 2000 pg/mL, about 3000 pg/mL, about 4000 pg/mL, about 5000 pg/mL, about 6000 pg/mL, about 7000 pg/mL, about 8000 pg/mL, about 9000 pg/mL, or about 10000 pg/mL.

[0153] In some embodiments, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, are administered at a dose and frequency to provide a C _ss of about 10 pg/mL, about 20 pg/mL, about 30 pg/mL, about 40 pg/mL, about 50 pg/mL, about 60 pg/mL, about 70 pg/mL, about 80 pg/mL, about 90 pg/mL, about 100 pg/mL, about 150 pg/mL, about 200 pg/mL, about 250 pg/mL, about 300 pg/mL, about 350 pg/mL, about 400 pg/mL, about 500 pg/mL, about 600 pg/mL, about 00 pg/mL, about 800 pg/mL, about 900 pg/mL, about 1000 pg/mL, about 2000 pg/mL, about 3000 pg/mL, about 4000 pg/mL, about 5000 pg/mL, about 6000 pg/mL, about 7000 pg/mL, about 8000 pg/mL, about 9000 pg/mL, or about 10000 pg/mL.

[0154] In further embodiments, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, are administered at a dose and frequency to provide an AUC of about 100 day*pg/mL, about 200 day*pg/mL, about 300 day*pg/mL, about 400 day*pg/mL, about 500 day*pg/mL, about 600 day*pg/mL, about 700 day*pg/mL, about 800 day*pg/mL, about 900 day*pg/mL, about 1000 day*pg/mL, about 2000 day*pg/mL, about 3000 day*pg/mL, about 4000 day*pg/mL, about 5000 day*pg/mL, about 6000 day*pg/mL, about 7000 day*pg/mL, about 8000 day*pg/mL, about 9000 day*pg/mL, 10000 day*pg/mL, 20000 day*pg/mL, 30000 day*pg/mL, 40000 day*pg/mL, 50000

day*pg/mL, 60000 day*pg/mL, 70000 day*pg/mL, 80000 day*pg/mL, 90000 day*pg/mL, or 100000 day*pg/mL.

[0155] In another aspect, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, produce desirable pharmacodynamics profiles as compared to existing bispecific antibodies. As discussed previously, a common phenomenon observed in antibody therapy is the occurrence of CRS. For example, the initial administration of blinatumomab provides a rapid increase in cytokine release with elevated levels of IL-10, IL-6, IFN-γ, TNFa, and IL-2 present on day 1. The initial cytokine release is also dose-dependent. This reported observation has led to a stepped dosing regimen for blinatumomab, where initial low dosing is required to reduce initial cytokine release.

[0156] It is further contemplated the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, provides for a controlled or gradual cytokine release in contrast to blinatumomab and other bispecific antibodies. Such cytokines include, but are not limited to, TNFa, IL-2, IL-4, IL-6, IL-8, IL-10, TGFP, and ΠΤΝΓγ. Furthermore, it is contemplated that the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, provides for controlled T cell expansion and/or activation. In some

embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, prevents short-term, burst-like T cell activation. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, promotes long term T cell activation and expansion.

[0157] In another aspect, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, reduce inflammation. Inflammation can be measured via markers such as C-reactive protein (CRP) levels in the blood or serum, or other tests such as erythrocyte sedimentation rate (ESR) or plasma viscosity (PV). A raised or elevated CRP (or ESR or PV) level is an indication of inflammation. Many subjects with cancer such as AML also have elevated CRP levels. In some embodiments, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, reduces CRP levels by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or by about 100%. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, reduces CRP levels to about 90 mg/L, about 80 mg/L, about 70 mg/L, about 60 mg/L, about 50 mg/L, about 40 mg/L, about 30 mg/L, about 20 mg/L, about 10 mg/L, about 5 mg/L, about 2 mg/L, or about 1 mg/L. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, reduces CRP levels to normal levels (e.g., about 5 to about 10 mg/L).

[0158] In certain cancers, the production of blood cells are markedly reduced or suppressed. In another aspect, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, promote, restore, or regenerate hematopoiesis. In another aspect, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, promote, restore, or regenerate myelopoiesis. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase hematopoietic stem cells. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase myeloid cells, which include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes, or platelets. In some embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase lymphoid cells (e.g., T cells, B cells, and K cells).

[0159] In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase absolute neutrophil counts by about 10%, about 120%, about 30%>, about 40%, about 50%, about 60%), about 70%), about 80%>, about 90%, or about 100%, or more. In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase absolute neutrophil counts to about 0.1 x l0 ⁹ /L, about 0.2 l0 ⁹ /L, about 0.3 l0 ⁹ /L, about 0.4 l0 ⁹ /L, about 0.5 x l 0 ⁹ /L, about 0.6 x l0 ⁹ /L, about 0.7 x l0 ⁹ /L, about 0.8 x l0 ⁹ /L, about 0.9 x l0 ⁹ /L, about 1 x l0 ⁹ /L, about 1.5 x l0 ⁹ /L, about 2 x l0 ⁹ /L, about 2.5 x l0 ⁹ /L, about 3 x l0 ⁹ /L, about 3.5 x l0 ⁹ /L, about 4 x l0 ⁹ /L, about 4.5 x l0 ⁹ /L, about 5 x l0 ⁹ /L, about 5.5 x l0 ⁹ /L, about 6 x l0 ⁹ /L, about 6.5 x l0 ⁹ /L, about 7 x l0 ⁹ /L, about 7.5 x 10 ⁹ /L, or about 8 x 10 ⁹ /L, or more. In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T- cell, e.g., CD33 and CD3, increase absolute neutrophil counts to normal levels (e.g., about 2 x l0 ⁹ /L to about 8 x l0 ⁹ /L).

[0160] In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase monocyte counts by about 10%, about 120%, about 30%, about 40%, about 50%, about 60%, about 70%), about 80%, about 90%, or about 100%, or more. In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase monocyte counts to about 0.05 x l0 ⁹ /L, about 0.1 x l0 ⁹ /L, about 0.15 x l0 ⁹ /L, about 0.2 x l 0 ⁹ /L, about 0.2.5 x l0 ⁹ /L, about 0.3 x l0 ⁹ /L, about 0.4 x l0 ⁹ /L, about 0.5 x l0 ⁹ /L, about 0.6 x l0 ⁹ /L, about 0.7 x l0 ⁹ /L, about 0.8 x l0 ⁹ /L, about 0.9 x l0 ⁹ /L, about 1 x l0 ⁹ /L, or more. In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T- cell, e.g., CD33 and CD3, increase absolute neutrophil counts to normal levels (e.g., about 0.2 l0 ⁹ /L to about 1 l07L).

[0161] In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase platelet levels. In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase platelet counts to about 40 x lOTL, about 50 x l07L, about 60 x lOTL, about 70x l07L, about 80 x l07L, about 90 x lOTL, about 100 x l07L, about 125 x l07L, about 150 x lOTL, about 175 x lOTL, about 200 x lOTL, about 225 x l07L, about 250 x lOTL, about 275 x l0 ⁹ /L, about 300 x l0 ⁹ /L, about 325 x l0 ⁹ /L, about 350 x l0 ⁹ /L, about 375 x l0 ⁹ /L, about 400 x l0 ⁹ /L, about 450 x l0 ⁹ /L, about 475 x l0 ⁹ /L, about 500 x l0 ⁹ /L, or more. In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase platelet counts to normal levels (e.g., about 150 x l0 ⁹ /L to about 450 x l0 ⁹ /L).

[0162] In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase erythrocyte, or red blood cell levels. Erythrocyte levels can be determined by hemoglobin concentration. In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase hemoglobin concentration by about 10%, about 120%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more. In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, increase hemoglobin

concentration to about 8 g/dL, about 8.5 g/dL, about 9 g/dL, about 9.5 g/dL, about 10 g/dL, about 10.5 g/dL, about 1 1 g/dL, about 11.5 g/dL, about 12 g/dL, about 12.5 g/dL, about 13 g/dL, about 13.5 g/dL, about 14 g/dL, about 14.5 g/dL, about 15 g/dL, about 15.5 g/dL, about 16 g/dL, about 16.5 g/dL, about 17 g/dL, about 17.5 g/dL, about 18 g/dL, about 18.5 g/dL, about 19 g/dL, about 19.5 g/dL, or about 20 g/dL, or more. In certain embodiments, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T- cell, e.g., CD33 and CD3, increase hemoglobin concentration to normal levels (about 12 to about 18 g/dL).

[0163] In another aspect, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, reduce the level of myeloblasts in subjects having AML. In certain embodiments, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, reduce the level of myeloblasts by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%), about 70%, about 80%, about 90%, or by about 100%. In certain embodiments, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T- cell, e.g., CD33 and CD3, control the level of myeloblasts, wherein the myeloblasts do not increase in their levels.

[0164] In another aspect, the administration of the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, reduce the level of myeloid-derived suppressor cells (MDSCs). In certain embodiments, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, reduce the level of MDSCs by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or by about 100%.

Further Combinations

[0165] In further embodiments, the bispecific antibodies to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, are administered in combination with a standard therapy to cancers, diseases or conditions. Standard therapies include

chemotherapies, immunotherapies, hormone therapies, radiation, surgery, gene therapies and the like. In certain instances, the bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, is administered in combination with a standard AML therapy. In certain instances, the bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, is administered in combination with cytarabine, azacitidine, decitabine, an anthracycline (e.g., daunorubicin, idarubicin, doxorubicin, and the like), amsacrine, fludarabine, clofarabine, cladribine, nelarabine, methotrexate, bortezomib, carfilzomib, melphalan, ibrutinib, thalidomide, lenalidomide, pomalidomide, apremilast, an epipodophyllotoxin (e.g., etoposide, teniposide, and the like), an anthracenedione (e.g., mitoxantrone, pixantrone, losoxantrone, piroxantrone, ametantrone and the like) an anti-CD20 agent (e.g., rituximab, ocrelizumab, ofatumumab, or combinations thereof. In certain instances, the bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, is administered in combination with cytarabine (ara-C). In certain instances, the bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, is administered in combination with azacitidine. In certain instances, the bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, is administered in combination with decitabine. In further instances, the bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, is administered in combination with an anthracycline (e.g., daunorubicin, idarubicin, doxorubicin, and the like). In other instances, the bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, is administered in combination with a checkpoint inihibitor (e.g., PD-1 inhibitor, CTLA-4 inhibitor, and the like). In yet other instances, the bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, is administered in combination with an

epipodophyllotoxin (e.g., etoposide, teniposide, and the like). In yet other instances, the bispecific antibody to an antigen expressed on a target cell and an antigen expressed on a T-cell, e.g., CD33 and CD3, is administered in combination with an anthracenedione (e.g.,

mitoxantrone, pixantrone, losoxantrone, piroxantrone, ametantrone and the like).

[0166] The examples below further illustrate the described embodiments without limiting the scope of the invention.

EXAMPLE 1

Cloning of DNA expression constructs encoding single-chain Fv antibodies

[0167] For bacterial expression of anti-CD33 single-chain Fv (scFv) antibodies m E. coli, DNA coding sequences of all molecules were cloned into a bacterial expression vector. All expression constructs were designed to contain coding sequences for an N-terminal signal peptide and C- terminal hexa-histidine (6xHis)-tag to facilitate antibody secretion into the periplasm and purification, respectively. The amino acid sequences of the VL and VH-domains from all anti-

CD33 scFv clones are shown in Table 3 and Table 4.

Expression of recombinant anti-CD33 single-chain Fv antibodies in E. coli

[0168] Recombinant scFv antibodies were expressed as soluble secreted proteins in the E. coli periplasm. In a first step a small medium culture supplemented with ampicillin was inoculated with transformed bacteria and incubated for 16 h at 28°C. Subsequently, optical density was adjusted by adding a second medium supplemented with ampicillin and incubated once more at

28°C until an optical density in the range of 0.6 - 0.8 at 600 nm was reached. Protein expression was induced through addition of 50 μΜ IPTG and incubation of cultures at 21-28°C and 200 rpm for up to 16 h. Following incubation, cells were pelleted (30 min, 4°C, 7500 rpm) and stored at -20°C until further processing.

Purification of anti-CD33 single-chain Fv antibodies

[0169] Recombinant scFv were extracted from E. coli periplasm following centrifugation of bacterial cell cultures by resuspending cell pellets in buffer and incubation for 30 min at room temperature with gentle agitation. Cells were pelleted and supematants containing recombinant proteins were kept. The procedure was repeated once more before supematants were pooled and homogenized by ultrasonication. Homogenates were diluted, supplemented with low concentrations of imidazole and loaded onto a prepacked immobilized metal affinity

chromatography (EVIAC) column (GE Healthcare). The column was washed until baseline was reached and bound protein was then eluted with an imidazole buffer. Antibody containing fractions were pooled and subsequently purified by size-exclusion chromatography (SEC). Finally, protein eluates were concentrated by ultrafiltration and dialysed against storage buffer. Subsequent to low pH treatment (incubation at pH 3.0 for 20-24 h at 37°C), samples were neutralized using Tris. Purified proteins were stored as aliquots at -80°C until use.

EXAMPLE 2

Cloning of DNA expression constructs encoding 2x2 T-cell Engagers

[0170] For expression of bispecific 2x2 T-cell engagers in CHO cells, coding sequences of all molecules were cloned into a mammalian expression vector system. The anti-CD33 scFv domains of Example 1 were used to construct CD33/CD3 2x2 T-cell engagers in combination with an anti-CD3 scFv domain, with domains organized as shown in Table 7 and Figure 3. In brief, gene sequences encoding anti-CD33 VH and VL domains separated by a peptide linker (VH-linker-VL or VL-linker-VH) were synthesized and subcloned. The resulting construct was digested to generate separate VH and VL coding sequences utilizing a Bam HI restriction site located within the linker sequence. These VH and VL fragments were then ligated with a DNA fragment encoding VH and VL domains of anti-CD3 (VH-linker-VL or VL-linker-VH) to yield the final construct. Domain order variants 1 to 3 of CD33/CD3 2x2 T-cell engagers are shown in Figure 3. All expression constructs were designed to contain coding sequences for an N- terminal signal peptide and a C-terminal hexahistidine (6xHis)-tag to facilitate antibody secretion and purification, respectively.

Expression of 2x2 T-cell engagers in stably transfected CHO cells

[0171] A CHO cell expression system (Flp-In®, Life Technologies), a derivative of CHO-K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968;60(4): 1275-81), was used. Adherent cells were subcultured according to standard cell culture protocols provided by Life Technologies.

[0172] For adaption to growth in suspension, cells were detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells were cryopreserved in medium with 10% DMSO.

[0173] Recombinant CHO cell lines stably expressing secreted 2x2 T-cell engagers were generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities were measured twice a week, and cells were centrifuged and resuspended in fresh selection medium at a maximal density of 0. lxlO ⁶ viable cells/mL. Cell pools stably expressing 2x2 T-cell engagers were recovered after 2-3 weeks of selection at which point cells were transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins was confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools were cryopreserved in DMSO containing medium.

[0174] 2x2 T-cell engagers were produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant. Cell culture supernatants were harvested after 10 days at culture viabilities of typically >75%. Samples were collected from the production cultures every other day and cell density and viability were assessed. On day of harvest, cell culture supernatants were cleared by centrifugation and vacuum filtration before further use.

[0175] Protein expression titers and product integrity in cell culture supernatants were analyzed by SDS-PAGE.

Purification of 2x2 T-cell engagers

[0176] 2x2 T-cell engagers were purified from CHO cell culture supernatants in a two-step procedure. The His6-tagged constructs were subjected to Ni-NTA Superflow chromatography in a first step followed by preparative size exclusion chromatography (SEC) on Superdex 200 in a second step. Eluted 2x2 T-cell engagers were characterized with regards to their homodimer (2x2 T-cell engagers) content and pooled if the homodimer content was 90% or higher. Finally, pooled samples were buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL. Purity and homogeneity (typically >90%) of final samples were assessed by SDS PAGE under reducing and non-reducing conditions, followed by

immunoblotting using an anti-His-Tag antibody as well as by analytical SEC, respectively. Purified proteins were stored at aliquots at -80°C until use.

[0177] Polypeptides of CD33/CD3 2x2 T-cell engagers are shown in Table 7 and Figure 3. Each 2x2 T-cell engager consists of two identical polypeptides (Figure 1). Both outer linkers LI and L3 were comprised of six amino acids GGSGGS (SEQ ID NO:95), whereas the central peptide linker 2 varied in length (4-6 amino acids) with the sequences GGSG (SEQ ID NO: 96), GGSGG (SEQ ID NO: 97), or GGSGGS (SEQ ID NO: 95), respectively.

[0178] Using a series of anti-CD33 variable domains and anti-CD3 variable domains a large number of 2x2 T-cell engager molecules was generated that could be stably produced in transfected cell lines and that maintained stability at body temperature as well as after repeated freeze/thaw cycles. To facilitate further development and preclinical toxicology studies, emphasis was placed on the selection of 2x2 T-cell engager molecules that showed binding to both human and cynomolgus monkey CD33. Examples of complete amino acid sequences are shown for the single-chain of 2 2 T-cell engagers 12 (SEQ ID NO: 109), 14 (SEQ ID NO: 111) and 16 (SEQ ID NO: 113) in Figures 10L, ION and 10P, respectively. In this example the order of the variable domains and their linkers for the structures is: VL (CD3)-L1-VH (CD33)-L2-VL (CD33)-L3-VH (CD3). The C-terminal hexa-histidine (6xHis)-tag is cleaved during purification. Complete amino acid sequences for the above mentioned 2x2 T-cell engagers, after removal of the hexa-histidine tag, are tandom diabody 12 (SEQ ID NO: 134), 2x2 T-cell engager 14 (SEQ ID NO: 136) and 2x2 T-cell engager 16 (SEQ ID NO: 138), as shown in Figures 11L, UN and 1 IP, respectively.

EXAMPLE 3

Determination of antibody affinity by flow cytometry

[0179] Cells were incubated with 100 of serial dilutions of CD33/CD3 2x2 T-cell engagers. After washing three times with FACS buffer the cells were incubated with 0.1 mL of 10 μg/mL mouse monoclonal anti-His antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells were incubated with 0.1 mL of 15 μg/mL FITC-conjugated goat anti- mouse IgG antibodies under the same conditions as before. As a control, cells were incubated with the anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG antibodies without anti-CD33 2x2 T-cell engagers. The cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg/mL propidium iodide (PI) in order to exclude dead cells. The fluorescence of lxlO ⁴ living cells was measured using a Beckman-Coulter FC500 MPL flow cytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using the Incyte software (Merck Millipore, Schwalbach, Germany). Mean fluorescence intensities of the cell samples were calculated using CXP software

(Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values of the cells stained with the secondary and tertiary reagents alone the values were used for calculation of the K _D values with the equation for one-site binding (hyperbola) of the GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla California USA).

[0180] The 2x2 T-cell engagers were tested for their binding affinities to human CD3 ⁺ and CD33 ⁺ cells and cynomolgus CD3 ⁺ and CD33 ⁺ cells. Exemplary binding data for selected 2x2 T-cell engagers are summarized in Table 8:

Table 8: CD3 and CD33 binding characteristics of CD33/CD3 2x2 T-cell engagers: 01 94.2 0.6 0.9 7.1 0.7 1.9

02 69.8 0.2 0.3 0.9 1.1 0.5

03 81.9 1.1 1.8 8.9 0.6 3.6

04 79.3 0.5 0.5 1.7 1.1 1.8

05 69.5 1.0 1.2 6.2 0.8 2.7

06 86.3 0.4 0.5 1.6 0.8 1.6

07 49.7 13.7 47.9 47.1 45.8 17.8

08 2.4 0.3 0.5 1.8 0.6 1.8

09 2.4 0.5 0.3 2.2 1.0 6.8

10 1.9 0.5 1.0 1.7 0.8 7.0

11 2.6 0.3 0.5 0.6 1.2 5.9

12 1.5 0.3 0.9 0.5 1.7 1.3

13 55.7 0.2 0.3 0.5 1.6 1.1

14 2.1 0.3 0.3 1.2 1.0 1.6

15 1.3 0.4 0.3 0.9 1.1 1.8

16 2.1 0.3 0.2 0.3 1.4 1.5

17 3.3 5.0 52.5 24.4 1.9 18.4

18 1.9 3.4 16.3 15.1 3.1 6.3

19 6.3 2.8 3.6 5.4 37.3 5.7

20 143.8 4.1 7.0 7.2 33.8 10.0

21 2.1 9.7 25.1 80.2 0.9 7.6

22 4.1 0.7 2.0 8.6 0.6 3.2

23 97.2 0.4 1.0 5,1 1.9 2.8

24 2.3 5.6 12.4 39.5 1.8 9.6

[0181] #K _D ratio cyno CD33 / human CD33 was calculated based on the K _D values measured on CHO cells expressing cynomolgus CD33 and human CD33, respectively. ^J K _D ratio hu CD3 / hu CD33 was calculated based on the K _D values measured on Jurkat cells (hu CD3) and the mean K _D of three human CD33 ⁺ tumor cell lines (HL-60, KG-1, U937).

[0182] CD3 binding affinity and crossreactivity were evaluated in titration and flow cytometric experiments on CD3 ⁺ Jurkat cells (provided by Dr. Moldenhauer, DKFZ Heidelberg; human acute T-cell leukemia) and the cynomolgus CD3 ⁺ HSC-F cell line (JCRB, cat. :JCRBl 164). CD33 binding and crossreactivity were assessed on the human CD33 ⁺ tumor cell lines: HL-60 (DSMZ, cat. :ACC 3, human B cell precursor leukemia), U-937 (DSMZ, cat.: ACC5; human histiocytic lymphoma), and KG-1 (DSMZ, cat.:ACC14; acute myeloid leukemia). The K _D ratio of crossreactivity was calculated using the K _D values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens. [0183] The 2x2 T-cell engagers exhibited a relatively high affinity to human CD33 ⁺ on most of the tested tumor cell lines below 1 nM. Affinities to human CD3 were determined to be equal or less than 2 nM.

EXAMPLE 4

Cytotoxicity Assay

[0184] For the cytoxicity assay target cells cultured under standard conditions were harvested, washed and resuspended in diluent C, provided in the PKH67 Green Fluorescent Cell Linker Mini Kit, to a density of 2xl0 ⁷ cells/mL. The cell suspension was then mixed with an equal volume of a double concentrated PKH67-labeling solution and incubated for 2-5 min at RT. The staining reaction was performed by adding an equal volume of FCS and incubating for 1 min. After washing the labeled target cells with complete RPMI medium, cells were counted and resuspended to a density of 2xl0 ⁵ cells/mL in complete RPMI medium. 2xl0 ⁴ target cells were then seeded together with enriched human T-cells as effector cells at an E:T ratio of 5: 1, in the presence of increasing concentrations of the indicated 2x2 T-cell engagers in individual wells of a microtiter plate, in a total volume of 200 μΕΛνεΙΙ. Spontaneous cell death and killing of targets by T-cells in the absence of antibodies were determined for at least three replicates on each plate. After centrifugation the assay plates were incubated for the indicated periods of time at 37°C in a humidified atmosphere with 5% C0 ₂ . After incubation, cultures were washed once with FACS buffer and then resuspended in 150 μΐ. FACS buffer supplemented with 2 μg/mL PI. The absolute amount of living target cells was measured by a positive green staining with PKH67 and negative staining for PI using a Beckman-Coulter FC500 MPL flow cytometer (Beckman-Coulter) or a Millipore Guava EasyCyte flow cytometer (Merck Millipore). Based on the measured remaining living target cells, the percentage of specific cell lysis was calculated according to the following formula: [1 -(number of living targetS _(Sam pie ₎ /number of living

targetS( _S pontaneous))] x 100%. Sigmoidal dose response curves and EC50 values were calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration were used to calculate sigmoidal dose-response curves by 4 parameter logistic fit analysis using the Prism software.

[0185] EC ₅₀ values were determined in 20-24 hour assay on CD33 ⁺ U-937 (DSMZ, cat.: ACC5; human histiocytic lymphoma) target cells with enriched human T-cells as effector cells at a ratio of 5 : 1. Some 2x2 T-cell engagers were also tested in cytotoxicity assays on CD33 ⁺ KG-1 (DSMZ, cat. :ACC14; acute myeloid leukemia) and HL-60 target cells. Specifically, HL-60 cells were chosen as a model of an AML with relatively high cell surface expression of CD33 (arbitrary MFI [mean±SEM]: 3,133±215; n=3), and KG-la was chosen as a model of an AML with very limited CD33 expression (arbitrary MFI: 277±11; n=3). Exemplary cytotoxicity data for selected 2x2 T-cell engagers are summarized in Table 9. Additional cytotoxicity data for HL-60 cell lines is found on Table 8, last column.

Table 9: In vitro potency of CD33/CD3 2x2 T-cell engagers on different CD33 ⁺ tumor cell lines:

[0186] EC ₅₀ values were determined in FACS-based cytotoxicity assays with primary human T- cells as effector cells at an E:T ratio of 5: 1 on the indicated target cell lines incubated for 20-24 hours Each 2x2 T-cell engager was tested on each tumor cell line in at least two independent experiments. Mean values are presented.

EXAMPLE 5

Further cytotoxicity screening experiments in human CD33+ AML cell lines at 48 hours

[0187] As described above significant cytotoxicity was detected as early as 24 hours, however higher levels of toxicity can be detected at 48 hours. For the subsequent assays a 48-hour time point was chosen. The impact of T-cell selection on 2x2 T-cell engager-induced cytotoxicity was tested. To accomplish this, unstimulated PBMCs from a healthy volunteer donor were obtained, and CD3 ⁺ cells were isolated both by simple "positive enrichment" via use of CD3 microbeads as well as by more complex "negative selection" via a microbead cocktail of antibodies against CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a. As depicted in Figure 4, 2x2 T-cell engager-induced cytotoxicity was greater with negatively selected healthy donor T-cells than positively selected T-cells. However, the relative cytotoxic activities of individual 2x2 T-cell engagers were unaffected by the method of T-cell selection. Therefore the subsequent assays were performed with positively enriched healthy donor T-cells.

[0188] Unstimulated mononuclear cells were collected from healthy adult volunteers via leukapheresis by the Fred Hutchinson Cancer Research Center (FHCRC) Hematopoietic Cell Processing Core (Core Center of Excellence) under research protocols approved by the FHCRC Institutional Review Board. T-cells were enriched through magnetic cell sorting either via CD3 Microbeads ("positive enrichment") or via Pan T-Cell Isolation Kit ("negative selection"; both from Miltenyi Biotec, Auburn, CA), and then frozen in aliquots and stored in liquid nitrogen. Thawed cell aliquots were labeled with 3 μΜ CellVue Burgundy (eBioscience, San Diego, CA) according to the manufacturer's instructions. Purified PBMCs were cultured in the presence of various concentrations of 2x2 T-cell engager molecules.

[0189] For the quantification of drug-induced cytotoxicity cells were incubated at 37°C (in 5% C0 ₂ and air), as in Example 4, at different E:T cell ratios. After 24-72 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined using a LSRII cytometer (BD Biosciences) and analyzed with FlowJo. AML cells were identified by forward/side scatter properties and, in experiments where healthy donor T-cells were added, negativity for CellVue Burgundy dye (Figure 5). Drug-induced specific cytotoxicity is presented as: % cytotoxicity = 100 x (1 - live target cells _t rea _t e _d /live target cells _CO ntroi)- Results from cytotoxicity assays are presented as mean values ± standard error of the mean (SEM). Spearman nonparametric correlation was used to compute correlations between continuous sample characteristics. All -values are two-sided. Statistical analyses were performed using GraphPad Prism software.

[0190] In the absence of healthy donor T-cells, neither of the CD33/CD 2x2 T-cell engagers exerted any noticeable cytotoxic effect on AML cell lines in the absence of T-cells, confirming the absolute requirement for T-cells for their cytotoxic effects (data not shown). In the presence of T-cells, the extent of 2x2 T-cell engager-induced specific cytotoxicity was dependent on the concentration of the 2x2 T-cell engager as well as the E:T cell ratio. Direct head-to-head comparisons between the CD33/CD3-directed 2x2 T-cell engager molecules and one control 2x2 T-cell engager (00) indicated considerable differences in antibody-induced cytotoxicity in both HL-60 cells (Figure 6A/B and Table 10) and KG- la cells (Figure 6C/D and Table 10), with results being highly reproducible in repeat experiments. Overall, the degree of 2x2 T-cell engager-induced cytotoxicity correlated with the binding affinity for CD3 on primary human T- cells (for cytotoxicity in KG-la cells at 25 pM (approx. 2.5 ng/mL) and E:T=5: 1 : r=-0.542, p=0.009; for cytotoxicity in HL-60 cells at 25 pM and E:T=5: 1 : r=-0.391, p=0.07). The 2x2 T- cell engagers 12, 14, 16 were highly cytotoxic for both HL-60 and KG-la cells.

TABLE 10: CD25 and CD69 induction and cytotoxicity at 48 h of CD33/CD3 2x2 T-cell engagers

Cytotoxicit

2x2 T- CD3 K _D CD33 K _D T cell y

CD25 CD69 Cytotoxicity

cell (nM) (nM) Proliferation KG-la

Induction Induction HL-60 cells

engager Human HL-60 in PBMC cells 1 ECso (pM) ² EC ₅₀ (pM) ² (% + SEM) ⁴

T-cells cells EC ₅₀ (pM) ³ (% +

SEM) ⁴

15 1.3 0.4 6 7 7 82.9 + 3.7 80.2 + 1.9 12 1.5 0.3 6 3 2 84.7 + 2.3 85.6+1.6

10 1.9 0.5 10 6 6 48.0 + 2.4 78.6 + 2.3

14 2.1 0.3 10 7 6 86.0 + 0.4 69.8 + 5.7 1 2.1 9.7 ND 225 500 12.4 + 1.0 0.0+0.2 4 2.3 5.6 ND 57 264 24.5 + 1.9 1.1+0.2 9 2.4 0.5 11 7 9 43.2+15.8 74.6 + 3.2

11 2.6 0.3 11 5 6 52.7 + 8.1 84.7+1.4

17 3.3 5.0 30 114 30 4.2 + 0.2 0.7+0.4 2 4.1 0.7 10 4 7 74.2 + 7.4 44.4 + 5.3

16 5.1 0.3 1 2 3 86.0 + 1.4 81.3 ± 1.5

19 6.3 2.8 9 5 6 79.4 + 3.5 83.8 + 2.9 7 49.7 13.7 134 65 50 6.3+3.3 2.1+0.7

13 55.7 0.2 30 22 23 70.4 + 2.5 1.3+0.4 5 69.5 1 116 74 74 23.8 + 6.9 0.3+0.3 2 69.8 0.2 42 27 4 80.9 + 3.6 4.6+2.1 4 79.3 0.5 94 62 44 24.1+4.0 0.7+0.8 3 81.9 1.1 117 87 63 13.1+3.6 0.0+0.5 6 86.3 0.4 39 21 48 45.7 + 6.4 1.4+0.2 1 94.2 0.6 92 91 89 8.0 + 1.6 0.4+0.4 3 97.2 0.4 41 17 37 73.7 + 2.6 1.5+0.3 0 143.8 4.1 98 75 38 31.2 + 3.9 1.1+0.3

^: 2x2 T-cell engagers are listed in order of increasing CD3 affinity.

² CD25 and CD69 induction was measured after 24 hours in unfractionated PBMC cultures.

³ T cell proliferation induced by CD33/CD32^2 T-cell engagers in unfractionated PBMC with CD33+ cells present.

⁴ Cytotoxicity (%) after 48 hours of DAPI+ cells at a 2x2 T-cell engager concentration of 25 pM in the presence of healthy donor T-cells at an E:T cell ratio of 5: 1 from 3 independent experiments performed in duplicate wells.

ND: no CD25 activation detectable

EXAMPLE 6

Further characterization of 2x2 T-cell engagers in primary human AML specimens

[0191] For a comprehensive characterization of the cytotoxic properties of these candidates, specimens from AML patients were obtained for the studies from a FHCRC specimen repository.

[0192] Frozen aliquots of Ficoll -isolated mononuclear cells from pretreatment ("diagnostic") peripheral blood or bone marrow specimens from adult patients with AML were obtained from repositories at FHCRC. We used the 2008 WHO criteria to define AML (Vardiman et al.; Blood. 2009; 114(5):937-951) and the refined United Kingdom Medical Research Council (MRC) criteria to assign cytogenetic risk (Grimwalde et al.; Blood. 2010; 116(3):354-365). Patients provided written informed consent for the collection and use of their biospecimens for research purposes under protocols approved by the FHCRC Institutional Review Board.

Clinical data were de-identified in compliance with Health Insurance Portability and

Accountability Act regulations. After thawing, cells were stained with directly labeled antibodies recognizing CD33 (clone P67.6; PE-Cy7-conjugated), CD3 (clone SK7; PerCP- conjugated), CD34 (clone 8G12; APC-conjugated; all from BD Biosciences, San Jose, CA), and CD45 (clone HI30; APC-eFluor®780-conjugated; eBioscience). To identify nonviable cells, samples were stained with 4',6-diamidino-2-phenylindole (DAPI). At least 10,000 events were acquired on a Canto II flow cytometer (BD Biosciences), and DAPI- cells analyzed using FlowJo (Tree Star, Ashland, OR).

[0193] After thawing, specimens had >58% AML blasts, as determined by flow cytometry based on CD45/side-scatter properties. Specimens had >50% viable cells immediately after thawing and >50% viable cells after 48 hours in cytokine-containing liquid culture (Figure 7). Median age of the patients was 58.1 (range: 23.9-76.2) years; cytogenetic disease risk was favorable in 2, intermediate in 18, and adverse in 7. Information on the mutation status of NPM1, FLT3, and CEBPA was incomplete; however, one sample was known to be CEBPA ^{iouUe' mutant} , and another sample was NPMl ^pos /FL T3-ITD ^aeg . The median percentage of myeloid blasts and CD3 ⁺ T-cells in the studied specimens was 86.1% (range: 58.4-97.0%) and 2.0% (range: 0- 11.9%), respectively, and the median sample viability after 48 hours in culture was 80.1% (range: 53.6-93.6%)). Fifteen of the patients had newly diagnosed AML, whereas 12 either had relapsed (n=7) or refractory (n=5) disease at the time of specimen collection. As summarized in Table 11, basic characteristics of the specimens from patients with newly diagnosed AML were similar to those with relapsed/refractory disease with regard to CD33 expression on myeloid blasts, amount of autologous T-cells, proportion of myeloid blasts, and culture viability.

[0194] The addition of 2x2 T-cell engager molecules to AML specimen cultures resulted in modest, dose-dependent cytotoxicity (Figure 8A), demonstrating that autologous T-cells, contained in the specimens from patients with active AML, can be engaged to lyse leukemic cells. In the presence of healthy donor T-cells, the cytotoxic activity of individual 2x2 T-cell engagers was strictly dependent on the drug dose and the E:T cell ratio (Figure 8B/C).

However, high activity of 2x2 T-cell engagers was observed even in some specimens with very low CD33 expression on AML blasts. Among the 2x2 T-cell engager molecules, 12 appeared to be the most active, since it had the highest cytotoxicity at low concentrations (2.5 pM (approx. 250 ng/mL) and, to a less pronounced degree, also 10 pM (approx. 1 ng/mL)) at both E:T=1 :3 and E:T=l : l .

[0195] The CD33/CD3 2x2 T-cell engagers have been screened in representative AML patient blood samples, which varied in terms of patient sex, age, disease stage (newly diagnosed, relapsed, refractory), degree of CD33 expression and cytogenic risk (Table 1 1). Remarkably, a number of examined 2x2 T-cell engagers (e.g., 02, 08, 09, 1 1, 12, 14, 16, 19, 22 and 23) were highly active in nearly all patient samples across the disease spectrum as shown in Figure 17. Moreover, the extent and scope of activity is similar in all stages of AML, including newly- diagnosed, relapsed and refractory patients.

TABLE 11. Characteristics of primary AML specimens

EXAMPLE 7

Potency and efficacy of CD33/CD3 2x2 T-cell engager 12 and 2x2 T-cell engager 16 on different CD33 ⁺ cell lines of various origin expressing different levels of CD33

[0196] In order to assess whether potency and efficacy of CD33/CD3 2x2 T-cell engagers depend on the CD33 density on the target cells, various human CD33 ⁺ tumor cell lines and CHO cells expressing recombinant human CD33 were tested for their CD33 expression levels using the QIFIKIT quantification kit and anti-CD33 mAb WM53. The results in Table 12 show that the CD33 densities on the tumor cell lines were in the range between -1300 SABC

(standardized antibody binding capacity) and -46000 SABC. The expression on CHO-CD33 cells was -197000 SABC, substantially higher than on the tumor cell lines. All tested CD33 ⁺ cell lines were used as target cells in at least 3 independent FACS-based cytotoxicity assays with human T-cells as effector cells at an effector-to-target ratio of 5: 1 in the presence of serial dilutions of CD33/CD3 2x2 T-cell engager 12 and 2x2 T-cell engager 16. In each assay EC ₅₀ and 2x2 T-cell engager-mediated lysis values were calculated by non-linear regression. The results demonstrate that neither the potency (EC ₅₀ values) nor the efficacy (% lysis) of 12 and 16 correlates with the CD33 density on the surface of target cells.

[0197] Noteworthy, at least 12 and 16 exhibit their cytotoxic activity also against cells like SEM with very low CD33 densities of below 1500 SABC.

Table 12: CD33 target cell surface expression and cytotoxic potency of CD33/CD3 2x2 T- cell engager 12 and 2x2 T-cell engager 16:

[0198] The standardized antibody binding capacity (SABC) on CD33 ⁺ cell lines was determined using QIFIKIT and the anti-CD33 mAb WM53. EC ₅₀ values for 2 2 T-cell engager 12 and 2x2 T-cell engager 16 redirected target cell lysis were determined in FACS-based cytotoxicity assays with human primary T-cells as effector cells at E:T ratios of 5: 1 and 20-24 h incubation; assays with CD33 -expressing CHO cells were incubated for 40-48 h. Mean and SD of at least 3 independent assays are shown.

EXAMPLE 8

2x2 T-cell engager-activation of T-cells and in vitro killing of AML cells

[0199] 2x2 T-cell engagers were incubated with purified human T cells and a VPD -450 -labeled human CD33 ⁺ leukemia cell line, KG-1, or the CD33 ^" human ALL cell line, G2 (E:T 5: 1). Flow cytometry was used to evaluate target cell lysis by 2x2 T-cell engagers (10 ^"15 to 10 ^"8 M; 24h,

37°C).

[0200] Incubation of 2x2 T-cell engagers 12, 16, and 19 with human T cells efficiently lysed KG-1 cells (IC50 -0.01, 0.5, and 5 pM respectively). Up to 40% of T cells were activated (CD25+) rising with cytotoxic activity. A control 2x2 T-cell engager with an irrelevant target, 00 (>10 ^"v M), did not result in significant killing of KG-1 in vitro. Separately, 16 induced lysis of KG-1 cells (IC50 = 5 x 10 ^"12 M) while 1 x 10 ^"8 M had no effect on CD33- G2 cells. The results indicate thats T cells become activated and potently lyse tumor cells when targeted to CD33+ leukemic cells (KG-1) and primary CD33+ AML blasts by CD33/CD3 2 2 T-cell

EXAMPLE 9

Epitope mapping

[0201] 2x2 T-cell engagers containing different CD33 binding moieties were subjected to epitope mapping using CLIPS Technology (Pepscan) in order to identify CD33-binding epitopes.

[0202] CLIPS Technology facilitates the structuring of peptides into single loops, double-loops, triple loops, sheet-like folds, helix-like folds, and combinations thereof, offering the possibility to map discontinuous epitopes of the target molecule.

[0203] An array of more than 7000 independent peptides was synthesized and the binding of each antibody to the peptides was tested in an ELISA.

[0204] The 2x2 T-cell engagers 12, 14, 16 and 22 bind to the stretch ₆₂ DQEVQEETQ ₇ o (SEQ ID NO:94) in the first Ig like domain of human CD33. The respective amino acid stretches are shown underlined and in bold in Figures 10 and 11. It is contemplated that 2x2 T-cell engagers 01, 02, 04, 06, 08, 09, 13 and 23 also bind to this epitope as these 2x2 T-cell engagers share the same CD33 binding domains (SEQ ID NOs:2 and 12, 3 and 13, 5 and 15, 9 and 19) as 2x2 T- cell engagers 12, 14 16 and 12.

EXAMPLE 10

Dose-response in a prophylactic in vivo tumor model

[0205] 2x2 T-cell engagers 12 and 16 are compared at different dose levels in a prophylactic HL-60 tumor xenograft model in NOD/scid mice reconstituted with human T-cells. In order to achieve a dose-response three dose levels at 10, 1 and O. ^g (0.5, 0.05, and 0.005 mg/kg) were selected.

[0206] Eight experimental groups of immunodeficient NOD/scid mice were xenotransplanted by subcutaneous injection with a suspension of 4xl0 ⁶ HL-60 cells. Prior to injection cells were mixed with 3xl0 ⁶ T-cells isolated from buffy coats (healthy donors) employing negative selection. To account for potential donor variability of the T-cells, each of the experimental groups was subdivided into three cohorts each receiving T-cells of one individual donor only. All animals of the experimental groups transplanted with tumor cells and T-cells received an intravenous bolus on days 0, 1, 2, 3 and 4 (qdxd5) of either vehicle (control) or 16 or 12 at three different dose levels as indicated (0.1 μg, 1 μg, and 10 μg). One group without effector cells and vehicle treatment served as an additional control. Table 13 summarizes group allocation and dosing schedule.

Table 13

[0207] Treatment groups for the in vivo dose-response study in a HL-60 xenograft model. All animals in the control groups reliably developed a tumor and exhibited homogeneous tumor growth. The presence of T-cells had no influence on tumor development. No difference in HL- 60 growth was observed in the presence or absence of T-cells in the vehicle-treated control groups.

[0208] Treatment with both test items revealed a clear dose-dependent anti-tumor effect (Figure 12). No substantial difference was found between the two 2x2 T-cell engagers. Plotting of mean tumor volumes in Figure 12 was restricted to day 29 when most of the treatment groups were complete. The study was continued until day 45 and animals were observed for tumor-free survival. In the groups treated with 10 or 1 μg of 16, 6 of 9 animals were tumor-free at the end of the observation period and 5 of 9 animals receiving 10 μg of 12 were tumor-free on day 45. One animal remained tumor-free when treated with 1 μg of 12.

[0209] All animals in the control groups reliably developed a tumor and exhibited homogeneous tumor growth. Treatment with either of the 2x2 T-cell engagers revealed a dose-dependent antitumor effect and no substantial difference was found between the two 2x2 T-cell engagers until day 29. [0210] Detectable differences were observed only after prolonged observation (day 45), at which time the low dose and control groups had already been terminated due to the growth of large tumors. Groups treated with 16 had more tumor-free animals.

EXAMPLE 11

Established tumor model

[0211] A xenograft model in NOD/scid mice with pre-established HL-60 tumors employing 16 was developed to demonstrate proof of concept.

[0212] In brief, female immune-deficient NOD/scid mice were sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4xl0 ⁶ HL-60 cells. On day 9 the animals received a single bolus injection of anti-asialo GM1 rabbit antibody (Wako, Neuss, Germany) to deplete murine natural killer (NK) cells. On day 10, when the tumor reached a volume between 50-150 mm ³ (mean 73 ± 11 mm ³ ) animals were allocated to 3 treatment groups. Groups 2 and 3 (8 animals each) were intraperitoneally injected with 1.5xl0 ⁷ activated human T-cells. Prior to injection T- cells were isolated from buffy coats (healthy donors) employing negative selection. T-cells were expanded and activated with the T-Cell Activation / Expansion Kit according to the manufacturer's specification (Miltenyi Biotech). In order to address potential donor variability Groups 2 and 3 were subdivided into two cohorts each receiving expanded and activated T-cells from an individual donor. Each cohort received T-cells from one individual T-cell donor only.

Table 14: Treatment groups for the established HL-60 xenograft model.

[0213] Starting on day 13 animals in Group 3 displayed a mean tumor volume of 105 mm ³ and were treated with a total of 9 intravenous doses of 50 μg 2x2 T-cell engager 16 (qdx9d). Table

14 illustrates group allocation and dosing schedule. Groups 1 and 2 were only treated with the vehicle. Body weight and tumor volume were determined until day 27.

[0214] All animals reliably developed a tumor, which was palpable on day 6. The mean tumor volume of vehicle-treated Group 1 and 2 (HL-60) animals continually increased until study termination on day 27 (Figure 13). In Group 2 animals that received primary activated human T-cells in addition to HL-60 tumor cells, the mean tumor volume increased faster compared to Group 1 (HL-60 only).

[0215] Repeated intravenous treatment from days 13 to 21 (qdxd9) with 2x2 T-cell engager 16 (50 μg/animal; 2.5 mg/kg) in the presence of human T-cells (Group 3) rapidly delayed tumor growth relative to Group 1 and Group 2. 2x2 T-cell engager 16 delayed tumor growth in Group 3 by approximately 4 - 5 days compared to vehicle-treated control group (Group 2). Statistically significant differences in the time period from day 6 to day 27 were identified between Group 2 (HL-60, T-cells, vehicle) and Group 3 (HL-60, T-cells, 16) on day 22 (p<0.05), day 23 (p<0.01) and day 27 (p<0.01) (Two-way Repeated Measures ANOVA with Bonferroni post-tests). No statistically significant differences were present between Group 1 and Group 3 due to unusual slow growth of the tumor in Group 1.

[0216] No donor variability with regard to T-cell activity was observed, when comparing tumor development in Cohort 1 and Cohort 2 within a group, which received T-cells from different donors (see Table 14).

[0217] Example 10 shows that a xenograft model in NOD/scid mice with a pre-established HL- 60 tumor (AML) and intraperitoneally-engrafted human T-cells was successfully developed. Repeated dosing with 2x2 T-cell engager 16 at a single dose level lead to a statistically significant delay in tumor growth in comparison to the respective vehicle-treated control group. The data generated are comparable to results published for a similar study with a CD33/CD3 BiTE™ (Aigner et al, 2012; Leukemia, 2013, Apr;27(5): 1107-15).

EXAMPLE 12

Efficacy of CD33/CD3 2x2 T-cell engagers in an AML PDX model in NSG mice

[0218] Cryopreserved cells from an AML patient whose CD33 ⁺ leukemia contained 2-4% CD3 ⁺ T-cells were used to establish an AML PDX model in NSG mice. One hour post-injection of tumor cells into irradiated (250 cGy) NSG mice, CD33/CD3 2x2 T-cell engagers, 16 or 12, at either of two i.v. doses (50 μg or 5 μg; n= 8 mice/group) were injected in a 200 μΕ bolus.

Additional injections of 2x2 T-cell engagers were performed on each of the following 4 days. Mice were weighed once weekly, and subsequently were sacrificed on day 38 to permit collection of peripheral blood, bone marrow, and spleen for analysis by flow cytometry

(huCD33, huCD34, huCD45, muCD45, huCD14, huCD3, huCD4, huCD8, and 7 A AD). The results are shown in Figure 14.

[0219] Figure 14 shows that untreated mice had substantial amounts of human blasts in the bone marrow and spleen after 38 days. In contrast, mice treated with daily i.v. injections of 2x2 T- cell engagers 12 or 16 exhibited substantially lower numbers of human AML blasts in the bone marrow and in the spleen. The strong anti-AML effect of the CD33/CD3 2x2 T-cell engager was observed at both dose levels (5 and 50 μg/injection).

[0220] The observed anti-AML effect for both CD33/CD3 2x2 T-cell engagers, 12 and 16, was much stronger than the effect of a CD123/CD3 DART® antibody targeting AML in an identical mouse model (Hussaini et al. :" Targeting CD123 In Leukemic Stem Cells Using Dual Affinity Re-Targeting Molecules (DARTs®) November 15, 2013; Blood: 122 (21)). In contrast to the CD33/CD3 2x2 T-cell engagers which eliminated nearly all AML blasts in bone marrow and spleen, Hussaini et al. reported that the CD123/CD3 DART® reduced the number of AML blasts in the bone marrow and spleen in the PDX model only by factor 50-1000 at 2.5 and 0.25 mg/kg, the authors further reported that the CD123/CD3 DART™ reduced the number of AML blasts in bone marrow and spleen in the PDX model only by 40-78% at 0.5 mg/kg.

EXAMPLE 13

Fast onset of CD33/CD3 2x2 T-cell engager 16-mediated target cell lysis

[0221] In order to assess the kinetics of CD33/CD3 2x2 T-cell engager-mediated target cell lysis, calcein-release cytotoxicity assays with different incubation times were performed.

Calcein-labeled CD33 ⁺ HL-60 target cells were incubated with serial dilutions of 2x2 T-cell engager 16 in the presence of primary human T cells as effector cells at an E:T ratio of 25: 1 for 30 min, 1 h, 2 h, 3 h, 4 h, or 5 h. At each time point the calcein that was released from lysed target cells was used to calculate the EC ₅₀ value and 2x2 T-cell engager 16-mediated target cell lysis using non-linear regression/sigmoidal dose-response. Figure 15 shows an unexpected fast onset of 2x2 T-cell engager-mediated target cells lysis with more than 40% lysis after 30 min incubation at saturating 2x2 T-cell engager concentrations. After 4 hours incubation more than 90% target cell lysis was reached. Table 15 and Figure 16 summarize the EC ₅₀ and specific lysis values determined for 2x2 T-cell engager 16 at incubation times between 30 min and 5 hours. The results further demonstrate that under the used assay conditions maximal potency (lowest EC ₅₀ value) was reached after 2 hours incubation and that after 5 hours incubation almost all target cells were lysed. Altogether these results demonstrate a very fast, potent and efficacious target cell lysis mediated by CD33/CD3 2x2 T-cell engagers.

TABLE 15: Kinetics of EC ₅₀ and lysis values determined for 2x2 T-cell engager 16

EXAMPLE 14

Clinical Trial Protocol for Dose Escalation Study of a CD33/CD3 2 2 T-cell engager to A ML Patients

[0222] This is a Phase I clinical trial to characterize the safety and tolerability of CD33/CD3 2x2 T-cell engager 16 (AMV 564).

[0223] Study Outcomes:

[0224] Primary: Dose Escalation Stage: 1) To characterize the safety and tolerability, including dose-limiting toxicity (DLT), of CD33/CD3 2x2 T-cell engager 16 when administered via continuous intravenous infusion; 2) To identify the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D)

[0225] Secondary: To characterize the pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of CD33/CD3 2x2 T-cell engager 16 when administered continuous intravenous infusion (CIV);

[0226] Study Design: This study is a first in human, Phase 1, open label, multicenter, dose escalation study with expansion at the RP2D, to evaluate the safety, tolerability and preliminary antileukemic activity of CD33/CD3 2x2 T-cell engager 16 in patients with relapsed or refractory acute myeloid leukemia (AML). Approximately 50 patients are enrolled at approximately 6 centers in the US or EU; the total number of patients will depend on the dose level at which the RP2D is defined.

[0227] CD33/CD3 2x2 T-cell engager 16 is given via CIV administration for a total of 14 days per cycle, for 1 or 2 induction cycles. Patients undergo bone marrow assessments at Screening, on Day 15 (within 24 hours of end of infusion), on Day 29 (+ 5 days), and at time of hematologic recovery during each CD33/CD3 2x2 T-cell engager 16 induction cycle, and at other times if clinically indicated.

[0228] Dose Escalation Stage

[0229] A standard 3 + 3 dose escalation scheme is employed to determine the MTD and will follow the scheme outlined in the below table: Dose Dose Treatment Regimen*

Level

1 0.5 μg/day 0.5 μ /d x 14d

2 1.5 μg /day 1.5 μ _§ Λ1 χ 14d

3 5 μg /day 5 μg /d x 14d

4 15 μg /day 15 μ _§ Λ1 χ 14d

5 50 μg /day 50 μ /d x 14d

6 100 μg /day 100 μ _§ Λ1 χ 14d

7 150 μg /day 150 μ χ 14d

8 200 μg /day 200 μ /d x 14d

9 250 μg /day 250 μ /d x 14d

10 300 μg /day 300 μ /d x 14d

Abbreviations: d = day

* If cytokine release syndrome (CRS) is observed in > 2 patients in

the study (at any dose), the Study Evaluation Team (SET) will

designate a lead-in dose (lower than that at which CRS was observed)

for the first 3 days, followed by 11 days at the assigned dose level ibility:

a. > 18 years of age at the time of signing informed consent

b. Diagnosis of AML according to the World Health Organization (WHO) 2008 criteria

c. Relapsed or refractory disease meeting the following criteria: (a) Primary

refractory, ie, refractory to induction with a standard anthracycline-based regimen or a hypomethylating agent (e.g. decitabine or azacitidine) for patients ineligible for anthracycline-based therapy; (b) First relapse after a first complete remission (CR) lasting less than 12 months; or (c) Second or later relapse. Relapse is defined as the reappearance of leukemic blasts in the peripheral blood or > 5% leukemic blasts in the bone marrow after prior achievement of a CR or CRi. d. No more than 2 prior induction regimens for newly diagnosed AML, no more than 1 prior stem cell transplant, and no more than 2 prior salvage regimens for relapsed or refractory AML. Any number of continuous cycles of therapy with an individual hypomethylating agent count as one induction or salvage regimen. e. Blasts at least 5% in bone marrow

f. Peripheral white blood cell (WBC) count: no upper limit at Screening, but must be < 10 x 10 ⁹ /L on Day 1 prior to treatment; patients with excessive blasts may be treated with hydroxyurea to bring counts down.

g. Chemistry laboratory parameters within the following range: Aspartate

aminotransferase (AST) and alanine aminotransferase (ALT) < 2x the upper limit of normal (ULN); Total bilirubin < 1.5x the ULN; patients with Gilbert' s syndrome can enroll if conjugated bilirubin is within normal limits; Creatinine clearance > 50 mL/min (measured or calculated by Cockcroft-Gault method) h. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

Patients with ECOG score of 2 may be included, after discussion with the Sponsor Medical Monitor, if score is influenced by symptoms attributable to underlying AML disease.

[0231] Clinical Pharmacokinetics:

TABLE 16: Pharmacokinetic Parameters of Subjects dosed with AMV 564

[0232] Figure 18 depicts the serum concentration of CD33/CD3 2x2 T-cell engager 16 (AMV 564) in subjects 02-001, -002, and -003 at a dose of 0.5 μg/day in a first 14 day cycle (left panel). Subject 02-002 received a second cycle at the same concentration.

[0233] Figure 19 shows serum concentrations following continuous intravenous administration of AMV564 for 14 days at the 0.5, 1.5, 5, 15, or 50 meg dose levels to patients with

relapsed/refractory acute myeloid leukemia. It was observed that concentrations gradually increase and reach steady-state in 3 - 7 days. The terminal half-life is approximately 2 days. N = 3 per dose level except 50 meg where N = 1. Concentrations were measured from serum collected at specified timepoints using a validated method.

[0234] Pharmacodynamics:

[0235] In a subject dosed at 0.5 μg/day with AMV 564 for 14 days, it was observed that myeloblast counts were controlled, CRP levels were reduced and increased hematopoiesis was observed as evidenced by sustained and increased hemoglobin and neutrophils (Figure 20). The subject received a transfusion on day 3 of the treatment.

[0236] A similar response was observed in a second subject dosed at 1.5 μg/day with AMV 564 for 14 days which shows dramatic improvement in blood counts (Figure 21). Neutrophils and erythrocyte counts improved and recovered (upper panels) following administration of

AMV564. Blood samples were collected at specified timepoints and processed to determine these blood parameters. Interleukin-6 and C-reactive protein both decreased following administration of AMV564 at a 1.5 meg dose level for 14 days (lower panels). Samples were collected at specified timepoints. Interleukin-6 was measured in a validated multiplex immunoassay. C-reactive protein was measured using a qualified instrument. It is contemplated that recovery is driven by elimination of MDSCs in the subject.

[0237] Additional subjects dosed at 1.5 μg/day with AMV 564 for 14 days attained similar results in blood counts. Figure 22 (upper panels) shows one subject that had improved hemoglobin, neutrophils, platelet, and monocyte counts following administration and another subject (lower panels) having improved hemoglobin, neutrophils, platelet, and monocyte counts as well as decreased C-reactive protein. Blood samples were collected at specified timepoints and processed to determine these blood parameters. C-reactive protein was measured using a qualified instrument.

[0238] The improvements observed in the above subjects have not been seen with other bispecific antibody treatments.

Additional Observations

[0239] Antileukemic activity was additionally observed in initial subjects. Figure 23 depicts best relative change in percent bone marrow leukemic blasts from baseline following administration of AMV564 for 14 days to patients with relapsed/refractory acute myeloid leukemia. Each bar represents an individual patient response. Bone marrow samples were taken periodically during the clinical study period and the percent bone marrow blasts determined by cellular morphology. The x-axis denotes the dose administered in the units of meg.

[0240] In one of the subjects, spleen size was reduced from 18 cm to 11 cm. This observation provides evidence of a clinical benefit and lends support toward the study endpoints.

[0241] While certain embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.