CROSS REFERENCE TO RELATED APPLICATIONS

[ 0001 ] This application claims priority to U.S. Provisional Application No.

63/034,889, filed June 4, 2020. The above-identified application is hereby incorporated herein by reference for all purposes.

REFERENCE TO THE SEQUENCE LISTING

[ 0002 ] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 28, 2021, is named A-2636-WO-PCT_ST25.txt and is 135,950 bytes in size.

FIELD OF THE INVENTION

[ 0003] The invention is in the field of protein engineering.

BACKGROUND

[ 0004 ] Bispecific binding constructs have shown therapeutic promise in recent years. For example, a bispecific binding construct that targets both CD3 and CD19 in a Bispecific T cell Engager (BiTE") format has shown impressive efficacy at low doses. Bargou et al. (2008), Science 321: 974-978. This BiTE” format comprises two scFv's, one of which targets CD3 and one of which targets a tumor antigen, CD19, joined by a flexible linker. This unique design allows the bispecific binding construct to bring activated T-cells into proximity with target cells, resulting in cytolytic killing of the target cells. See, for example,

WO 99/54440A1 (U.S. Patent No. 7,112,324 Bl) and WO 2005/040220 (U.S. Patent Appl. Publ. No. 2013/0224205A1). Later developments were bispecific binding constructs binding to a context independent epitope at the N-terminus of the CD3s chain (see WO 2008/119567; U.S. Patent Appl. Publ. No. 2016/0152707A1). [0005] In the biopharmaceutical industry, molecules are typically produced in a large-scale fashion in order to meet the commercial needs of supplying a large number of patients and can be assessed for a number of attributes to mitigate the risk that the molecule is not amenable to large-scale production and purification. Efficient expression of these complex, recombinant polypeptides can be an ongoing challenge. Further, even once expressed, the polypeptides are often not as stable as desired for a pharmaceutical composition. Accordingly, there is a need in the art for bispecific therapeutics with favorable pharmacokinetic properties, as well as therapeutic efficacy, and a format that provides efficient production and increased stability.

SUMMARY

[0006] Described herein are several new formats of bispecific binding constructs. In one embodiment, the invention provides a bispecific binding construct comprising a polypeptide chain comprising an amino acid sequence having a structural format selected from:

[0007] VHl-linker-VLl-linker-VH2-linker-VL2 ("H1L1H2L2"),

[0008] VHl-linker-VH2-linker-VLl-linker-VL2 ("H1H2L1L2"),

[0009] VHl-linker-VLl-linker-VL2-linker-VH2 ("H1L1L2H2"),

[0010] VHl-linker-VH2-linker-VL2-linker-VLl ("H1H2L2L1"),

[0011] VHl-linker-VL2-linker-VLl-linker-VH2 ("H1L2L1H2"),

[0012] VHl-linker-VL2-linker-VH2-linker-VLl ("H1L2H2L1"),

[0013] VLl-linker-VHl-linker-VH2-linker-VL2 ("L1H1H2L2"),

[0014 ] VLl-linker-VH2-linker-VHl-linker-VL2 ("L1H2H1L2"),

[0015] VLl-linker-VHl-linker-VL2-linker-VH2 ("L1H1L2H2"),

[0016] VLl-linker-VH2-linker-VL2-linker-VHl ("L1H2L2H1"),

[0017] VLl-linker-VL2-linker-VHl-linker-VH2 ("L1L2H1H2"),

[0018] VLl-linker-VL2-linker-VH2-linker-VHl ("L1L2H2H1"),

[0019] VH2-linker-VL2-linker-VLl-linker-VHl ("H2L2L1H1"),

[ 0020 ] VH2-linker-VLl-linker-VL2-linker-VHl ("H2L1L2H1"),

[0021] VH2-linker-VL2-linker-VHl-linker-VLl ("H2L2H1L1"),

[0022] VH2-linker-VLl-linker-VHl-linker-VL2 ("H2L1H1L2"),

[0023] VH2-linker-VHl-linker-VL2-linker-VLl ("H2H1L2L1"), [ 0024 ] VH2-linker-VHl-linker-VLl-linker-VL2 ("H2H1L1L2"),

[0025] VL2-linker-VH2-linker-VLl-linker-VHl ("L2H2L1H1"),

[0026] VL2-linker-VLl-linker-VH2-linker-VHl ("L2L1H2H1"),

[0027] VL2-linker-VH2-linker-VHl-linker-VLl ("L2H2H1L1"),

[0028] VL2-linker-VLl-linker-VHl-linker-VH2 ("L2L1H1H2"),

[0029] VL2-linker-VHl-linker-VH2-linker-VLl ("L2H1H2L1"), or

[0030] VL2-linker-VHl-linker-VLl-linker-VH2 ("L2H1L1H2")

[0031] wherein VH1 and VH2 are immunoglobulin heavy chain variable regions, VL1 and VL2 are immunoglobulin light chain variable regions, wherein the linker is at least 10 amino acids, and wherein the bispecific binding construct can bind to an immune effector cell and a target cell.

[0032] In another embodiment, the invention provides a nucleic acid encoding the bispecific binding constructs described herein, and vectors comprising these nucleic acids. Further, the invention provides a host cell comprising the vectors described herein.

[0033] In yet other embodiments, the invention provides a method of manufacturing the bispecific constructs described herein comprising (1) culturing a host cell under conditions to express the bispecific construct and (2) recovering the construct from the cell mass or cell culture supernatant, wherein the host cell comprises one or more nucleic acid(s) encoding any of the bispecific constructs described herein.

[0034 ] In other embodiments, the invention provides a method of treating a cancer patient comprising administering to the patient a therapeutically effective amount of the bispecific binding constructs described herein.

[0035] In other embodiments, the invention provides a method of treating a patient having an infectious disease comprising administering to the patient a therapeutically effective amount of the bispecific binding constructs described herein.

[0036] In other embodiments, the invention provides a method of treating a patient having an autoimmune, inflammatory, or fibrotic condition comprising administering to the patient a therapeutically effective amount of the bispecific binding constructs described herein.

[0037 ] In another embodiment, the invention provides a pharmaceutical composition comprising the bispecific binding constructs described herein. BRIEF DESCRIPTION OF DRAWINGS

[ 0038 ] Figure 1. A representative diagram of the 24 different construct formats comprising Fll, LI, H2, L2 binding domains and the linkers.

[ 0039] Figure 2. A representative diagram of the 24 different formats comprising HI, LI, H2, L2 and the linkers, and indicating which formats utilize a single linker to join the H1L1 to the H2L2 binding domains.

[ 0040 ] Figure 3. A representative diagram of the 24 different formats comprising HI, LI, H2, L2 and the linkers, and indicating which formats utilize two linkers to join the H1L1 to the H2L2 binding domains.

[ 0041 ] Figure 4. A representative diagram of the 24 different formats comprising HI, LI, H2, L2 and the linkers, and indicating which formats utilize three linkers to join the H1L1 to the H2L2 binding domains.

[ 0042 ] Figure 5. This figure provides a graphical depiction of expression data for various constructs (H1L1H2L2, H1H2L1L2, H1L2L1H2, L1H2H1L2, L1L2H1H2, L2H1H2L1) based on expression yields of each construct from a 1L flask of HEK293 cells after 6 days. [ 0043] Figures 6A and 6B. Figures 6A and 6B provide a graphical depiction and table, respectively, of chemical stability data for the various constructs.

[ 0044 ] Figure 7. Figures 7A and 7B provide a graphical depiction and tables, respectively, of melting temperature (Tm) (°C) for the various constructs.

[ 0045] Figure 8. This figure provides a graphical depiction of accelerated stability data at 40°C of the various constructs at time 0, 2 weeks, and 4 weeks.

[ 0046] Figure 9. This figure provides a graphical depiction of levels of clipping measured for the various constructs at time 0, 2 weeks, and 4 weeks at 40°C and showing that within the variants the H1H2L1L2 has the lowest levels of clipping as measured by rCE after 1 month.

[ 0047 ] Figure 10. This figure provides a graphical depiction of relative potency of the various constructs compared to the "wild type" (WT) H1L1H2L2 construct. DETAILED DESCRIPTION

[ 0048 ] Described herein are novel formats for bispecific binding constructs. As depicted in Figure 1, these formats comprise the 24 various permutations created from different linker placement in relation to the four VH and VL polypeptide chains (i.e., two VH- VL binding domains) that form the bispecific construct. This bispecific construct comprises a single polypeptide chain that comprises two immunoglobulin variable heavy chain (VH) regions, two immunoglobulin variable light chain (VL) regions, and optionally, an Fc region (e.g., an scFc), arranged in the following orders or formats:

[ 0049] 1. VHl-linker-VLl-linker-VH2-linker-VL2 ("H1L1H2L2")

[ 0050 ] 2. VHl-linker-VH2-linker-VLl-linker-VL2 ("H1H2L1L2")

[ 0051 ] 3. VHl-linker-VLl-linker-VL2-linker-VH2 ("H1L1L2H2")

[ 0052 ] 4. VHl-linker-VH2-linker-VL2-linker-VLl ("H1H2L2L1")

[ 0053] 5. VHl-linker-VL2-linker-VLl-linker-VH2 ("H1L2L1H2")

[ 0054 ] 6. VHl-linker-VL2-linker-VH2-linker-VLl ("H1L2H2L1")

[ 0055] 7. VLl-linker-VHl-linker-VH2-linker-VL2 ("L1H1H2L2")

[ 0056] 8. VLl-linker-VH2-linker-VHl-linker-VL2 ("L1H2H1L2")

[ 0057 ] 9. VLl-linker-VHl-linker-VL2-linker-VH2 ("L1H1L2H2")

[ 0058 ] 10. VLl-linker-VH2-linker-VL2-linker-VHl ("L1H2L2H1")

[ 0059] 11. VLl-linker-VL2-linker-VHl-linker-VH2 ("L1L2H1H2")

[ 0060 ] 12. VLl-linker-VL2-linker-VH2-linker-VHl ("L1L2H2H1")

[ 0061 ] 13. VH2-linker-VL2-linker-VLl-linker-VHl ("H2L2L1H1")

[ 0062 ] 14. VH2-linker-VLl-linker-VL2-linker-VHl ("H2L1L2H1")

[ 0063] 15. VH2-linker-VL2-linker-VHl-linker-VLl ("H2L2H1L1")

[ 0064 ] 16. VH2-linker-VLl-linker-VHl-linker-VL2 ("H2L1H1L2")

[ 0065] 17. VH2-linker-VHl-linker-VL2-linker-VLl ("H2H1L2L1")

[ 0066] 18. VH2-linker-VHl-linker-VLl-linker-VL2 ("H2H1L1L2")

[ 0067 ] 19. VL2-linker-VH2-linker-VLl-linker-VHl ("L2H2L1H1")

[ 0068 ] 20. VL2-linker-VLl-linker-VH2-linker-VHl ("L2L1H2H1")

[ 0069] 21. VL2-linker-VH2-linker-VHl-linker-VLl ("L2H2H1L1")

[ 0070 ] 22. VL2-linker-VLl-linker-VHl-linker-VH2 ("L2L1H1H2") [0071] 23. VL2-linker-VHl-linker-VH2-linker-VLl ("L2H1H2L1")

[0072] 24. VL2-linker-VHl-linker-VLl-linker-VH2 ("L2H1L1H2")

[0073] These bispecific binding construct formats in certain embodiments can provide both enhanced stability and increased in vitro expression as compared to, for example, VH-linker-VL-linker-VH-linker-VL ("HLHL") binding construct format, yet maintain the intended function of binding the desired targets on the immune effector cell and the target cell. Accordingly, in certain embodiments the binding construct formats herein provide bispecific binding constructs that can be produced more efficiently and have greater stability, characteristics that are sought after in a pharmaceutical composition.

[ 0074 ] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term "portion" can include part of a moiety or the entire moiety.

[0075] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references.

[0076] Polynucleotide and polypeptide sequences are indicated using standard one- or three-letter abbreviations. Unless otherwise indicated, polypeptide sequences have their amino termini at the left and their carboxy termini at the right, and single-stranded nucleic acid sequences, and the top strand of double-stranded nucleic acid sequences, have their 5' termini at the left and their 3' termini at the right. A particular section of a polypeptide can be designated by amino acid residue number such as amino acids 1 to 50, or by the actual residue at that site such as asparagine to proline. A particular polypeptide or polynucleotide sequence also can be described by explaining how it differs from a reference sequence.

Definitions

[ 0077 ] The term "isolated" in reference to a molecule (where the molecule is, for example, a polypeptide, a polynucleotide, antigen binding protein, an antibody, or a bispecific binding construct) is a molecule that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be "isolated" from its naturally associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using H PLC or other means well known in the art for purification.

[ 0078 ] The terms "polynucleotide," "oligonucleotide" and "nucleic acid" are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The nucleic acid molecule can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules of the invention comprise a contiguous open reading frame encoding a binding construct, or a fragment, derivative, mutein, or variant thereof, of the invention. [0079] A "vector" is a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid/' which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), wherein additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a chosen polynucleotide.

[ 0080 ] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked. The regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid). Examples of regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals).

[0081] A "host cell" is a cell that can be used to express a nucleic acid, e.g., a nucleic acid of the invention. A host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0082] A "single-chain variable fragment" ("scFv") is a fusion protein in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., Science 242:423-26 (1988) and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83 (1988)). When in the context of other additional moieties (e.g., an Fc region), the scFv can be arranged VH-linker-VL, or VL-linker-VH, for example.

[0083] The term "CDR" refers to the complementarity determining region (also termed "minimal recognition units" or "hypervariable region") within antibody variable sequences, and these CDRs can be used to generate the bispecific binding constructs of the invention. The CDRs permit the antibody or bispecific binding construct to specifically bind to a particular antigen of interest. There are three heavy chain variable region CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable region CDRs (CDRL1, CDRL2 and CDRL3). The CDRs in each of the two chains typically are aligned by the framework regions to form a structure that binds specifically to a specific epitope or domain on the target protein. From N-terminus to C-terminus, naturally-occurring light and heavy chain variable regions both typically conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody may be identified using this system. Other numbering systems for the amino acids in immunoglobulin chains include IMGT ^® (the international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203;

2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). One or more CDRs may be incorporated into a molecule either covalently or noncovalently to generate a bispecific binding construct. [ 0084 ] The "binding domain" of a construct according to the invention may, e.g., comprise the above referred groups of CDRs. Preferably, those CDRs are comprised in the framework of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Or in the context of the terminology used herein, the "L" and "H" variable regions (e.g., "H1H2L1L2" or "H1L1L2H2").

[ 0085] The term "human antibody" includes antibodies having antibody regions such as variable and constant regions or domains which correspond substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991). The human antibodies referred to herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, in CDR3. The human antibodies can have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence. The definition of human antibodies as used herein also contemplates fully human antibodies, which include only non-a rtificia lly and/or genetically altered human sequences of antibodies as those can be derived by using technologies or systems known in the art, such as for example, phage display technology or transgenic mouse technology, including but not limited to the Xenomouse' ^* . In the context of the present invention, the variable regions from a human antibody can be used in the bispecific construct formats contemplated.

[0086] A humanized antibody has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non human species antibody, when it is administered to a human subject. In one embodiment, certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody. In another embodiment, the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immunospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293. In the context of the present invention, the variable regions from a humanized antibody can be used in the bispecific construct formats contemplated.

[ 0087 ] The term "chimeric antibody" refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the CDRs are derived from a human antibody. In another embodiment, all of the CDRs are derived from a human antibody. In another embodiment, the CDRs from more than one human antibodies are mixed and matched in a chimeric antibody. For instance, a chimeric antibody may comprise a CDR1 from the light chain of a first human antibody, a CDR2 and a CDR3 from the light chain of a second human antibody, and the CDRs from the heavy chain from a third antibody. Further, the framework regions may be derived from one of the same antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and/or light chain is identical with, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with, homologous to, or derived from an antibody or antibodies from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies that exhibit the desired biological activity. In the context of the present invention, the variable regions from a chimeric antibody can be used in the bispecific binding construct formats contemplated.

[ 0088 ] The invention provides bispecific binding constructs that comprise the 24 different H-L permutations set forth in Figure 1: 1. VHl-linker-VLl-linker-VH2-linker-VL2 ("H1L1H2L2"), 2. VHl-linker-VH2-linker-VLl-linker-VL2 ("H1H2L1L2"), 3. VHl-linker-VLl- linker-VL2-linker-VH2 ("H1L1L2H2"), 4. VHl-linker-VH2-linker-VL2-linker-VLl ("H1H2L2L1"), 5. VHl-linker-VL2-linker-VLl-linker-VH2 ("H1L2L1H2"), 6. VHl-linker-VL2-linker-VH2-linker- VL1 ("H1L2H2L1"), 7. VLl-linker-VHl-linker-VH2-linker-VL2 ("L1H1H2L2"), 8. VLl-linker- VH2-linker-VHl-linker-VL2 ("L1H2H1L2"), 9. VLl-linker-VHl-linker-VL2-linker-VH2 ("L1H1L2H2"), 10. VLl-linker-VH2-linker-VL2-linker-VHl ("L1H2L2H1"), 11. VL1-Iinker-VL2- linker-VHl-linker-VH2 ("L1L2H1H2"), 12. VLl-linker-VL2-linker-VH2-linker-VHl ("L1L2H2H1"), 13. VH2-linker-VL2-linker-VLl-linker-VHl ("H2L2L1H1"), 14. VH2-Iinker-VL1- linker-VL2-linker-VHl ("H2L1L2H1"), 15. VH2-linker-VL2-linker-VHl-linker-VLl ("H2L2H1L1"), 16. VH2-linker-VLl-linker-VHl-linker-VL2 ("H2L1H1L2"), 17. VH2-Iinker-VH1- linker-VL2-linker-VLl ("H2H1L2L1"), 18. VH2-linker-VHl-linker-VLl-linker-VL2 ("H2H1L1L2"), 19. VL2-linker-VH2-linker-VLl-linker-VHl ("L2H2L1H1"), 20. VL2-linker-VLl-linker-VH2- linker-VHl ("L2L1H2H1"), 21. VL2-linker-VH2-linker-VHl-linker-VLl ("L2H2H1L1"), 22. VL2- linker-VLl-linker-VHl-linker-VH2 ("L2L1H1H2"), 23. VL2-linker-VHl-linker-VH2-linker-VLl ("L2H1H2L1"), and 24. VL2-linker-VHl-linker-VLl-linker-VH2 ("L2H1L1H2").

[0089] In the most general sense, a bispecific construct as described herein comprises several polypeptide chains having different amino acid sequences, which, when linked together, can bind to two different antigens. See also, for example, U.S. Patent Application Nos. 62/858,509 (filed June 7, 2019), 62/858,630 (filed June 7, 2019), PCT/US20/36464 (filed June 5, 2020), and PCT/US20/36474 (Filed June 5, 2020). Optionally, the bispecific constructs further comprise a half-life extending moiety. In some embodiments, the half-life extending moiety is an Fc polypeptide chain. In other embodiments, the half-life extending moiety is a single-chain Fc. In yet other embodiment, the half-life extending moiety is a hetero-Fc. In yet other embodiments, the half-life extending moiety is human albumin.

Linkers

[0090] Between the immunoglobulin variable regions (e.g. VH1, VL1, VH2, VL2 in the various permutations) is a peptide linker, which can be the same linker or different linkers of different lengths. The linkers can play a critical role in the structure of the bispecific constructs and the invention described herein provides not only the appropriate linker sequences, but also the appropriate linker lengths for each position in the bispecific constructs of the invention. If the linker is too short, it will not allow enough flexibility for the appropriate variable regions on a single polypeptide chain to interact to form an antigen binding site. If the linker is the appropriate length, it will allow a variable region to interact with another variable region on the same polypeptide chain to form an antigen binding site, or a "binding domain". In certain embodiments, the various formats may comprise disulfide bonds - both intra-domain (within HI, LI) and inter-domain (between HI and LI). In order to achieve proper expression and conformation of the bispecific constructs of the invention, in certain embodiments specific linkers are used between the various immunoglobulin regions (see, e.g., Fig. 1 herein). Exemplary linkers are provided in Table 1 herein. In certain embodiments, increasing linker length might result in increased protein clipping, an undesirable property. Accordingly, it is desirable to achieve the appropriate balance between linker length to allow proper polypeptide structure and activity, yet not result in increased clipping.

[ 0091 ] A "linker," as meant herein, is a peptide that links two polypeptides. In certain embodiments such as those provided herein, a linker can link two immunoglobulin variable regions in the context of a bispecific construct. A linker can be from 2-30 amino acids in length. In some embodiments, a linker can be 2-25, 2-20, or 3-18 amino acids long. In some embodiments, a linker can be a peptide no more than 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids long. In other embodiments, a linker can be 5-25, 5-15, 4-11, 10-20, or 20- 30 amino acids long. In other embodiments, a linker can be about, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In a preferred embodiment, a linker is 20 amino acids long. In a further preferred embodiment, all three linkers utilized in the bispecific binding constructs of the invention are 20 amino acids long.

[ 0092 ] Exemplary linkers include, for example, the amino acid sequences GGGGS (SEQ ID NO: 1), GGGGSGGGGS (SEQ ID NO: 2), GGGGSGGGGSGGGGS (SEQ ID NO: 3), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 5), GGGGQ (SEQ ID NO: 6), GGGGQGGGGQ (SEQ ID NO: 7), GGGGQGGGGQGGGGQ (SEQ ID NO: 8), GGGGQGGGGQGGGGQGGGGQ (SEQ ID NO: 9),

GGGGQGGGGQGGGGQGGGGQGGGGQ (SEQ ID NO: 10), GGGGSAAA (SEQ ID NO: 11), TVAAP (SEQ ID NO: 12), ASTKGP (SEQ ID NO: 13), and AAA, among others, including repeats of the aforementioned amino acid sequences or subunits of amino acid sequences (e.g., GGGGS or GGGGQ repeats).

[ 0093] In certain embodiments, the linker sequence of Linker 1 is at least 10 amino acids. In other embodiments, Linker 1 is at least 15 amino acids. In other embodiments, Linker 1 is at least 20 amino acids. In other embodiments, Linker 1 is at least 25 amino acids. In other embodiments, Linker 1 is at least 30 amino acids. In other embodiments, Linker 1 is 10-30 amino acids. In other embodiments, Linker 1 is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet other embodiments, Linker 1 is greater than 30 amino acids. In a preferred embodiment, Linker 1 is 20 amino acids long.

[ 0094 ] In certain embodiments, the linker sequence of Linker 2 is at least 15 amino acids. In other embodiments, Linker 2 is at least 20 amino acids. In other embodiments, Linker 2 is at least 25 amino acids. In other embodiments, Linker 2 is at least 30 amino acids. In other embodiments, Linker 2 is 15-30 amino acids. In other embodiments, Linker 2 is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet other embodiments, Linker 2 is greater than 30 amino acids. In a preferred embodiment, Linker 2 is 20 amino acids long.

[ 0095] In certain embodiments, the linker sequence of Linker 3 is at least 15 amino acids. In other embodiments, Linker 3 is at least 20 amino acids. In other embodiments, Linker 3 is at least 25 amino acids. In other embodiments, Linker 3 is at least 30 amino acids. In other embodiments, Linker 3 is 15-30 amino acids. In other embodiments, Linker 3 is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet other embodiments, Linker 3 is greater than 30 amino acids. In a preferred embodiment, Linker 3 is 20 amino acids long.

[ 0096] In certain embodiments, the linker sequence of Linker 4 is at least 5 amino acids. In other embodiments, Linker 4 is at least 10 amino acids. In other embodiments, Linker 4 is at least 15 amino acids. In other embodiments, Linker 4 is at least 20 amino acids. In other embodiments, Linker 4 is at least 25 amino acids. In other embodiments, Linker 4 is at least 30 amino acids. In other embodiments, Linker 4 is 5-30 amino acids. In other embodiments, Linker 4 is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In yet other embodiments, Linker 4 is greater than 30 amino acids.

[ 0097 ] In certain embodiments, the linker sequences and positions are set forth in the following Table 1, with linker positions corresponding to those set forth in Figure 1 where Linker 1 is the first linker as read from the N-terminal end of the molecule, Linker 2 is the second linker as read from the N-terminal end of the molecule, Linker 3 is the third linker as read from the N-terminal end of the molecule, and with Linker 4 being optionally used if an Fc region is also attached to the bispecific molecule and being the fourth linker as read from the N-terminal end of the molecule.

Table 1

*numerical subscript indicates the number of repeats, e.g., (GGGGS) ₂ = GGGGSGGGGS

Amino Acid Sequences of Binding Regions

[ 0098 ] In the exemplary embodiments described herein, the bispecific constructs maintain desired binding to the various desired targets which results from their assuming the proper conformation to allow this binding. The immunoglobulin variable region comprises a VH and a VL domain, which associate to form the variable domain that binds the desired target. In all of the various 24 permutations disclosed herein, the linkers of appropriate length allow this association to occur.

[ 0099] The variable domains can be obtained from any immunoglobulin with the desired characteristics, and the methods to accomplish this are further described herein. In one embodiment, VH1 and VL1 associate and bind CD3s, and VH2 and VL2 associate and bind a different target. In another embodiment, the VH2 and VL2 associate and bind CD3s and the VH1 and VL1 associate and bind a different target.

[ 00100] In another embodiment, the light-chain variable domain comprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of a light chain variable domain set forth herein.

[ 00101] In another embodiment, the light chain variable domain comprises a sequence of amino acids that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequence set forth herein. In another embodiment, the light chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide that encodes a light chain variable domain selected from the sequences set forth herein. In another embodiment, the light chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide that encodes a light chain variable domain selected from the group consisting of the sequences set forth herein.

[ 00102] In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of a heavy chain variable domain selected from the sequences set forth herein. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence that encodes a heavy chain variable domain selected from the sequences set forth herein. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide that encodes a heavy chain variable domain selected from the sequences set forth herein. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide that encodes a heavy chain variable domain selected from the sequences set forth herein.

Substitutions

[00103] It will be appreciated that a bispecific binding construct of the present invention may have at least one amino acid substitution, providing that the bispecific binding construct retains the same or better desired binding specificity (e.g., binding to CD3). Therefore, modifications to the bispecific binding construct structures are encompassed within the scope of the invention. In one embodiment, the bispecific binding construct comprises sequences that each independently differ by 5, 4, 3, 2, 1, or 0 single amino acid additions, substitutions, and/or deletions from a CDR sequence of those set forth herein. As used herein, a CDR sequence that differs by no more than a total of, for example, four amino acid additions, substitutions and/or deletions from a CDR sequence set forth herein refers to a sequence with 4, 3, 2, 1 or 0 single amino acid additions, substitutions, and/or deletions compared with the sequences set forth herein. These may include amino acid substitutions, which may be conservative or non-conservative that do not destroy the desired binding capability of a binding construct. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties. A conservative amino acid substitution may also involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.

[ 00104 ] Non-conservative substitutions may involve the exchange of a member of one class of amino acids or amino acid mimetics for a member from another class with different physical properties (e.g. size, polarity, hydrophobicity, charge). In certain embodiments, such substituted residues may be introduced into regions of a human antibody (or region of the antibody used in the bispecific binding constructs) that are homologous with non-human antibodies, or into the non-homologous regions of the molecule. [00105] Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

[00106] A skilled artisan will be able to determine suitable variants of the bispecific binding construct as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides as has been describe above. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[00107] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[ 00108] In some embodiments, one skilled in the art may identify residues that may be changed that result in enhanced properties as desired. For example, an amino acid substitution (conservative or non-conservative) may result in enhanced binding affinity to a desired target.

[ 00109] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody (or those regions used in the bispecific binding constructs of the invention) with respect to its three-dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. A number of scientific publications have been devoted to the prediction of secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al., Nucl. Acid. Res., 27(l):244-247 (1999). Additional methods of predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)), "profile analysis" (Bowie et al., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al.,

Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage" (See Holm, supra (1999), and Brenner, supra (1997)).

[ 00110] In certain embodiments, variants of the bispecific binding construct include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide. In certain embodiments, variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protein. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N- linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. Additional variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants may be useful when constructs must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[ 00111] Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify important residues of binding constructs to the target of interest, or to increase or decrease the affinity of the binding constructs to the target of interest described herein.

[00112] According to certain embodiments, desired amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation,

(3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or modify other physiochemical or functional properties on such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally- occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). In certain embodiments, a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991), which are each incorporated herein by reference.

Half-life extension and Fc regions

[00113] In certain embodiments, it is desirable to extend the in vivo half-life of the bispecific binding constructs of the invention. This can be accomplished by including a half- life extending moiety as part of the bispecific construct. Nonlimiting examples of half-life extending moieties include an Fc polypeptide, albumin, an albumin fragment, a moiety that binds to albumin or to the neonatal Fc receptor (FcRn), a derivative of fibronectin that has been engineered to bind albumin or a fragment thereof, a peptide, a single domain protein fragment, or other polypeptide that can increase serum half-life. In alternate embodiments, a half-life-extending moiety can be a non-polypeptide molecule such as, for example, polyethylene glycol (PEG).

[00114] The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization also are included. In addition to other properties described herein, polypeptides comprising Fc moieties offer the advantage of purification by affinity chromatography over, e.g., Protein A or Protein G columns.

[00115] In certain embodiments, the half-life extending moiety is an Fc region of an antibody. The Fc region can be located at the N-terminal end of the bispecific construct, or it can be located at the C-terminal end of the bispecific construct. There can be, but need not be, a linker between the bispecific construct and the Fc region. As explained above, an Fc polypeptide chain may comprise all, or part of a hinge region followed by a CH2 and a CH3 region. The Fc polypeptide chain can be of mammalian (for example, human, mouse, rat, rabbit, dromedary, or new or old world monkey), avian, or shark origin. In addition, as explained above, an Fc polypeptide chain can include a limited number alterations. For example, an Fc polypeptide chain can comprise one or more heterodimerizing alterations, one or more alteration that inhibits or enhances binding to FcyR, or one or more alterations that increase binding to FcRn.

[00116] In a specific embodiment, the Fc utilized for half-life extension is a single chain Fc ("scFc").

[00117] In some embodiments the amino acid sequences of the Fc polypeptides can be mammalian, for example a human, amino acid sequences. The isotype of the Fc polypeptide can be IgG, such as IgGl, lgG2, lgG3, or lgG4, IgA, IgD, IgE, or IgM. Table 2 below shows an alignment of the amino acid sequences of human IgGl, lgG2, lgG3, and lgG4 Fc polypeptide chains.

[00118] Sequences of human IgGl, lgG2, lgG3, and lgG4 Fc polypeptides that could be used are provided in SEQ ID NOs: 42-45. Variants of these sequences containing one or more heterodimerizing alterations, one or more Fc alteration that extends half-life, one or more alteration that enhances ADCC, and/or one or more alteration that inhibits Fc gamma receptor (FcyR) binding are also contemplated, as are other close variants containing not more than 10 deletions, insertions, or substitutions of a single amino acid per 100 amino acids of sequence.

Table 2: Amino acid sequences of human IgG Fc polypeptide chains

IgGl - lgG2 - lgG3 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP lgG4 -

225 235 245 255 265 275

IgGl EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF lgG2 ERKCCVE— CPPCPAPPVA-GPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQF lgG3 EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQF lgG4 ESKYG— PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQF

285 295 305 315 325 335

IgGl NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT lgG2 NWYVDGMEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKT lgG3 KWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT lgG4 NWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT

345 355 365 375 385 395

IgGl ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP lgG2 ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP lgG3 ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTP lgG4 ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP

405 415 425 435 445

IgGl PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42) lgG2 PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 43) lgG3 PMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 44) lgG4 PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 45)

[00119] The numbering shown in Table 2 is according the EU system of numbering, which is based on the sequential numbering of the constant region of an IgGl antibody. Edelman et al. (1969), Proc. Natl. Acad. Sci. 63: 78-85. Thus, it does not accommodate the additional length of the lgG3 hinge well. It is nonetheless used here to designate positions in an Fc region because it is still commonly used in the art to refer to positions in Fc regions The hinge regions of the IgGl, lgG2, and lgG4 Fc polypeptides extend from about position 216 to about 230. It is clear from the alignment that the lgG2 and lgG4 hinge regions are each three amino acids shorter than the IgGl hinge. The lgG3 hinge is much longer, extending for an additional 47 amino acids upstream. The CH2 region extends from about position 231 to 340, and the CH3 region extends from about position 341 to 447.

[ 00120] Naturally occurring amino acid sequences of Fc polypeptides can be varied slightly. Such variations can include no more than 10 insertions, deletions, or substitutions of a single amino acid per 100 amino acids of sequence of a naturally occurring Fc polypeptide chain. If there are substitutions, they can be conservative amino acid substitutions, as defined above. The Fc polypeptides on the first and second polypeptide chains can differ in amino acid sequence. In some embodiments, they can include "heterodimerizing alterations," for example, charge pair substitutions, as defined above, that facilitate heterodimer formation. Further, the Fc polypeptide portions of the PABP can also contain alterations that inhibit or enhance FcyR binding. Such mutations are described above and in Xu et al. (2000), Cell Immunol. 200(1): 16-26, the relevant portions of which are incorporated herein by reference. The Fc polypeptide portions can also include an "Fc alteration that extends half-life," as described above, including those described in, e.g., US Patents 7,037,784, 7,670,600, and 7,371,827, US Patent Application Publication 2010/0234575, and International Application PCT/US2012/070146, the relevant portions of all of which are incorporated herein by reference. Further, an Fc polypeptide can comprise "alterations that enhance ADCC," as defined above.

[ 00121] Another suitable Fc polypeptide, described in PCT application WO 93/10151 (hereby incorporated by reference), is a single chain polypeptide extending from the N- terminal hinge region to the native C-terminus of the Fc region of a human IgGl antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,457,035 and in Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

[00122] The effector function of an antibody, or region used in the bispecific binding constructs of the invention (e.g., Fc), can be increased, or decreased, by introducing one or more mutations into the Fc. Embodiments of the invention include IL-2 mutein Fc fusion proteins having an Fc engineered to increase effector function (U.S. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both incorporated herein by reference in its entirety). For certain therapeutic indications, it may be desirable to increase effector function. For other therapeutic indications, it may be desirable to decrease effector function.

[ 00123] Exemplary IgGl Fc molecules having increased effector function include those having the following substitutions:

S239D/I332E

S239D/A330S/I332E

S239D/A330L/I332E

S298A/D333A/K334A

P247I/A339D

P247I/A339Q

D280H/K290S

D280H/K290S/S298D

D280H/K290S/S298V

F243L/R292P/Y300L

F243L/R292P/Y300L/P396L

F243L/R292P/Y300L/V305I/P396L

G236A/S239D/I332E

K326A/E333A

K326W/E333S

K290E/S298G/T299A

K290N/S298G/T299A

K290E/S298G/T299A/K326E

K290N/S298G/T299A/K326E [ 00124 ] Another method of increasing effector function of IgG Fc-containing proteins is by reducing the fucosylation of the Fc. Removal of the core fucose from the biantennary complex-type oligosachharides attached to the Fc greatly increased ADCC effector function without altering antigen binding or CDC effector function. Several ways are known for reducing or abolishing fucosylation of Fc-containing molecules, e.g., binding constructs. These include recombinant expression in certain mammalian cell lines including a FUT8 knockout cell line, variant CFIO line Lecl3, rat hybridoma cell line YB2/0, a cell line comprising a small interfering RNA specifically against the FUT8 gene, and a cell line coexpressing a-l,4-/V-acetylglucosaminyltransferase III and Golgi a-mannosidase II. Alternatively, the Fc-containing molecule may be expressed in a non-mammalian cell such as a plant cell, yeast, or prokaryotic cell, e.g., E. coli.

[ 00125] In certain embodiments of the invention, the bispecific binding constructs comprise an Fc engineered to decrease effector function. Exemplary Fc molecules having decreased effector function include those having the following substitutions:

N297A or N297Q (lgGl)

L234A/L235A ( IgG 1)

V234A/G237A (lgG2)

L235A/G237A/E318A (lgG4)

H268Q/V309L/A330S/A331S (lgG2)

C220S/C226S/C229S/P238S (IgGl)

C226S/C229S/E233P/L234V/L235A (IgGl)

L234F/L235E/P331S (IgGl)

S267E/L328F (IgGl)

[ 00126] It is known that human IgGl has a glycosylation site at N297 (EU numbering system) and glycosylation contributes to the effector function of IgGl binding constructs.

An exemplary IgGl sequence is provided in SEQ ID NO: 42. N297 can be mutated to make aglycosylated binding constructs. For example, mutations can substitute N297 with amino acids that resemble asparagine in physiochemical nature such as glutamine (N297Q), or with alanine (N297A), which mimics asparagines without polar groups. [00127] In certain embodiments, mutation of amino acid N297 of human IgGl to glycine, i.e., N297G, provides far superior purification efficiency and biophysical properties over other amino acid substitutions at that residue. See, for example, U.S. Patent Nos. 9,546,203 and 10,093,711. In a specific embodiment, the bispecific binding constructs of the invention comprise a human IgGl Fc having an N297G substitution.

[00128] A bispecific construct of the invention comprising a human IgGl Fc having the N297G mutation may also comprise further insertions, deletions, and substitutions. In certain embodiments the human IgGl Fc comprises the N297G substitution and is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 42. In a particularly preferred embodiment, the C-terminal lysine residue is substituted or deleted.

[00129] In certain instances, aglycosylated IgGl Fc-containing molecules can be less stable than glycosylated IgGl Fc-containing molecules. Accordingly, the Fc region may be further engineered to increase the stability of the aglycosylated molecule. In some embodiments, one or more amino acids are substituted to cysteine so to form di-sulfide bonds in the dimeric state. In specific embodiments, residues V259, A287, R292, V302,

L306, V323, or 1332 of the amino acid sequence set forth in SEQ ID NO: 36 may be substituted with cysteine. In other embodiments, specific pairs of residues are substitution such that they preferentially form a di-sulfide bond with each other, thus limiting or preventing di-sulfide bond scrambling. In specific embodiments, pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. [00130] As discussed herein above in the Linker section, in certain embodiments, the bispecific binding constructs of the invention comprise a linker between the Fc and the bispecific construct, specifically, e.g., linking the Fc to the last domain at the C-terminal end of the construct or the first domain at the N-terminal end of the construct. In certain embodiments, one or more copies of a peptide consisting of GGGGS (SEQ ID NO: 1), GGNGT (SEQ ID NO: 15), or YGNGT (SEQ ID NO: 16) between the Fc and the bispecific construct polypeptide. In some embodiments, the polypeptide region between the Fc region and the bispecific construct polypeptide comprises a single copy of GGGGS (SEQ ID NO: 1), GGNGT (SEQ ID NO: 15), or YGNGT (SEQ ID NO: 16). In certain embodiments, the linkers GGNGT (SEQ ID NO: 15) or YGNGT (SEQ ID NO: 16) are glycosylated when expressed in the appropriate cells and such glycosylation may help stabilize the protein in solution and/or when administered in vivo. Accordingly, in certain embodiments, a bispecific construct of the invention comprises a glycosylated linker between the Fc region and the bispecific construct polypeptide.

Nucleic acids encoding the bispecific binding constructs

[00131] In another embodiment, the present invention provides isolated nucleic acid molecules that encode the bispecific binding constructs of the present invention. In addition, provided are vectors comprising the nucleic acids, cell comprising the nucleic acids, and methods of making the bispecific binding constructs of the invention. The nucleic acids comprise, for example, polynucleotides that encode all or part of bispecific construct, for example, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing. The nucleic acids can be any length as appropriate for the desired use or function, and can comprise one or more additional sequences, for example, regulatory sequences, and/or be part of a larger nucleic acid, for example, a vector. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and/or DNA nucleotides, and artificial variants thereof (e.g., peptide nucleic acids).

[00132] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chain, variable domain only, or full length) may be isolated from B-cells of mice that have been immunized with antigen. The nucleic acid may be isolated by conventional procedures such as polymerase chain reaction (PCR). These nucleic acids may then be used to further generate the bispecific constructs of the invention.

[00133] Nucleic acid sequences encoding the variable regions of the heavy and light chain variable regions are included herein. The skilled artisan will appreciate that, due to the degeneracy of the genetic code, each of the polypeptide sequences disclosed herein is encoded by a large number of other nucleic acid sequences. The present invention provides each degenerate nucleotide sequence encoding each bispecific binding construct of the invention.

[00134 ] The invention further provides nucleic acids that hybridize to other nucleic acids under particular hybridization conditions. Methods for hybridizing nucleic acids are well-known in the art. See, e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. As defined herein, for example, a moderately stringent hybridization condition uses a prewashing solution containing 5X sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6X SSC, and a hybridization temperature of 55° C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42° C), and washing conditions of 60° C, in 0.5X SSC, 0.1% SDS. A stringent hybridization condition hybridizes in 6X SSC at 45° C, followed by one or more washes in 0.1X SSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art can manipulate the hybridization and/or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% identical to each other typically remain hybridized to each other. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by, for example, Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the DNA. Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., a bispecific binding construct) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues is changed using, for example, a random mutagenesis protocol. However, it is made, a mutant polypeptide can be expressed and screened for a desired property. [ 00135] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. In one embodiment, a nucleotide sequence provided herein for of the binding constructs of the present invention, or a desired fragment, variant, or derivative thereof, is mutated such that it encodes an amino acid sequence comprising one or more deletions or substitutions of amino acid residues that are shown herein for the light chains of the binding constructs of the present invention or the heavy chains of the binding constructs of the present invention to be residues where two or more sequences differ. In another embodiment, the mutagenesis inserts an amino acid adjacent to one or more amino acid residues shown herein for the light chains of the binding constructs of the present invention or the heavy chains of the binding constructs of the present invention to be residues where two or more sequences differ. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively change the biological activity of a polypeptide that it encodes. [ 00136] In another embodiment, the present invention provides vectors comprising a nucleic acid encoding a polypeptide of the invention or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors.

[ 00137] The recombinant expression vectors of the invention can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter and cytomegalovirus promoter), those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences, see Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science 236:1237, incorporated by reference herein in their entireties), and those that direct inducible expression of a nucleotide sequence in response to particular treatment or condition (e.g., the metallothionin promoter in mammalian cells and the tet-responsive and/or streptomycin responsive promoter in both prokaryotic and eukaryotic systems (see id.). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

[00138] In another embodiment, the present invention provides host cells into which a recombinant expression vector of the invention has been introduced. A host cell can be any prokaryotic cell or eukaryotic cell. Prokaryotic host cells include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DXB-11, which is deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20). Additional CHO cell lines include CHO-K1 (ATCC#CCL-61), EM9 (ATCC# CRL-1861), and UV20 (ATCC# CRL-1862). Additional host cells include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,

Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), AM-l/D cells (described in U.S. Patent No. 6,210,924), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[00139] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Additional selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods.

[00140] The transformed cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide recovered by conventional protein purification procedures. Polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian polypeptides substantially free of contaminating endogenous materials.

[00141] Cells containing the nucleic acid encoding the bispecific binding constructs of the present invention also include hybridomas. The production and culturing of hybridomas are discussed herein.

[00142] In some embodiments, a vector comprising a nucleic acid molecule as described herein is provided. In some embodiments, the invention comprises a host cell comprising a nucleic acid molecule as described herein.

[00143] In some embodiments, a nucleic acid molecule encoding the bispecific binding constructs as described herein is provided.

[00144] In some embodiments, a pharmaceutical composition comprising at least one bispecific construct described herein is provided.

METHODS OF PRODUCING

[00145] The bispecific binding constructs of the invention can be produced by any method known in the art for the synthesis of proteins (e.g., antibodies or bispecific binding constructs), in particular, by chemical synthesis or preferably, by recombinant expression techniques.

[ 00146] Recombinant expression of the bispecific binding constructs requires construction of an expression vector containing a polynucleotide that encodes the bispecific construct. Once a polynucleotide encoding the bispecific construct has been obtained, the vector for the production of the bispecific construct may be produced by recombinant DNA technology. An expression vector is constructed containing the bispecific construct coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. [ 00147] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce a bispecific construct of the invention.

[ 00148] A variety of host-expression vector systems may be utilized to express the bispecific binding constructs of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express a molecule of the invention in situ. Bacterial cells such as E. coli, and eukaryotic cells are commonly used for the expression of a recombinant polypeptide molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies or other binding constructs (Foecking et al., Gene 45:101 (1986); Cockett et a I., Bio/Technology 8:2 (1990)).

[ 00149] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, COS, 293, 3T3, or myeloma cells.

[ 00150] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the bispecific binding molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the bispecific construct. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the bispecific construct.

[00151] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk, hgprt or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

[00152] The expression levels of a bispecific construct can be increased by vector amplification (for a review, see Bebbington and Hentschel, "The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells" (DNA Cloning, Vol. 3. Academic Press, New York, 1987)). When a marker in the vector system expressing the bispecific construct is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the construct gene, production of the construct will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[ 00153] The host cell may be co-transfected with multiple expression vectors of the invention. The vectors may contain identical selectable markers which enable equal expression of the expressed polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, for example, the polypeptides of the invention. The coding sequences may comprise cDNA or genomic DNA.

[ 00154 ] Once a construct of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule or bispecific binding construct, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and size-exclusion chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the binding constructs of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification. The purification techniques may be varied, depending on whether an Fc region (e.g., an scFc) is attached to the bispecific binding constructs of the invention.

[ 00155] In some embodiments, the present invention encompasses binding constructs recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide. Fused or conjugated binding constructs of the present invention may be used for ease in purification. See e.g., Flarbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., Proc. Natl. Acad. Sci. 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991).

[ 00156] Moreover, the binding constructs or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.

GENERATION OF BISPECIFIC BINDING CONSTRUCTS

[00157] The bispecific binding constructs of the invention, in a general sense, are constructed by selecting VH and VL regions from desired antibodies and linking them using polypeptide linkers as described herein to form the bispecific construct, optionally with an Fc region attached. More specifically, the nucleic acids encoding the VH, VL and linkers, and optionally the Fc, are combined to create the nucleic acid constructs that encode the bispecific binding constructs of the invention.

Generation of antibodies

[00158] In certain embodiments, prior to generation of the bispecific binding constructs of the invention, monospecific antibodies are first generated with binding specificities to desired targets.

[ 00159] Antibodies, and in particular, VH and VL domains for use in generating the bispecific binding constructs of the invention may be prepared by techniques that are well known to those skilled in the art. For example, by immunizing an animal (e.g., a mouse or rat or rabbit) and then by immortalizing spleen cells harvested from the animal after completion of the immunization schedule. The spleen cells can be immortalized using any technique known in the art, e.g., by fusing them with myeloma cells to produce hybridomas. See, for example, Antibodies; Harlow and Lane, Cold Spring Harbor Laboratory Press, 1st Edition, e.g. from 1988, or 2nd Edition, e.g. from 2014).

[ 00160] A humanized monoclonal antibody comprises the variable domain of a murine antibody (or all or part of the antigen binding site thereof) and a constant domain derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of engineered monoclonal antibodies that may be used to generate the bispecific binding constructs of the invention include those described in Riechmann et al., 1988, Nature 332:323, Liu et al., 1987, Proc. Nat. Acad. Sci. USA 84:3439, Larrick et al., 1989, Bio/Technology 7:934, and Winter et al., 1993, TIPS 14:139. In one embodiment, the chimeric antibody is a CDR grafted antibody. Techniques for humanizing antibodies that may then be used to generate the bispecific binding constructs of the invention are discussed in, e.g., U.S. Pat. Nos. 5,869,619; 5,225,539; 5,821,337; 5,859,205; 6,881,557, Padlan et al., 1995, FASEB J. 9:133-39, Tamura et al., 2000, J. Immunol. 164:1432-41, Zhang, W., et al., Molecular Immunology. 42(12):1445-1451, 2005; Hwang W. et al., Methods. 36(l):35-42, 2005; Dall'Acqua WF, et al., Methods 36(l):43-60, 2005; and Clark, M., Immunology Today. 21(8):397-402, 2000.

[ 00161] A bispecific binding construct of the present invention may also be generated using regions from a fully human monoclonal antibody. Fully human monoclonal antibodies that may be used to generate the bispecific binding constructs of the invention may be generated by any number of techniques with which those having ordinary skill in the art will be familiar. Such methods include, but are not limited to, Epstein Barr Virus (EBV) transformation of human peripheral blood cells (e.g., containing B lymphocytes), in vitro immunization of human B-cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V region phage libraries, or other procedures as known in the art and based on the disclosure herein.

[ 00162] Procedures have been developed for generating human monoclonal antibodies in non-human animals. For example, mice in which one or more endogenous immunoglobulin genes have been inactivated by various means have been prepared.

Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, human immunoglobulin transgenes may be mini-gene constructs, or transloci on yeast artificial chromosomes, which undergo B-cel l-specific DNA rearrangement and hypermutation in the mouse lymphoid tissue.

[ 00163] Antibodies produced in the animal incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. In one embodiment, a non-human animal, such as a transgenic mouse, is immunized with a suitable immunogen. [ 00164 ] Examples of techniques for production and use of transgenic animals for the production of human or partially human antibodies, which can then be used to generate the bispecific binding constructs of the invention, are described in U.S. Patents 5,814,318, 5,569,825, and 5,545,806, Davis et al., Production of human antibodies from transgenic mice in Lo, ed. Antibody Engineering: Methods and Protocols, Humana Press, NJ:191-200 (2003), Kellermann et al., 2002, Curr Opin Biotechnol. 13:593-97, Russel et al., 2000, Infect Immun. 68:1820-26, Gallo et al., 2000, Eur J Immun. 30:534-40, Davis et al., 1999, Cancer Metastasis Rev. 18:421-25, Green, 1999, J Immunol Methods. 231:11-23, Jakobovits, 1998, Advanced Drug Delivery Reviews 31:33-42, Green et al., 1998, J Exp Med. 188:483-95, Jakobovits A, 1998, Exp. Opin. Invest. Drugs. 7:607-14, Tsuda et al., 1997, Genomics. 42:413- 21, Mendez et al., 1997, Nat Genet. 15:146-56, Jakobovits, 1994, Curr Biol. 4:761-63, Arbones et al., 1994, Immunity. 1:247-60, Green et al., 1994, Nat Genet. 7:13-21, Jakobovits et al., 1993, Nature. 362:255-58, Jakobovits et al., 1993, Proc Natl Acad Sci U S A. 90:2551- 55. Chen, J., M. Trounstine, F. W. Alt, F. Young, C. Kurahara, J. Loring, D. Huszar. "Immunoglobulin gene rearrangement in B-cell deficient mice generated by targeted deletion of the JH locus." International Immunology 5 (1993): 647-656, Choi et al., 1993, Nature Genetics 4: 117-23, Fishwild et al., 1996, Nature Biotechnology 14: 845-51, Harding et al., 1995, Annals of the New York Academy of Sciences, Lonberg et al., 1994, Nature 368: 856-59, Lonberg, 1994, Transgenic Approaches to Human Monoclonal Antibodies in Handbook of Experimental Pharmacology 113: 49-101, Lonberg et al., 1995, Internal Review of Immunology 13: 65-93, Neuberger, 1996, Nature Biotechnology 14: 826, Taylor et al., 1992, Nucleic Acids Research 20: 6287-95, Taylor et al., 1994, International Immunology 6: 579-91, Tomizuka et al., 1997, Nature Genetics 16: 133-43, Tomizuka et al., 2000, Proceedings of the National Academy of Sciences USA 97: 722-27, Tuaillon et al., 1993, Proceedings of the National Academy of Sciences USA 90: 3720-24, and Tuaillon et al., 1994, Journal of Immunology 152: 2912-20.; Lonberg et al., Nature 368:856, 1994; Taylor et al.,

Int. Immun. 6:579, 1994; U.S. Patent No. 5,877,397; Bruggemann et al., 1997 Curr. Opin. Biotechnol. 8:455-58; Jakobovits et al., 1995 Ann. N. Y. Acad. Sci. 764:525-35. In addition, protocols involving the XenoMouse ^® (Abgenix, now Amgen, Inc.) are described, for example in U.S. 05/0118643 and WO 05/694879, WO 98/24838, WO 00/76310, and US Patent 7,064,244. [00165] Lymphoid cells from the immunized transgenic mice are fused with myeloma cells for example to produce hybridomas. Myeloma cells for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for use in such fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bui; examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

[ 00166] The lymphoid (e.g., spleen) cells and the myeloma cells may be combined for a few minutes with a membrane fusion-promoting agent, such as polyethylene glycol or a nonionic detergent, and then plated at low density on a selective medium that supports the growth of hybridoma cells but not unfused myeloma cells. One selection media is HAT (hypoxanthine, aminopterin, thymidine). After a sufficient time, usually about one to two weeks, colonies of cells are observed. Single colonies are isolated, and antibodies produced by the cells may be tested for binding activity to desired targets using any one of a variety of immunoassays known in the art and described herein. The hybridomas are cloned (e.g., by limited dilution cloning or by soft agar plaque isolation) and positive clones that produce an antibody specific to a desired target is selected and cultured. The monoclonal antibodies from the hybridoma cultures may be isolated from the supernatants of hybridoma cultures. These hybridomas can be cultured according to methods described herein and known in the art, and the polynucleotides that encode the monoclonal antibodies can be isolated and further used to generate the bispecific binding constructs of the invention.

[00167] Another method for generating human antibodies that may be used to generate the bispecific binding constructs of the invention includes immortalizing human peripheral blood cells by EBV transformation. See, e.g., U.S. Patent No. 4,464,456. Such an immortalized B-cell line (or lymphoblastoid cell line) producing a monoclonal antibody that specifically binds to a desired target can be identified by immunodetection methods as provided herein, for example, an ELISA, and then isolated by standard cloning techniques. The stability of the lymphoblastoid cell line producing an antibody may be improved by fusing the transformed cell line with a murine myeloma to produce a mouse-human hybrid cell line according to methods known in the art (see, e.g., Glasky et al., Hybridoma 8:377-89 (1989)). Still another method to generate human monoclonal antibodies that may be used to generate the bispecific binding constructs of the invention is in vitro immunization, which includes priming human splenic B-cells with antigen, followed by fusion of primed B-cells with a heterohybrid fusion partner. See, e.g., Boerner et al., 1991 J. Immunol. 147:86-95. [ 00168] In certain embodiments, a B-cell that is producing a desired antibody is selected and the light chain and heavy chain variable regions are cloned from the B-cell according to molecular biology techniques known in the art (WO 92/02551; U.S. patent 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)) and described herein. B-cells from an immunized animal may be isolated from the spleen, lymph node, or peripheral blood sample by selecting a cell that is producing a desired antibody. B-cells may also be isolated from humans, for example, from a peripheral blood sample. Methods for detecting single B-cells that are producing an antibody with the desired specificity are well known in the art, for example, by plaque formation, fluorescence-activated cell sorting, in vitro stimulation followed by detection of specific antibody, and the like. Methods for selection of specific antibody-producing B-cells include, for example, preparing a single cell suspension of B-cells in soft agar that contains antigen. Binding of the specific antibody produced by the B-cell to the antigen results in the formation of a complex, which may be visible as an immunoprecipitate. After the B-cells producing the desired antibody are selected, the specific antibody genes may be cloned by isolating and amplifying DNA or mRNA according to methods known in the art and described herein, and then utilized to generate the bispecific constructs of the invention.

[ 00169] An additional method for obtaining antibodies that may be used to generate the bispecific binding constructs of the invention is by phage display. See, e.g., Winter et al., 1994 Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol. 57:191-280.

Human or murine immunoglobulin variable region gene combinatorial libraries may be created in phage vectors that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that bind specifically to TGF-beta binding protein or variant or fragment thereof. See, e.g., U.S. Patent No. 5,223,409; Huse et al., 1989 Science 246:1275-81; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol. 227:381-388; Schlebusch et al., 1997 Hybridoma 16:47-52 and references cited therein. For example, a library containing a plurality of polynucleotide sequences encoding Ig variable region fragments may be inserted into the genome of a filamentous bacteriophage, such as M13 or a variant thereof, in frame with the sequence encoding a phage coat protein. A fusion protein may be a fusion of the coat protein with the light chain variable region domain and/or with the heavy chain variable region domain. According to certain embodiments, immunoglobulin Fab fragments may also be displayed on a phage particle (see, e.g., U.S. Patent No. 5,698,426).

[00170] Heavy and light chain immunoglobulin cDNA expression libraries may also be prepared in lambda phage, for example, using AlmmunoZapTM(H) and AlmmunoZapTM(L) vectors (Stratagene, La Jolla, California). Briefly, mRNA is isolated from a B-cell population, and used to create heavy and light chain immunoglobulin cDNA expression libraries in the AlmmunoZap(H) and AlmmunoZap(L) vectors. These vectors may be screened individually or co-expressed to form Fab fragments or antibodies that may be used to generate the bispecific binding constructs of the invention (see Huse et al., supra; see also Sastry et al., supra). Positive plaques may subsequently be converted to a non-lytic plasmid that allows high level expression of monoclonal antibody fragments from E. coli.

[00171] In one embodiment, in a hybridoma the variable regions of a gene expressing a monoclonal antibody of interest are amplified using nucleotide primers. These primers may be synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources. (See, e.g., Stratagene (La Jolla, California), which sells primers for mouse and human variable regions including, among others, primers for VHa, VHb, VHc, VHd, CHI, VL and CL regions.) These primers may be used to amplify heavy or light chain variable regions, which may then be inserted into vectors such as ImmunoZAPTMH or ImmunoZAPTML (Stratagene), respectively. These vectors may then be introduced into E. coli, yeast, or mammalian-based systems for expression. Large amounts of a single-chain protein containing a fusion of the VH and VL domains may be produced using these methods (see Bird et al., Science 242:423-426, 1988). These can then be used to generate the bispecific binding constructs of the invention.

[00172] In certain embodiments, the binding constructs of the invention are obtained from transgenic animals (e.g., mice) that produce "heavy chain only" antibodies or "HCAbs." HCAbs are analogous to naturally occurring camel and llama single-chain VHH antibodies. See, for example, U.S. Patent Nos. 8,507,748 and 8,502,014, and U.S. Patent Application Publication Nos. US2009/0285805A1, US2009/0169548A1, US2009/0307787A1, US2011/0314563A1, US2012/0151610A1, WO2008/122886A2, and W02009/013620A2. [ 00173] Once cells producing antibodies that may be used to generate the bispecific binding constructs according to the invention have been obtained using any of the above-described immunization and other techniques, the specific antibody genes may be cloned by isolating and amplifying DNA or mRNA therefrom according to standard procedures as described herein and then used to generate the bispecific constructs of the present invention. The antibodies produced therefrom may be sequenced and the CDRs identified and the DNA coding for the CDRs may be manipulated as described previously to generate the bispecific binding constructs according to the invention.

[ 00174 ] Molecular evolution of the complementarity determining regions (CDRs) in the center of the antibody binding site also has been used to isolate antibodies with increased affinity, for example, those as described by Schier et a I., 1996, J. Mol. Biol.

263:551. Accordingly, such techniques are useful in preparing the bispecific binding constructs of the invention.

[ 00175] Although human, partially human, or humanized antibodies will be suitable for many applications, particularly those of the present invention, other types of bispecific binding constructs will be suitable for certain applications. These non-human antibodies can be, for example, derived from any antibody-producing animal, such as mouse, rat, rabbit, goat, donkey, or non-human primate (for example, monkey such as cynomologous or rhesus monkey) or ape (e.g., chimpanzee)) and may be used to generate the bispecific binding constructs of the invention. An antibody from a particular species can be made by, for example, immunizing an animal of that species with the desired immunogen or using an artificial system for generating antibodies of that species (e.g., a bacterial or phage display- based system for generating antibodies of a particular species), or by converting an antibody from one species into an antibody from another species by replacing, e.g., the constant region of the antibody with a constant region from the other species, or by replacing one or more amino acid residues of the antibody so that it more closely resembles the sequence of an antibody from the other species. In one embodiment, the antibody is a chimeric antibody comprising amino acid sequences derived from antibodies from two or more different species. Then, the desired binding region sequences can be used to generate the bispecific binding molecules of the present invention. [00176] Where it is desired to improve the affinity of antibodies or binding constructs according to the invention containing one or more of the above-mentioned CDRs can be obtained by a number of affinity maturation protocols including maintaining the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et a I., Bio/Technology, 10, 779-783, 1992), use of mutation strains of E. coli. (Low et al., J. Mol. Biol., 250, 350-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 7-88, 1996) and additional PCR techniques (Crameri, et al., Nature, 391, 288-291, 1998). All of these methods of affinity maturation are discussed by Vaughan et al. (Nature Biotechnology, 16, 535-539, 1998).

[00177] In certain embodiments, to generate the bispecific binding constructs of the present invention it may first be desirable to generate a more typical single chain antibody which may be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VH). The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Techniques developed for the production of single chain antibodies include those described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf et al., 2002, Methods Mol Biol. 178:379-87. These single chain antibodies are distinct from and differ from the bispecific binding constructs of the invention.

[00178] Antigen binding fragments that may be used to generate the bispecific binding constructs of the invention can be obtained from an antibody, for example, by proteolytic hydrolysis of the antibody, for example, pepsin or papain digestion of whole antibodies according to conventional methods. By way of example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment termed F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Patent No. 4,331,647, Nisonoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., in Methods in Enzymology 1:422 (Academic Press 1967); and by Andrews, S.M. and Titus, J.A. in Current Protocols in Immunology (Coligan J.E., et al., eds), John Wiley & Sons, New York (2003), pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as separating heavy chains to form monovalent light-heavy chain fragments (Fd), further cleaving of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. [00179] In certain embodiments, the bispecific binding constructs comprise one or more complementarity determining regions (CDRs) of an antibody. CDRs can be obtained by constructing polynucleotides that encode the CDR of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc. 1995)). The antibody fragment further may comprise at least one variable region domain of an antibody described herein. Thus, for example, the V region domain may be monomeric and be a VH or VL domain, which is capable of independently binding a desired target (e.g., human CD3) with an affinity at least equal to 10-7M or less as described herein. [00180] The variable region may be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region that has been created using recombinant DNA engineering techniques. Such engineered versions include those created, for example, from a specific antibody variable region by insertions, deletions, or changes in or to the amino acid sequences of the specific antibody. One of ordinary skill in the art can use any known methods for identifying amino acid residues appropriate for engineering. Additional examples include engineered variable regions containing at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody. Engineered versions of antibody variable domains may be generated by any number of techniques with which those having ordinary skill in the art will be familiar. These engineered domains can then be used to generate the bispecific constructs of the invention.

[00181] The variable region may be covalently attached at a C-terminal amino acid to at least one other binding domain or a fragment thereof. Thus, for example, a VH that is present in the variable region may be linked to an immunoglobulin CHI domain. Similarly, a VL domain may be linked to a CK domain. In this way, for example, the binding domain may be a Fab fragment wherein the binding domain contains associated VH and VL domains covalently linked at their C-termini to a CHI and CK domain, respectively. The CHI domain may be extended with further amino acids, for example to provide a hinge region or a portion of a hinge region domain as found in a Fab' fragment, or to provide further domains, such as antibody CH2 and CH3 domains.

Binding Specificity

[00182] An antibody or bispecific binding construct "specifically binds" to an antigen if it binds to the antigen with a tight binding affinity as determined by an equilibrium dissociation constant (KD, or corresponding KD, as defined below) value of 10-7 M or less. [00183] Affinity can be determined using a variety of techniques known in the art, for example but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or by a surface plasmon resonance assay or other mechanism of kinetics-based assay (e.g., BIACORE ^® analysis or Octet ^® analysis (forteBIO)), and other methods such as indirect binding assays, competitive binding assays fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott- Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen with the antibody or bispecific binding construct of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody or bispecific construct bound to the labeled antigen. The affinity of the antibody or bispecific construct of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody or bispecific construct can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody or bispecific construct of interest conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody or bispecific construct.

[ 00184 ] Further embodiments of the invention provide bispecific binding constructs that bind to desired targets with an equilibrium dissociation constant or KD (koff/kon) of less than 10 ⁷ M, or of less than 10 ⁸ M, or of less than 10 ⁹ M, or of less than 10 ¹⁰ M, or of less than 10 ¹¹ M, or of less than 10 ¹² M, or of less than 10 ¹³ M, or of less than 5x10 ¹³ M (lower values indicating tighter binding affinity). Yet further embodiments of the invention are bispecific binding constructs that bind to desired targets with an with an equilibrium dissociation constant or KD (koff/kon) of less than about 10 ⁷ M, or of less than about 10 ⁸ M, or of less than about 10 ⁹ M, or of less than about 10 ¹⁰ M, or of less than about 10 ¹¹ M, or of less than about 10 ¹² M, or of less than about 10 ¹³ M, or of less than about 5x10 ¹³ M. [00185] In still another embodiment, bispecific binding constructs that bind to desired targets have an equilibrium dissociation constant or KD (koff/kon) of between about 10 ⁷ M and about 10 ⁸ M, between about 10 ⁸ M and about 10 ⁹ M, between about 10 ⁹ M and about 10 ¹⁰ M, between about 10 ¹⁰ M and about 10 ¹¹ M, between about 10 ¹¹ M and about 10 ¹² M, between about 10 ¹² M and about 10 ¹³ M. In still another embodiment, a bispecific construct of the invention have an equilibrium dissociation constant or KD (koff/kon) of between 10 ⁷ M and 10 ⁸ M, between 10 ⁸ M and 10 ⁹ M, between 10 ⁹ M and 10 ¹⁰ M, between 10 ¹⁰ M and 10 ¹¹ M, between 10 ¹¹ M and 10 ¹² M, between 10 ¹² M and 10 ¹³ M.

Molecule Stability

[00186] Various aspects of molecule stability may be desired, particularly in the context of a biopharmaceutical therapeutic molecule. For example, stability at various temperatures ("thermostability") may be desired. In some embodiments, this can encompass stability at physiologic temperature ranges, e.g., at or about 37°C, or from 32°C to 42°C. In other embodiments, this can encompass stability at higher temperature ranges, e.g., 42°C to 60°C. In other embodiments, this can encompass stability at cooler temperature ranges, e.g. 20°C to 32°C. In yet other embodiments, this can encompass stability while in the frozen state, e.g. 0°C or lower.

[00187] Assays to determine thermostability of protein molecules are known in the art. For example, the fully automated UNcle platform (Unchained Labs) which allowed for simultaneous acquisition of intrinsic protein fluorescence during thermal ramp was used and is further described herein in the Examples. Additionally, thermal stability assays, such as differential scanning fluorimetry (DSF) can also be used to measure thermal melting (Tm). [00188] Alternatively, and as described herein in the Examples, accelerated stress studies can be performed on the molecules. Briefly, this involves incubating the protein molecules at a particular temperature (e.g., 40°C) and then measuring aggregation by size exclusion chromatography (SEC) at various timepoints, where lower levels of aggregation indicate better protein stability.

[00189] Alternatively, the thermostability parameter can be determined in terms of molecule aggregation temperature as follows: molecule solution at a concentration 250 pg/ml is transferred into a single use cuvette and placed in a Dynamic Light Scattering (DLS) device. The sample is heated from 40°C to 70°C at a heating rate of 0.5°C/min with constant acquisition of the measured radius. Increase of radius indicating melting of the protein and aggregation is used to calculate the aggregation temperature of the molecule. [00190] Alternatively, temperature melting curves can be determined by Differential Scanning Calorimetry (DSC) to determine intrinsic biophysical protein stabilities of the binding constructs. These experiments are performed using a MicroCal LLC (Northampton, MA, U.S.A) VP-DSC device. The energy uptake of a sample containing a binding construct is recorded from 20°C to 90°C compared to a sample containing only the formulation buffer. The binding constructs are adjusted to a final concentration of 250 pg/ml e.g. in SEC running buffer. For recording of the respective melting curve, the overall sample temperature is increased stepwise. At each temperature T energy uptake of the sample and the formulation buffer reference is recorded. The difference in energy uptake Cp (kcal/mole/°C) of the sample minus the reference is plotted against the respective temperature. The melting temperature is defined as the temperature at the first maximum of energy uptake. [00191] In a further embodiment the bispecific binding constructs according to the invention is stable at or about physiologic pH, i.e., about pH 7.4. In other embodiments, the bispecific binding constructs are stable at a lower pH, e.g., down to pH 6.0. In other embodiments, the bispecific binding constructs are stable at a higher pH, e.g., up to pH 9.0. In one embodiment, the bispecific binding constructs are stable at a pH of 6.0 to 9.0. In another embodiment, the bispecific binding constructs are stable at a pH of 6.0 to 8.0. In another embodiment, the bispecific binding constructs are stable at a pH of 7.0 to 9.0. [ 00192] In certain embodiments, the more tolerant the bispecific binding construct is to unphysiologic pH (e.g., pH 6.0), the higher the recovery of the binding construct eluted from an ion exchange column is relative to the total amount of loaded protein. In one embodiment, recovery of the binding construct from an ion (e.g., cation) exchange column is > 30%. In another embodiment, recovery of the binding construct from an ion (e.g., cation) exchange column is > 40%. In another embodiment, recovery of the binding construct from an ion (e.g., cation) exchange column is > 50%. In another embodiment, recovery of the binding construct from an ion (e.g., cation) exchange column is > 60%. In another embodiment, recovery of the binding construct from an ion (e.g., cation) exchange column is > 70%. In another embodiment, recovery of the binding construct from an ion (e.g., cation) exchange column is > 80%. In another embodiment, recovery of the binding construct from an ion (e.g., cation) exchange column is > 90%. In another embodiment, recovery of the binding construct from an ion (e.g., cation) exchange column is > 95%. In another embodiment, recovery of the binding construct from an ion (e.g., cation) exchange column is > 99%.

[ 00193] In certain embodiments, it may be desired to determine the chemical stability of the molecules. Determination of bispecific binding construct chemical stability can be carried out via isothermal chemical denaturation ("ICD") by monitoring intrinsic protein fluorescence, as further described herein in the Examples. ICD yields Cl/2 and AG which can be good metrics for protein stability. Cl/2 is the amount of chemical denaturant required to denature 50% of the protein and is used to derive AG (or unfolding energy). [ 00194 ] Clipping of protein chains is another critical product quality attribute that is carefully monitored and reported for biologic drugs. Typically, a longer and/or a less structured linker is expected to result in increased clipping as a function of incubation time and temperature. Clipping is a critical issue for bispecific binding constructs as clips to linkers connecting either the target or T-cell engaging domains have terminal detrimental impact on drug potency and efficacy. Clips to additional sites including the scFc may impact pharmaco-dynamic/kinetic properties. Increased clipping is an attribute to be avoided in a pharmaceutical product. Accordingly, in certain embodiments, protein clipping can be assayed as described herein in the Examples, with results depicted in Figure 9.

Immune Effector Cells and Effector Cell Proteins

[00195] A bispecific binding construct can bind to a molecule expressed on the surface of an immune effector cell (called "effector cell protein" herein) and to another molecule expressed on the surface of a target cell (called a "target cell protein" herein). The immune effector cell can be a T cell, an NK cell, a macrophage, or a neutrophil. In some embodiments the effector cell protein is a protein included in the T cell receptor (TCR)-CD3 complex. The TCR-CD3 complex is a heteromultimer comprising a heterodimer comprising TCRa and TCRp or TCRy and TCR5 plus various CD3 chains from among the CD3 zeta (0ϋ3z) chain, CD3 epsilon (CD3s) chain, CD3 gamma (CD3y) chain, and CD3 delta (CD35) chain. [00196] The CD3 receptor complex is a protein complex and is composed of four chains. In mammals, the complex contains a CD3y (gamma) chain, a CD35 (delta) chain, and two CD3 (epsilon) chains. These chains associate with the T cell receptor (TCR) and the so- called z (zeta) chain to form the T cell receptor CD3 complex and to generate an activation signal in T lymphocytes. The CD3y (gamma), CD35 (delta), and CD3 (epsilon) chains are highly related cell-surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. The intracellular tails of the CD3 molecules contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM for short, which is essential for the signaling capacity of the TCR. The CD3 epsilon molecule is a polypeptide which in humans is encoded by the CD3e gene which resides on chromosome 11. The most preferred epitope of CD3 epsilon is comprised within amino acid residues 1-27 of the human CD3 epsilon extracellular domain. It is envisaged that the bispecific binding construct constructs according to the present invention typically and advantageously show less unspecific T cell activation, which is not desired in specific immunotherapy. This translates to a reduced risk of side effects.

[00197] In some embodiments the effector cell protein can be the human CD3 epsilon (CD3s) chain (the mature amino acid sequence of which is disclosed in SEQ ID NO: 40), which can be part of a multimeric protein. Alternatively, the effector cell protein can be human and/or cynomolgus monkey TCRa, TCRp, TCR5, TCRy, CD3 beta (CD3P) chain, CD3 gamma (CD3y) chain, CD3 delta (CD35) chain, or CD3 zeta (0ϋ3z) chain.

[00198] Moreover, in some embodiments, a bispecific binding construct can also bind to a CD3s chain from a non-human species, such as mouse, rat, rabbit, new world monkey, and/or old-world monkey species. Such species include, without limitation, the following mammalian species: Mus musculus; Rattus rattus; Rattus norvegicus; the cynomolgus monkey, Macaca fascicularis; the hamadryas baboon, Papio hamadryas; the Guinea baboon, Papio papio; the olive baboon, Papio anubis; the yellow baboon, Papio cynocephalus; the Chacma baboon, Papio ursinus; Callithrix jacchus; Saguinus Oedipus; and Saimiri sciureus. The mature amino acid sequence of the CD3s chain of cynomolgus monkey is provided in SEQ ID NO: 41. Having a therapeutic molecule that has comparable activity in humans and species commonly used for preclinical testing, such as mice and monkeys, can simplify, accelerate, and ultimately provide improved outcomes in drug development. In the long and expensive process of bringing a drug to market, such advantages can be critical.

[00199] In certain embodiments, the bispecific binding construct can bind to an epitope within the first 27 amino acids of the CD3s chain (SEQ ID NO: 43), which may be a human CD3s chain or a CD3s chain from different species, particularly one of the mammalian species listed above. The epitope can contain the amino acid sequence Gln- Asp-Gly-Asn-Glu. The advantages of a binding construct that binds such an epitope are explained in detail in U.S. Patent Application Publication 2010/0183615A1, the relevant portions of which are incorporated herein by reference. The epitope to which a binding construct binds can be determined by alanine scanning, which is described in, e.g., U.S. Patent Application Publication 2010/0183615A1, the relevant portions of which are incorporated herein by reference. In other embodiments, the bispecific binding construct can bind to an epitope within the extracellular domain of CD3s (SEQ ID NO: 42).

[00200] Alternative exemplary sequences of binding constructs that bind to CD3 are provided herein in SEQ ID NOs: 50 and 51, and in WO 2008/119567, for example. Additional alternative CD3 binding constructs are well known in the art and can be readily applied for use in the bispecific constructs of the present invention. [ 00201] In embodiments where a T cell is the immune effector cell, effector cell proteins to which a bispecific binding construct can bind include, without limitation, the CD3s chain, the CD3y, the CD35 chain, the CD3z chain, TCRa, TCRp, TCRy, and TCR5. In embodiments where an NK cell or a cytotoxic T cell is an immune effector cell, NKG2D, CD352, NKp46, or CD16a can, for example, be an effector cell protein. In embodiments where a CD8+ T cell is an immune effector cell, 4-1BB or NKG2D, for example, can be an effector cell protein. Alternatively, in other embodiments a bispecific binding construct could bind to other effector cell proteins expressed on T cells, NK cells, macrophages, or neutrophils.

Target Cells and Target cell proteins Expressed on Target Cells

[ 00202] As explained above, a bispecific binding construct can bind to an effector cell protein and a target cell protein. The target cell protein can, for example, be expressed on the surface of a cancer cell, a cell infected with a pathogen, or a cell that mediates a disease, for example an inflammatory, autoimmune, and/or fibrotic condition. In some embodiments, the target cell protein can be highly expressed on the target cell, although high levels of expression are not necessarily required.

[ 00203] Where the target cell is a cancer cell, a bispecific binding construct as described herein can bind to a cancer cell antigen as described above. A cancer cell antigen can be a human protein or a protein from another species. For example, a bispecific binding construct may bind to a target cell protein from a mouse, rat, rabbit, new world monkey, and/or old-world monkey species, among many others. Such species include, without limitation, the following species: Mus musculus; Rattus rattus; Rattus norvegicus; cynomolgus monkey, Macaca fascicularis; the hamadryas baboon, Papio hamadryas; the Guinea baboon, Papio papio; the olive baboon, Papio anubis; the yellow baboon, Papio cynocephalus; the Chacma baboon, Papio ursinus, Callithrix jacchus, Saguinus oedipus, and Saimiri sciureus.

[ 00204 ] In some examples, the target cell protein can be a protein selectively expressed on an infected cell. For example, in the case of an FIBV or FICV infection, the target cell protein can be an envelope protein of FIBV or FICV that is expressed on the surface of an infected cell. In other embodiments, the target cell protein can be gpl20 encoded by human immunodeficiency virus (HIV) on HIV-infected cells.

[00205] In other aspects, a target cell can be a cell that mediates an autoimmune or inflammatory disease. For example, human eosinophils in asthma can be target cells, in which case, EGF-like module containing mucin-like hormone receptor (EMR1), for example, can be a target cell protein. Alternatively, excess human B cells in a systemic lupus erythematosus patient can be target cells, in which case CD19 or CD20, for example, can be a target cell protein. In other autoimmune conditions, excess human Th2 T cells can be target cells, in which case CCR4 can, for example, be a target cell protein. Similarly, a target cell can be a fibrotic cell that mediates a disease such as atherosclerosis, chronic obstructive pulmonary disease (COPD), cirrhosis, scleroderma, kidney transplant fibrosis, kidney allograft nephropathy, or a pulmonary fibrosis, including idiopathic pulmonary fibrosis and/or idiotypic pulmonary hypertension. For such fibrotic conditions, fibroblast activation protein alpha (FAP alpha) can, for example, be a target cell protein.

[ 00206] Exemplary sequences of a binding domain that binds to an exemplary target cell proteins (mesothelin) is provided herein in SEQ ID NOs: 52 and 53.

Therapeutic methods and compositions

[00207] Bispecific binding constructs can be used to treat a wide variety of conditions including, for example, various forms of cancer, infections, autoimmune or inflammatory conditions, and/or fibrotic conditions.

[00208] Accordingly, in an embodiment provided herein are bispecific binding constructs for use in the prevention, treatment, or amelioration of a disease.

[00209] Another embodiment provides the use of the binding construct of the invention (or of the binding construct produced according to the process of the invention) in the manufacture of a medicament for the prevention, treatment or amelioration of a disease.

[ 00210] Provided herein are pharmaceutical compositions comprising bispecific binding constructs. These pharmaceutical compositions comprise a therapeutically effective amount of a bispecific binding construct and one or more additional components such as a physiologically acceptable carrier, excipient, or diluent. In some embodiments, these additional components can include buffers, carbohydrates, polyols, amino acids, chelating agents, stabilizers, and/or preservatives, among many possibilities.

[00211] In some embodiments, a bispecific binding construct can be used to treat cell proliferative diseases, including cancer, which involve the unregulated and/or inappropriate proliferation of cells, sometimes accompanied by destruction of adjacent tissue and growth of new blood vessels, which can allow invasion of cancer cells into new areas, i.e. metastasis. Included within conditions treatable with a bispecific binding construct are non- malignant conditions that involve inappropriate cell growth, including colorectal polyps, cerebral ischemia, gross cystic disease, polycystic kidney disease, benign prostatic hyperplasia, and endometriosis. A bispecific binding construct can be used to treat a hematologic or solid tumor malignancy. More specifically, cell proliferative diseases that can be treated using a bispecific binding construct are, for example, cancers including mesotheliomas, squamous cell carcinomas, myelomas, osteosarcomas, glioblastomas, gliomas, carcinomas, adenocarcinomas, melanomas, sarcomas, acute and chronic leukemias, lymphomas, and meningiomas, Hodgkin's disease, Sezary syndrome, multiple myeloma, and lung, non-small cell lung, small cell lung, laryngeal, breast, head and neck, bladder, ovarian, skin, prostate, cervical, vaginal, gastric, renal cell, kidney, pancreatic, colorectal, endometrial, and esophageal, hepatobiliary, bone, skin, and hematologic cancers, as well as cancers of the nasal cavity and paranasal sinuses, the nasopharynx, the oral cavity, the oropharynx, the larynx, the hypolarynx, the salivary glands, the mediastinum, the stomach, the small intestine, the colon, the rectum and anal region, the ureter, the urethra, the penis, the testis, the vulva, the endocrine system, the central nervous system, and plasma cells.

[ 00212] Among the texts providing guidance for cancer therapy is Cancer, Principles and Practice of Oncology, 4th Edition, DeVita et al., Eds. J. B. Lippincott Co., Philadelphia, PA (1993). An appropriate therapeutic approach is chosen according to the particular type of cancer, and other factors such as the general condition of the patient, as is recognized in the pertinent field. A bispecific binding construct can be added to a therapy regimen using other anti-neoplastic agents in treating a cancer patient.

[ 00213] In some embodiments, a bispecific binding construct can be administered concurrently with, before, or after a variety of drugs and treatments widely employed in cancer treatment such as, for example, chemotherapeutic agents, non-chemotherapeutic, anti-neoplastic agents, and/or radiation. For example, chemotherapy and/or radiation can occur before, during, and/or after any of the treatments described herein. Examples of chemotherapeutic agents are discussed above and include, but are not limited to, cisplatin, taxol, etoposide, mitoxantrone (Novantrone ^® ), actinomycin D, cycloheximide, camptothecin (or water soluble derivatives thereof), methotrexate, mitomycin (e.g., mitomycin C), dacarbazine (DTIC), anti-neoplastic antibiotics such as adriamycin (doxorubicin) and daunomycin, and all the chemotherapeutic agents mentioned above.

[ 00214 ] A bispecific binding construct can also be used to treat infectious disease, for example a chronic hepatis B virus (HBV) infection, a hepatis C virus (HCV) infection, a human immunodeficiency virus (HIV) infection, an Epstein-Barr virus (EBV) infection, or a cytomegalovirus (CMV) infection, among many others.

[00215] A bispecific binding construct can find further use in other kinds of conditions where it is beneficial to deplete certain cell types. For example, depletion of human eosinophils in asthma, excess human B cells in systemic lupus erythematosus, excess human Th2 T cells in autoimmune conditions, or pathogen-infected cells in infectious diseases can be beneficial. In a fibrotic condition, it can be useful to deplete cells forming fibrotic tissue. [00216] Therapeutically effective doses of a bispecific binding construct can be administered. The amount of bispecific binding construct that constitutes a therapeutically dose may vary with the indication treated, the weight of the patient, the calculated skin surface area of the patient. Dosing of a bispecific binding construct can be adjusted to achieve the desired effects. In many cases, repeated dosing may be required.

[00217] A bispecific binding construct, or a pharmaceutical composition containing such a molecule, can be administered by any feasible method. Protein therapeutics will ordinarily be administered by a parenteral route, for example by injection, since oral administration, in the absence of some special formulation or circumstance, would lead to hydrolysis of the protein in the acid environment of the stomach. Subcutaneous, intramuscular, intravenous, intraarterial, intralesional, or peritoneal bolus injection are possible routes of administration. A bispecific binding construct can also be administered via infusion, for example intravenous or subcutaneous infusion. Topical administration is also possible, especially for diseases involving the skin. Alternatively, a bispecific binding construct can be administered through contact with a mucus membrane, for example by intra-nasal, sublingual, vaginal, or rectal administration or administration as an inhalant. Alternatively, certain appropriate pharmaceutical compositions comprising a bispecific binding construct can be administered orally.

[ 00218] The term "treatment" encompasses alleviation of at least one symptom or other embodiment of a disorder, or reduction of disease severity, and the like. A bispecific binding construct according to the present invention need not effect a complete cure, or eradicate every symptom or manifestation of a disease, to constitute a viable therapeutic agent. As is recognized in the pertinent field, drugs employed as therapeutic agents may reduce the severity of a given disease state, but need not abolish every manifestation of the disease to be regarded as useful therapeutic agents. Simply reducing the impact of a disease (for example, by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease will occur or worsen in a subject, is sufficient. One embodiment of the invention is directed to a method comprising administering to a patient a bispecific binding construct of the invention in an amount and for a time sufficient to induce a sustained improvement over baseline of an indicator that reflects the severity of the particular disorder.

[00219] The term "prevention" encompasses prevention of at least one symptom or other embodiment of a disorder, and the like. A prophy lactical ly administered treatment incorporating a bispecific binding construct according to the present invention need not be completely effective in preventing the onset of a condition in order to constitute a viable prophylactic agent. Simply reducing the likelihood that the disease will occur or worsen in a subject, is sufficient.

[ 00220] As is understood in the pertinent field, pharmaceutical compositions comprising the bispecific binding construct are administered to a subject in a manner appropriate to the indication and the composition. Pharmaceutical compositions may be administered by any suitable technique, including but not limited to parenterally, topically, or by inhalation. If injected, the pharmaceutical composition can be administered, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes, by bolus injection, or continuous infusion. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the bispecific binding construct in aerosol form, and the like. Other alternatives include oral preparations including pills, syrups, or lozenges. [00221] The bispecific binding constructs can be administered in the form of a composition comprising one or more additional components such as a physiologically acceptable carrier, excipient or diluent. Optionally, the composition additionally comprises one or more physiologically active agents. In various particular embodiments, the composition comprises one, two, three, four, five, or six physiologically active agents in addition to one or more bispecific binding constructs.

[00222] Kits for use by medical practitioners are provided including one or more bispecific binding construct and a label or other instructions for use in treating any of the conditions discussed herein. In one embodiment, the kit includes a sterile preparation of one or more bispecific binding constructs which may be in the form of a composition as disclosed herein, and may be in one or more vials.

[00223] Dosages and the frequency of administration may vary according to such factors as the route of administration, the particular bispecific binding construct employed, the nature and severity of the disease to be treated, whether the condition is acute or chronic, and the size and general condition of the subject.

[ 00224 ] Having described the invention in general terms above, the following examples are offered by way of illustration and not limitation.

EXAMPLES

[ 00225] Example 1: Expression and Purification of Bispecific Constructs

[00226] Msln Chain Permutation

[00227] Designed, expressed and purified were a panel of bispecific binding constructs that target mesothelin (MSLN) in all 24 possible configurations depicted in Figure 1. These include: the canonical, or wildtype (WT) BiTE molecule (H1L1H2L2) and 23 different configurations. All the various heavy (HI or H2) or light (LI and L2) chains are connected with GlyGlyGlySer (G4S) (SEQ ID NO: 1) linkers to link the various chains that comprise the BiTE. In the WT BiTE the heavy and light chains (HI and LI) of the target domain were connected by three G4S repeats (SEQ ID NO: 3). The anti-CD3 domain also comprised a heavy and light chain pair (H2 and L2) and was connected by three G4S repeats (SEQ ID NO: 3). To connect the anti-target and anti-CD3 scFv domains, a single G4S linker (SEQ ID NO: 1) was needed. This can alternatively be described with the following nomenclature: H1-(G4S)3- L1-(G4S)I-H2-(G4S)3-L2. TO accommodate for protein folding the remaining 23 chain permutations utilized the (G4S)4 linkers (SEQ ID NO: 4) at every linker position.

[ 00228] Plasmids:

[ 00229] Expression plasmids harboring the BiTE genes of interest with an N-terminal signal peptide were cloned into the pTT5 vector.

[ 00230] Expression and Purification:

[ 00231] All BiTE proteins were produced using transiently transfected HEK293-6E cells. Briefly, plasmid DNA encoding the BiTE target sequence with an N-terminal signal secretion peptide were introduced into cells at ~99.9% viability and 1.5e6 cell density with PEI MAX transfection reagent. Cells were maintained at 37°C, 5% CO2, 150 RPM for 6 days for protein overproduction. Cells were then harvested by centrifugation (4000 RPM for 30 mins) and then resulting cell media supernatants were filtered and stored for purification. [ 00232] BiTEs were purified using Protein L affinity chromatography (GE Healthcare, HiTrap Protein L). Protein L resin was equilibrated in binding buffer 25 mM Tris, 100 mM NaCI, pH 7.4, and proteins were eluted using 100 mM Sodium Acetate, pH 3.6. To rapidly remove BiTEs from the elution buffer a desalting step (GE Healthcare, HiPrep 26/10 Desalting) into 10 mM Potassium Phosphate, 75 mM Lysine, 4% Trehalose, pH 8.0 was carried out prior to separation by size exclusion chromatography (GE Healthcare, HiLoad Superdex 200). Purity was verified by SDS-PAGE. Titer was estimated by densitometry analysis done in triplicate as compared to expression standards. Results are depicted in Figure 5. Proteins were then formulated with 10 mM L-Glutamic Acid, 9% sucrose, 0.01% Polysorbate 80, pH 4.2 at a concentration of 1 mg/ml. Proteins were stored at -80°C prior to use.

[ 00233] Example 2: Chemical Stability of the Bispecific Constructs

[ 00234 ] Isothermal Chemical Denaturation

[ 00235] Determination of BiTE chemical stability was carried out via isothermal chemical denaturation ("ICD") by monitoring intrinsic protein fluorescence. ICD yields Cl/2 and AG which can be good metrics for protein stability. Cl/2 is the amount of chemical denaturant required to denature 50% of the protein and is used to derive AG (or unfolding energy). To monitor protein unfolding as a function of chemical denaturant we measured intrinsic protein fluorescence. This process was fully automated by utilizing the HUNK instrument (Unchained Labs). 32 independent denaturation data points ranging from 0 to 5.52 M Guanidine HCI, GuHCI, were generated and the resulting 350/330 nm fluorescence intensity ratio was plotted and fit to determine fraction denatured and derive Cl/2, and AG values. Results are depicted in Figure 6. Plots show normalized data fits. Data was fit to either a 2-state or 3-state model.

[ 00236] Example 3: Thermal Stability of the Bispecific Constructs

[ 00237] Differential Scanning Fluorimeter Tm Measurements

[00238] To determine the various Tm values of our BiTE proteins we utilized the fully automated UNcle platform (Unchained Labs) which allowed for acquisition of intrinsic protein fluorescence during thermal ramp. Briefly, protein samples at 1 mg/ml underwent a thermal ramp from 20-90°C during data acquisition. Tm values were derived from an average of three replicates using the UNcle analysis software and further validated by taking the first derivate of the dataset in Prism GraphPad. Results are depicted in Figure 7.

[00239] Accelerated Stress Studies

[00240] Performance of the various Msln chain permutation proteins in accelerated stress conditions was assessed. The panel of BiTEs was incubated at 40°C and aggregation was measured at timepoints TO, 2 weeks (2W) and 4 weeks (4W) by analytical size exclusion chromatography (SEC). UV absorbance was monitored at 220 nm. Separated species/peaks were quantified using the Chromeleon (Thermo Fisher Scientific). We quantified the level of aggregation by integrating the high molecular weight (HMW), main, and low molecular weight (LMW) peaks. Results are depicted in Figure 8. For simplicity, the percent main peak quantification is shown.

[00241] Example 4: Monitoring Protein Clipping by Reduced Capillary Electrophoresis

[00242] In addition to investigating accelerated stress induced aggregation, protein clipping was examined. Clipping is a critical issue for bispecific constructs as clips to linkers connecting either the target or T-cell engaging domains have terminal detrimental impact on drug potency and efficacy. For these reasons, the panel of bispecific constructs was assessed for protein clipping at TO, 2W, and 4W using reduced capillary electrophoresis (rCE). Briefly, in this assay we denature the proteins at 1 mg/mL in the presence of SDS- buffer supplemented with 2-Mercaptoethanol and heated to 70°C for 10 mins prior to separation by capillary electrophoresis (Beckman Coulter, PA800plus). Proteinaceous species were monitored by at 220 nm and peaks were integrated in Chromeleon (Thermo Fisher Sci). Results are depicted in Figure 9. For clarity, the quantified percent low molecular weight (LM W) species formation over the time course of the accelerated stress study is shown.

[ 00243] Example 5: Cell-Based Potency Assay

[ 00244 ] Target cell viability was determined via quantification of constitutively expressed firefly luciferase and was performed with the Steady-Glo Luciferase Assay System (Promega). Briefly in this assay, FluT-78 cells, a human cutaneous T cell lymphocyte cell line expressing CD3, were incubated with OVCAR-8-Luc cells, a human ovarian carcinoma cell line expressing mesothelin and engineered to constitutively express luciferase as a marker of cell number and viability. Msln BiTE proteins were diluted in triplicate ranging from 0.01 ng/mL to 5 ng/mL into a 96-well, full-area, flat bottom, tissue culture treated, sterile, white polystyrene plates (Costar, #3917). Cells were added to the plate in a 10:1 ratio of FlutT-78 T cells to OVCAR-8-Luc Msln expression cells respectively and allowed to incubate for a minimum of 24 hours prior to additional of Steady-Glo reagent. Per Promega's protocol, reconstituted Steady-Glo reagent was added (25 pL per well), and assay plates were incubated for 30 min at room temperature. Luminescence was quantified with an EnVision multilabel reader (Perkin Elmer) with an ultrasensitive luminescence detector. Data was normalized to 100% using Msln-WT as a reference/control. Results are depicted in Figure 10.

[ 00245] Each and every reference cited herein is incorporated herein by reference in its entirety for all purposes.

[ 00246] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual embodiments of the invention, and functionally equivalent methods and components of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the claims.

SEQUENCES

[ 00247] Exemplary Linker Sequences GGGGS (SEQ ID NO: 1)

GGGGSGGGGS (SEQ ID NO: 2)

GGGGSGGGGSGGGGS (SEQ ID NO: 3)

GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4)

GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 5)

GGGGQ (SEQ ID NO: 6)

GGGGQGGGGQ (SEQ ID NO: 7)

GGGGQGGGGQGGGGQ (SEQ ID NO: 8)

GGGGQGGGGQGGGGQGGGGQ (SEQ ID NO: 9)

GGGGQGGGGQGGGGQGGGGQGGGGQ (SEQ ID NO: 10)

GGGGSAAA (SEQ ID NO: 11)

TVAAP (SEQ ID NO: 12)

ASTKGP (SEQ ID NO: 13)

AAA

GGNGT (SEQ ID NO: 15)

YGNGT (SEQ ID NO: 16)

[ 00248] 24 MSLN Bispecific Binding Construct Permutation Protein Sequences

[ 00249] Note: The sequences contain an N-terminal signal peptide which is removed during expression. The Signal Peptide Sequence is: MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 17). [ 00250 ] H1L1H2L2 (WT)

[ 00251 ] Protein:

[ 00252 ] MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMT

WIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTA VYYCARDRNSHFD

YWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGIN TWLAWYQQK

PGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPR TFGQGTKVEIKSGGG

GSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKY NNYATYYADS

VKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVT VSSGGGGSGG

GGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGL IGGTKFLAPGT

PARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL* (SEQ ID NO: 18)

[ 00253] H1H2L1L2 [ 00254 ] Protein:

[ 00255] MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMT

WIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTA VYYCARDRNSHFD

YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAAS GFTFNKYAM

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTE DTAVYYCVRHG

NFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSAS VGDRVTITCR

ASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQAKSF

PRTFGQGTKVEIKSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCG SSTGAVTSGNY

PNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCV LWYSNRWVFGG

GTKLTVL* (SEQ ID NO: 19)

[ 00256] H1L2L1H2 [ 00257 ] Protein:

[ 00258 ] MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMT

WIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTA VYYCARDRNSHFD

YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSS TGAVTSGNYP

NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVL WYSNRWVFGG

GTKLTVLGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGIN TWLAWYQQKP

GKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRT FGQGTKVEIKSGGG

GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQA PGKGLEWV ARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWG QGTLVTVSS* (SEQ ID NO: 20)

[ 00259] L1H2H1L2 [ 00260 ] Protein:

MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCRASQGINTWLAW YQQKPGKAPK

LLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRTFGQGT KVEIKSGGGGSGGG

GSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGL EWVARIRSKY

NNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYW AYWGQGTLVTV

SSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMT WIRQAPGKGL

EWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDRNS HFDYWGQGTLVTV

SSGGGGSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVT SGNYPNWVQ

QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSN RWVFGGGTKLTV

L* (SEQ ID NO: 21)

[ 00261 ] L1L2H1H2 [ 00262 ] Protein:

[ 00263] MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCRASQGINTWLA

WYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQA KSFPRTFGQGTKV

EIKSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGN YPNWVQQKPG

QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVLGGG

GSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQA PGKGLEWLSYI

SSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDRNSHFDYWG QGTLVTVSSGGG

GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQA PGKGLEWV

ARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWG

QGTLVTVSS* (SEQ ID NO: 22)

[ 00264 ] L2H1H2L1 [ 00265] Protein:

[ 00266] MDMRVPAQLLGLLLLWLRGARCQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYS NRWVFGG GTKLTVLGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY MTWIRQA PGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDR NSHFDYWGQG

TLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFN KYAMNWVRQ

APGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSY

ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVT ITCRASQGIN

TWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKSFPRTFGQ

GTKVEIKS* (SEQ ID NO: 23)

[ 00267 ] H1L1L2H2 [ 00268 ] Protein:

[ 00269] MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMT

WIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTA VYYCARDRNSHFD

YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRA SQGINTWLA

WYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQA KSFPRTFGQGTKV

EIKSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGN YPNWVQQKPG

QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVLGGG

GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQA PGKGLEWV

ARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWG

QGTLVTVSS* (SEQ ID NO: 24)

[ 00270 ] H2L2L1H1 [ 00271 ] Protein:

[ 00272 ] MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAM

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTE DTAVYYCVRHG

NFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSP GGTVTLTCGS

STGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVL

WYSNRWVFGGGTKLTVLGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVT ITCRASQGI

NTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQAKSFPRTFG

QGTKVEIKSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFT FSDYYMTWIR

QAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYY CARDRNSHFDYWG

QGTLVTVSS* (SEQ ID NO: 25)

[ 00273] H2L2H1L1 [ 00274 ] Protein:

[ 00275] MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAM

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTE DTAVYYCVRHG

NFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSP GGTVTLTCGS

STGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVL

WYSNRWVFGGGTKLTVGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLS CAASGFTFS

DYYMTWIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLR AEDTAVYYCARDR

NSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVT ITCRASQGIN

TWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKSFPRTFGQ

GTKVEIKS* (SEQ ID NO: 26)

[ 00276] L1H1H2L2 [ 00277 ] Protein:

[ 00278 ] MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCRASQGINTWLA

WYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQA KSFPRTFGQGTKV

EIKSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY MTWIRQAPGK

GLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDR NSHFDYWGQGTLV

TVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPG

KGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISY

WAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCG SSTGAVTSG

NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYY CVLWYSNRWVF

GGGTKLTVL* (SEQ ID NO: 27)

[ 00279] L1H1L2H2 [ 00280 ] Protein:

[ 00281 ] MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCRASQGINTWLA

WYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQA KSFPRTFGQGTKV

EIKSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY MTWIRQAPGK

GLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDR NSHFDYWGQGTLV

TVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGN YPNWVQQKP

GQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWV FGGGTKLTVLGG

GGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQ APGKGLEW VARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNS YISYWAYWG QGTLVTVSS* (SEQ ID NO: 28)

[ 00282 ] L2H2L1H1 [ 00283] Protein:

[ 00284 ] MDMRVPAQLLGLLLLWLRGARCQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP

NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVL WYSNRWVFGG

GTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFN KYAMNWVRQ

APGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSY

ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVT ITCRASQGIN

TWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKSFPRTFGQ

GTKVEIKSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTF SDYYMTWIRQ

APGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYC ARDRNSHFDYWGQ

GTLVTVSS* (SEQ ID NO: 29)

[ 00285] L2H2H1L1 [ 00286] Protein:

[ 00287 ] MDMRVPAQLLGLLLLWLRGARCQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP

NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVL WYSNRWVFGG

GTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFN KYAMNWVRQ

APGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSY

ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRL SCAASGFTFS

DYYMTWIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLR AEDTAVYYCARDR

NSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVT ITCRASQGIN

TWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAKSFPRTFGQ

GTKVEIKS* (SEQ ID NO: 30)

[ 00288 ] H1H2L2L1 [ 00289] Protein:

[ 00290 ] MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMT WIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYY CARDRNSHFD YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAM NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTA VYYCVRHG

NFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSP GGTVTLTCGS

STGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVL

WYSNRWVFGGGTKLTVLGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVT ITCRASQGI

NTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQAKSFPRTFG

QGTKVEIKS* (SEQ ID NO: 31)

[ 00291 ] H1L2H2L1 [ 00292 ] Protein:

[ 00293] MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMT

WIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTA VYYCARDRNSHFD

YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSS TGAVTSGNYP

NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVL WYSNRWVFGG

GTKLTVGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQA

PGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYY CVRHGNFGNSYI

SYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTI TCRASQGINT

WLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAKSFPRTFGQG

TKVEIKS* (SEQ ID NO: 32)

[ 00294 ] H2H1L1L2 [ 00295] Protein:

[ 00296] MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAM

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTE DTAVYYCVRHG

NFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKP GGSLRLSCA

ASGFTFSDYYMTWIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLF LQMNSLRAEDTAVY

YCARDRNSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSAS VGDRVTITCR

ASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQAKSF

PRTFGQGTKVEIKSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCG SSTGAVTSGNY

PNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCV LWYSNRWVFGG

GTKLTVL* (SEQ ID NO: 33)

[ 00297 ] H2L1H1L2 [ 00298 ] Protein:

[ 00299] MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAM

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTE DTAVYYCVRHG

NFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSAS VGDRVTITCR

ASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQAKSF

PRTFGQGTKVEIKSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCA ASGFTFSDYYM

TWIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDT AVYYCARDRNSHF

DYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGS STGAVTSGNY

PNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCV LWYSNRWVFGG

GTKLTVL* (SEQ ID NO: 34)

[ 00300 ] L1H2L2H1 [ 00301 ] Protein:

[ 00302 ] MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCRASQGINTWLA

WYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQA KSFPRTFGQGTKV

EIKSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPG

KGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCV RHGNFGNSYISY

WAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCG SSTGAVTSG

NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYY CVLWYSNRWVF

GGGTKLTVLGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFT FSDYYMTWIR

QAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYY CARDRNSHFDYWG

QGTLVTVSS* (SEQ ID NO: 35)

[ 00303] L1L2H2H1 [ 00304 ] Protein:

[ 00305] MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCRASQGINTWLA

WYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQA KSFPRTFGQGTKV

EIKSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGN YPNWVQQKPG

QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVGGGG

SGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVA

RIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGN SYISYWAYWGQ

GTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTF SDYYMTWIRQ APGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARD RNSHFDYWGQ GTLVTVSS* (SEQ ID NO: 36)

[ 00306] L2H1L1H2 [ 00307 ] Protein:

[ 00308 ] MDMRVPAQLLGLLLLWLRGARCQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP

NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVL WYSNRWVFGG

GTKLTVLGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMTWIRQA

PGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCA RDRNSHFDYWGQG

TLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGIN TWLAWYQQKP

GKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRT FGQGTKVEIKSGGG

GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQA PGKGLEWV

ARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWG

QGTLVTVSS* (SEQ ID NO: 37)

[ 00309] L2L1H1H2 [ 00310 ] Protein:

[ 00311 ] MDMRVPAQLLGLLLLWLRGARCQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP

NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVL WYSNRWVFGG

GTKLTVLGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGIN TWLAWYQQKP

GKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRT FGQGTKVEIKSGGG

GSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQA PGKGLEWLSYI

SSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDRNSHFDYWG QGTLVTVSSGGG

GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQA PGKGLEWV

ARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWG

QGTLVTVSS* (SEQ ID NO: 38)

[ 00312 ] L2L1H2H1 [ 00313] Protein:

[ 00314 ] MDMRVPAQLLGLLLLWLRGARCQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYS NRWVFGG GTKLTVLGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGINTWL AWYQQKP GKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRTFGQ GTKVEIKSGGG

GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQA PGKGLEWV

ARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWG

QGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFT FSDYYMTWIR

QAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYY CARDRNSHFDYWG

QGTLVTVSS* (SEQ ID NO: 39)

[ 00315] H2H1L2L1 [ 00316] Protein:

[ 00317 ] MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAM

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTE DTAVYYCVRHG

NFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKP GGSLRLSCA

ASGFTFSDYYMTWIRQAPGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLF LQMNSLRAEDTAVY

YCARDRNSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSP GGTVTLTCGS

STGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVL

WYSNRWVFGGGTKLTVLGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVT ITCRASQGI

NTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQAKSFPRTFG

QGTKVEIKS* (SEQ ID NO: 40)

[ 00318 ] H2L1L2H1 [ 00319] Protein:

[ 00320 ] MDMRVPAQLLGLLLLWLRGARCEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAM

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTE DTAVYYCVRHG

NFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSAS VGDRVTITCR

ASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQAKSF

PRTFGQGTKVEIKSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCG SSTGAVTSGNY

PNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCV LWYSNRWVFGG

GTKLTVLGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMTWIRQA

PGKGLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCA RDRNSHFDYWGQG

TLVTVSS* (SEQ ID NO: 41)

[ 00321 ] IgGl Fc (SEQ ID NO: 42) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRE

EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSC

SVMHEALHNHYTQKSLSLSPGK

[ 00322] lgG2 Fc (SEQ ID NO: 43)

ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN WYVDGMEVHNA

KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSC

SVMHEALHNHYTQKSLSLSPGK

[ 00323] lgG3 Fc (SEQ ID NO: 44)

ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQF

KWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKT

ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYN TTP

PMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[ 00324 ] lgG4 Fc (SEQ ID NO: 45)

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNA

KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEE

MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCS

VMHEALHNHYTQKSLSLSLGK

[ 00325] Amino acid sequence of the mature human CD3s (SEQ ID NO: 46)

QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSD EDHLSLKEFSELE

QSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLV YYWSKNRKAKAKP

VTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

[ 00326] Amino acid sequence of the mature CD3s of cynomolgus monkey (SEQ ID

NO: 47)

QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKGDSGDQLFLPEF SEMEQSGYYVC

YPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNR KAKAKPVTRGAG

AGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI [ 00327] Amino acid sequence of the extracellular domain of human CD3s (SEQ ID NO: 48)

QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSD EDHLSLKEFSELE

QSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMS

[ 00328] Amino acids 1-27 of human CD3s (SEQ ID NO: 49)

QDGNEEMGGITQTPYKVSISGTTVILT

[ 00329] CD3 Binder Heavy (SEQ ID NO: 50)

EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNN YATYYADSVK

DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS

[ 00330] CD3 Binder Light (SEQ ID NO: 51)

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLA PGTPARFSGSLL

GGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL

[ 00331 ] MSLN Binder Heavy (SEQ ID NO: 52)

QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWLSYISSSGST IYYADSVKGRFTIS

RDNAKNSLFLQMNSLRAEDTAVYYCARDRNSHFDYWGQGTLVTVS

[ 00332] MSLN Binder Light (SEQ ID NO: 53)

DIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSG VPSRFSGSGSGT DFTLTISS LQP E D FATYYCQQAKSF P RTFG QGTKVE I K