BIESPECIFIC JOINT CONSTRUCTIONS WITH SELECTIVELY SEVERABLE CONNECTORS

Abstract

The present invention relates to a bispecific binding construct comprising a single polypeptide chain with the VH1-L1-scFcSubdomain1-L2-VH2-L3-VL1-L4-scFcSubdomain2-L5-VL2 molecular architecture. The molecular architecture incorporates variable regions of immunoglobulin heavy and light chains, two scFc subdomains derived from heavy chain constant domains, and a series of linkers of defined lengths, some of which include protease cleavage sites. The construct is designed to simultaneously bind to a human T lymphocyte (via a TCR-CD3 complex) and a cancerous target cell. Furthermore, it integrates at least one cysteine ​​clamp to stabilize the interactions between the subunits. Preferred variants include specific VH and VL sequences, as well as additional moieties linked by an L0 linker. Nucleic acids, vectors, and host cells for its expression, as well as a method of manufacturing, are described.The construct is useful in the preparation of drugs for the treatment of cancer, optionally in combination with chemotherapy, antineoplastic agents or radiotherapy, and can be formulated into pharmaceutical compositions.

Description

BIESPECIFIC JOINT CONSTRUCTIONS WITH SELECTIVELY SPLITTING LINKAGES REFERENCE TO RELATED APPLICATIONS This application claims priority over U.S. Provisional Application No. 62 / 858,509, filed on June 7, 2019, and U.S. Provisional Application No. 62 / 858,630, filed on June 7, 2019. Each of the above-identified applications is incorporated herein by reference for all purposes. REFERENCE TO THE LIST OF SEQUENCES This application contains a sequence list, which was submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on June 4, 2020, is named A-2406-WO-PCT_SL.txt and has a size of 164,621 bytes. FIELD The invention belongs to the field of protein engineering. BACKGROUND Bispecific binding constructs have shown therapeutic promise in recent years. For example, a bispecific binding construct targeting both CD3 and CD19 in a bispecific T-cell coupler (BiTE®) format has demonstrated impressive efficacy at low doses. Bargou et al. (2008), Science 321: 974-978. This BiTE® format comprises two scFvs, one targeting CD3 and the other targeting a tumor antigen, CD19, linked by a flexible linker. This unique design allows the bispecific binding construct to bring activated T cells closer to target cells, resulting in cytolytic cell death. See, for example, documents WO 99 / 54440A1 (U.S. Patent No. 7,112,324 B1) and WO 2005 / 040220 (U.S. Patent Application No. 2013 / 0224205A1).Later developments were bispecific binding constructs that bound to a context-independent epitope at the N-end of the CD3e string (see WO 2008 / 119567; U.S. Patent Sol. Pub. No. 2016 / 0152707A1). In the biopharmaceutical industry, molecules can have harmful and undesirable side effects in patients receiving treatment, particularly when the drug is active after administration. In small-molecule pharmaceutical agents, for example, these side effects can be minimized by administering inactive prodrugs that are activated upon metabolism. Bispecific binding constructs that mediate cellular cytotoxicity can exhibit some of these undesirable side effects. Therefore, there is a need in technology for bispecific therapies with favorable pharmacokinetic properties, therapeutic efficacy, and a formulation that provides efficient production, greater stability, and minimized side effects. SUMMARY This document describes several novel 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 the formula VH1-L1-VH2-L2-VL1-L3-VL2, wherein VH1 and VH2 comprise immunoglobulin heavy chain variable regions, VL1 and VL2 comprise immunoglobulin light chain variable regions, and L1, L2, and L3 are linkers, wherein L1 has at least 10 amino acids, L2 has at least 15 amino acids, and L3 has at least 10 amino acids, wherein L1 or L3 comprises a protease cleavage site, and wherein the bispecific binding construct can bind to an immune effector cell and a target cell. In another embodiment, the invention provides a bispecific linker construct comprising a polypeptide chain comprising an amino acid sequence having the formula VH1-L1-Fc-L2-VH2-L3-VL1-L4-Fc-L5-VL2, wherein VH1 and VH2 comprise immunoglobulin heavy chain variable regions, VL1 and VL2 comprise immunoglobulin light chain variable regions, Fc comprises an immunoglobulin heavy chain constant domain 2 and an immunoglobulin heavy chain constant domain 3, and L1, L2, L3, L4, and L5 are linkers, wherein L1 has at least 10 amino acids, L2 has at least 10 amino acids, L3 has at least 15 amino acids, L4 has at least 10 amino acids, and L5 has at least 10 amino acids, and wherein L1, L2, L4, and L5 further comprise a protease cleavage site of at least 5 amino acids, and wherein The bispecific junction construct can bind to an immune effector cell and a target cell. In further embodiments, the invention provides a nucleic acid encoding the bispecific junction constructs described herein, and vectors comprising these nucleic acids. The invention further provides a host cell comprising the vectors described herein. In other embodiments, the invention provides a method for manufacturing the bispecific junction constructs described herein comprising (1) growing a host cell under conditions expressing the bispecific junction construct and (2) recovering the bispecific junction construct from the supernatant of the cell mass or cell culture, wherein the host cell comprises one or more nucleic acids encoding any of the bispecific junction constructs described herein. In other embodiments, the invention provides a method for treating a cancer patient, comprising administering to the patient a therapeutically effective amount of the bispecific junction constructs described herein. In other embodiments, the invention provides a method for treating a patient having an infectious disease comprising administering to the patient a therapeutically effective amount of the bispecific junction constructs described herein. In other embodiments, the invention provides a method for treating a patient having an autoimmune, inflammatory, or fibrotic condition comprising administering to the patient a therapeutically effective amount of the bispecific junction constructs described herein. In another embodiment, the invention provides a pharmaceutical composition comprising the bispecific binding constructs described herein. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. A representative diagram of an example embodiment of HHLL formats A and B, and indicating where the protease cleavage sites, cysteine ​​clamps, and optional CD3c (for formats A and B) and scFc (for format A) residues are located. Figure 2. A representative diagram of an example embodiment of HHLL formats C and D, and indicating where the protease cleavage sites, cysteine ​​clamps and optional CD3e residues (for format C), optional HSA-CD3aai-6oi-27 (for format D) and scFc (for formats C and D) are located. Figure 3. A representative diagram of an example embodiment of an HHLL E format showing where the protease cleavage sites, cysteine ​​clamps, and optional scFc-CD3e moiety are located. Figure 4. A chromatography reading indicating adequate expression of the bispecific construct N4J. Figures 5. A chromatography and SDS-PAGE reading indicating adequate expression of the bispecific construct N7A. Figure 6. A chromatography and SDS-PAGE reading that indicates the expression of the bispecific construct V1E, but with a lower molecular weight than expected. Figure 7. A chromatography and SDS-PAGE reading indicating adequate expression of the bispecific construct B1U. Figure 8. A chromatography and SDS-PAGE reading indicating adequate expression of the bispecific Z9P construct. Figure 9. A chromatography and SDS-PAGE reading indicating adequate expression of the bispecific O7H construct. IVIA / a / ZUZ II Figure 10. A chromatography and SDS-PAGE reading that adequately shows the bispecific construct W9A. Figure 11. A chromatography and SDS-PAGE reading that adequately shows the bispecific B2P construct. Figure 12. A chromatography and SDS-PAGE reading that adequately shows the bispecific T7U construct. Figure 13. A chromatography and SDS-PAGE reading indicating the expression of the appropriate expression of the bispecific construct L2G. Figure 14A. SDS-PAGE of bispecific constructs (N4J, W2K, N7A, W9A and B2P) in the presence or absence of recombinant human MMP-9. Figure 14B. SDS-PAGE of bispecific constructs (W2K, Z9P, V1E, B1U, T7U and L2G) in the presence or absence of recombinant human MMP-9. Figures 15A and 15B. FACS analysis of binding to CD3-expressing cells (Figure 15A) and mesothelin-expressing cells (Figure 15B) using the bispecific N4J construct with and without protease activation. Figure 16. FACS analysis of binding to CD3 and mesothelin positive cells using the N7A bispecific construct with and without protease activation. Figure 17. FACS analysis of binding to CD3 and mesothelin positive cells using the bispecific constructs W2K, V1E without protease activation, B1U, Z9P with and without protease activation. Figure 18. FACS analysis of binding to CD3-positive cells using the bispecific constructs B2P, W9A, and N7A with and without protease activation. Figure 19. FACS analysis of binding to mesothelin-positive cells using the bispecific constructs B2P, W9A and N7A with and without protease activation. Figure 20. FACS-based in vitro cytotoxicity assay of the bispecific constructs N4J and W2K with and without protease activation. Figure 21. FACS-based in vitro cytotoxicity assay of the bispecific constructs N7A, W2K and negative control with and without protease activation. Figure 22. FACS-based in vitro cytotoxicity assay of bispecific constructs Z9P, V1E, B1U and negative control with and without protease activation. Figure 23. FACS-based in vitro cytotoxicity assay of bispecific constructs W9A, B2P, N7A with and without protease activation. Figure 24. FACS-based in vitro cytotoxicity assay of the bispecific constructs N7A, O7H and B2P with and without protease activation. Figure 25. FACS-based in vitro cytotoxicity assay of the bispecific constructs T7U, L2G, N7A and B2P with and without protease activation. Figure 26. Summary of CE50 ranges, CE50 shift factor, and number of in vitro cytotoxicity assays performed for each bispecific construct with and without protease activation. DETAILED DESCRIPTION This document describes novel formats for bispecific joint constructions. Figures 1-3 represent representative example formats (EA) of these constructions. In one embodiment, this format comprises a single polypeptide chain comprising two immunoglobulin variable heavy chain (VH) regions, two immunoglobulin variable light chain (VL) regions, a protease cleavage site, and optionally an Fe region, arranged in the following order: VH1-VH2-VL1-VL2 (HHLL) and more specifically, in a first format VH1-linker-VH2-linker-VL1-linkerVL2, optionally with another linker after VL2 and an scFc or other half-life extension residue, and a second format VH1-linker-CH2-CH3-linker-VH2-linker-VL1CH2-CH3-linker-VL2.This bispecific HHLL format provides both improved stability and increased in vitro expression compared to, for example, an HLHL format, while maintaining the intended function of binding the desired targets on the immune effector cell and the target cell. Therefore, the present HHLL format yields bispecific molecules that can be produced more efficiently and have greater stability—characteristics sought after in a pharmaceutical formulation. The specific numbered embodiments provided by the invention include, but are not limited to, the following: 1. A bispecific binding construct comprising a polypeptide chain comprising an amino acid sequence having the formula VH1-L1-VH2-L2-VL1-L3-VL2, wherein VH1 and VH2 comprise immunoglobulin heavy chain variable regions, VL1 and VL2 comprise immunoglobulin light chain variable regions, and L1, L2, and L3 are linkers, wherein L1 has at least 10 amino acids, L2 has at least 15 amino acids, and L3 has at least 10 amino acids, wherein L1 or L3 comprises a protease cleavage site, and wherein the bispecific binding construct can bind to an immune effector cell and a target cell. 2. A bispecific linker construct comprising a polypeptide chain comprising an amino acid sequence having the formula VH1-L1-scFcsubdomain1-L2-VH2L3-VL1-L4-scFcsubdomain2-L5-VL2, wherein VH1 and VH2 comprise immunoglobulin heavy chain variable regions, VL1 and VL2 comprise immunoglobulin light chain variable regions, scFc comprises either subdomain 1 or subdomain 2 of an immunoglobulin heavy chain constant domain 2 and an immunoglobulin heavy chain constant domain 3, and L1, L2, L3, L4, and L5 are linkers, wherein L1 has at least 10 amino acids, L2 has at least 10 amino acids, L3 has at least 15 amino acids, L4 has at least 10 amino acids, and L5 has at least 10 amino acids, and wherein L1, L2, L4 and L5 further comprise a protease cleavage site of at least 5 amino acids, and where the bispecific junction construct can bind to an immune effector cell and a target cell. 3. The bispecific junction construction of embodiment 1, wherein the protease cleavage site is present in both L1 and L3. 4. The bispecific joint construction of embodiment 1 or 3, further comprising at least one cysteine ​​clamp. 5. The bispecific junction construction of embodiment 4, wherein the cysteine ​​clamp is positioned to facilitate the junction between the VH1 and VL1 subunits, the VH2 and VL2 subunits, or the scFc subunits. 6. The bispecific joint construction of embodiment 2, further comprising at least one cysteine ​​clamp. 7. The bispecific junction construction of embodiment 6, wherein the cysteine ​​clamp is positioned to facilitate the junction between the VH1 and VL1 subunits, the VH2 and VL2 subunits and / or the scFc subunits. 8. The bispecific joint construction of any of embodiments 1-7, further comprising a half-life extension residue attached to the VL2 domain. 9. The bispecific binding construct of embodiment 8, wherein the half-life-prolonging remainder comprises an additional linker and a single-stranded immunoglobulin Fe (scFc) region encoding human lgG1, lgG2, or lgG4 antibody. 10. The bispecific linkage construction of embodiment 9, wherein the additional linker comprises a protease cleavage site. 11. The bispecific binding construct of embodiment 10, wherein the scFc polypeptide chain comprises one or more alterations that inhibit Fe gamma receptor (FcyR) binding and / or one or more alterations that prolong the half-life. 12. The bispecific junction construction of any of embodiments 1-11, wherein VH1, VH2, VL1 and VL2 have different sequences. 13. The bispecific joint construction of any of embodiments 1-12, wherein ML / a / zuzi / un i a. the sequence VH1 comprises SEQ ID NO: 65 or 67, and the sequence VL1 > your NC c comprises SEQ ID NO: 66 or 68, and the sequence VH2 comprises SEQ ID NO: 75 or 77, and the sequence VL2 comprises SEQ ID NO: 76 or 78, or b. the sequence VH1 comprises SEQ ID NO: 75 or 77, and the sequence VL1 comprises SEQ ID NO: 76 or 78, and the sequence VH2 comprises SEQ ID NO: 65 or 67, and the sequence VL2 comprises SEQ ID NO: 66 or 68. 14. The bispecific joint construction of any of embodiments 1-13, further comprising an additional remnant joined to VH1 with an additional linker (LO), wherein It has at least 5 amino acids in length. 15. The bispecific binding construct of embodiment 14, wherein the additional remainder is either a CD3e, or a human serum albumin-linker-CD3(aa 1-6) or a human serum albumin-linker-CD3(aa 1-27), or an scFc-linker-CD3c. 16. The bispecific junction construction of embodiment 14 or 15, wherein LO further comprises a protease site. 17. The bispecific joint construction of any of embodiments 1-16, wherein the linkers have different lengths. 18. The bispecific joint construction of any of embodiments 1-16, wherein the linkers have the same length. 19. The bispecific joint construction of any embodiment 1-16, wherein L1 and L2 have the same length. 20. The bispecific joint construction of any embodiment 1-16, wherein L1 and L3 have the same length. 21. The bispecific joint construction of any embodiment 1-16, wherein L2 and L3 have the same length. 22. The bispecific bonding construct of any embodiment 1-16, wherein the amino acid sequence of L1 is at least 10 amino acids in length, the amino acid sequence of L2 is at least 15 amino acids in length, and the amino acid sequence of L3 is at least 15 amino acids in length. 23. The bispecific junction construct of any embodiment 1-22, wherein the effector cell expresses an effector cell protein that is part of a human T lymphocyte receptor (TCR)-CD3 complex. 24. The bispecific binding construct of any embodiment 1-22, wherein the effector cell protein is the CD3e chain 25. A nucleic acid encoding the bispecific junction construct of any of embodiments 1-24. 26. A vector comprising the nucleic acid of embodiment 25. 27. A host cell comprising the vector of realization 26. 28. A method for manufacturing the bispecific junction construct of any of embodiments 1-24 comprising (1) growing a host cell under conditions expressing the bispecific junction construct and (2) recovering the bispecific junction construct from the supernatant of the cell mass or cell culture, wherein the host cell comprises one or more nucleic acids encoding the bispecific junction construct of any of embodiments 1-24. 29. A method for treating a cancer patient, comprising administering to the patient a therapeutically effective amount of the bispecific junction construct of any of embodiments 1-24. 30. The method of realization 29, wherein the patient is administered a chemotherapeutic agent, a non-chemotherapeutic antineoplastic agent and / or radiation at the same time, before or after administration of the bispecific junction construct. 31. A pharmaceutical composition comprising the bispecific bonding construct of any embodiment 1-24. 32. The use of the bispecific junction construction of any of embodiments 124 in the manufacture of a medicament for the prevention, treatment or improvement of a disease. 33. The bispecific bond construct of any embodiment 1-24, wherein the amino acid sequence of the bond construct comprises a sequence selected from SEQ ID NO: 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98. It should be understood that both the foregoing general description and the following detailed description are merely illustrative and explanatory 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 otherwise stated. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is not limiting. In addition, terms such as "element" or "component" encompass both elements and components comprising a unit and elements and components comprising more than one subunit, unless specifically stated otherwise. Likewise, the use of the term "portion" may include part of a remainder or the entire remainder. Unless otherwise defined herein, the scientific and technical terms used in connection with this application shall have the meanings commonly assigned to them by those skilled in the art. Furthermore, unless the context otherwise requires, singular terms shall include plurals and plural terms shall include singulars. Generally, the nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and chemistry and hybridization of The proteins and nucleic acids described herein are those already known and commonly used in the art. The methods and techniques of the present invention are generally carried out in accordance with conventional methods well known in the art and as described in various general and more specific references. Polynucleotide and polypeptide sequences are indicated using standard one- or three-letter abbreviations. Unless otherwise stated, polypeptide sequences have their amino ends on the left and their carboxy ends on the right, and single-stranded nucleic acid sequences and the top strand of double-stranded nucleic acid sequences have their 5' ends on the left and their 3' ends on the right. A particular section of a polypeptide may be designated by the number of amino acid residues, 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 may also be described by explaining how it differs from a reference sequence. DEFINITIONS The term "isolated" in reference to a molecule (where the molecule is, for example, a polypeptide, a polynucleotide, a bispecific junction construct, or an antibody) is a molecule that, by virtue of its origin or source of derivation, (1) is not associated with naturally occurring components that accompany it in its native state, (2) is substantially free of other molecules of the same species, (3) is expressed by a cell of a different species, or (4) is not present in nature. Therefore, a molecule that is chemically synthesized or expressed in a cell system different from the cell from which it naturally originates will be isolated from its naturally occurring components. A molecule can also be made substantially free of its naturally occurring components through isolation, using well-established purification techniques.The purity or homogeneity of a molecule can be assessed using several well-established methods. For example, the purity of a polypeptide sample can be tested using polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide, employing techniques well-established in the field. For certain purposes, higher resolution can be achieved using HPLC or other well-established methods for purification. The terms polynucleotide, oligonucleotide, and nucleic acid are used interchangeably and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally derived nucleotide analogs), and hybrids thereof. The nucleic acid molecule may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules of the invention comprise a contiguous open reading frame encoding an antibody, or a fragment, derivative, mutain, or variant thereof, of the invention. A vector is a nucleic acid that can be used to introduce another nucleic acid attached 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), into which additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication within a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the host cell's genome upon introduction and thus replicate along with the host genome.An expression vector is a type of vector that can direct the expression of a chosen polynucleotide. A nucleotide sequence is operatively 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 another nucleic acid to which it is operatively linked. The regulatory sequence may, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more 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). A host cell is a cell that can be used to express a nucleic acid, for example, a nucleic acid of the invention. A host cell may be a prokaryote, for example, E. coli, or it may be a eukaryote, for example, a unicellular eukaryote (for example, yeast or another fungus), a plant cell (for example, a tobacco or tomato plant cell), an animal cell (for example, 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 nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The term recombinant host cell may be used to indicate a host cell that has been transformed or transfected with a nucleic acid to be expressed.A host cell may also be a cell that contains nucleic acid but does not express it at the desired level unless a regulatory sequence is introduced into the host cell so that it operatively binds to the nucleic acid. The term host cell is understood to refer not only to the particular cell in question but also to the progeny or potential progeny of that cell. Because certain modifications may occur in successive generations due, for example, to 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. A single-stranded variable fragment (scFv) is a fusion protein in which a VL region and a VH region are joined by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain where 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. Nati. Acad. Sci. USA 85:5879-83 (1988)). When in the context of other additional residues (e.g., an Fe region), the scFv may be arranged VH-linker-VL or VL-linker-VH, for example. The term CDR refers to the complementarity-determining region (also called minimal recognition units or hypervariable regions) within variable sequences of the antibody. CDRs allow the antibody or bispecific binding construct to bind specifically to a particular antigen of interest, and the bispecific binding structures provided herein may comprise heavy-chain and / or light-chain CDRs. 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 are typically aligned by framework regions to form a structure that binds specifically to a specific epitope or domain on the target protein.From the N-terminus to the C-terminus, naturally occurring light- and heavy-chain variable regions typically conform to the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids occupying positions within 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 and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity-determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using this system. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (the ImMunoGeneTics International 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 can be incorporated into a molecule covalently or non-covalently to convert it into a bispecific bonding construct. The term "human antibody" includes antibodies that have antibody regions such as variable and constant regions or domains that substantially correspond to known human germline immunoglobulin sequences, including, for example, those described by Kabat et al. (1991) (Ibid.). 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. Human antibodies may have at least one, two, three, four, five, or more positions replaced by an amino acid residue not encoded by the human germline immunoglobulin sequence.The definition of human antibodies, as used herein, also includes fully human antibodies, comprising only human antibody sequences that are not artificially and / or genetically altered, and which can be obtained using technologies or systems known in the art, such as, for example, phage presentation technology or transgenic mouse technology, including, but not limited to, the Xenomouse. In the context of the present invention, the variable regions of a human antibody can be used in the contemplated bispecific binding construct formats. A humanized antibody has a sequence that differs from the sequence of an antibody obtained from a non-human species by one or more substitutions, deletions, and / or additions of amino acids, such that the humanized antibody is less likely to induce an immune response, and / or induces a less severe immune response, compared to the non-human species antibody, when 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 fused to produce the humanized antibody. In another embodiment, the hinge constant domain(s), the CH2 and CH3 domains, of a human antibody are fused with the variable domain(s) of a non-human species antibody.In another embodiment, one or more amino acid residues in one or more CDR sequences of a nonhuman antibody are changed to reduce the likely immunogenicity of the nonhuman antibody when administered to a human subject, wherein the changed amino acid residues are not critical for the immunospecific binding of the antibody to its antigen, or the changes in the amino acid sequence are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the nonhuman antibody to the antigen. Examples of how to produce humanized antibodies can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293. In the context of the present invention, the variable regions of a humanized antibody can be used in the contemplated bispecific binding construct formats. The term chimeric antibody refers to an antibody containing 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 the CDRs are derived from a human antibody. In yet another embodiment, the CDRs from more than one human antibody are mixed and paired into a chimeric antibody. For example, a chimeric antibody might comprise a light chain CDR1 from a first human antibody, a light chain CDR2 and CDR3 from a second human antibody, and the heavy chain CDRs from a third antibody. Furthermore, the framework regions can 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 an example of a chimeric antibody, a portion of the heavy and / or light chain is identical, homologous, or derived from an antibody of a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain or chains are identical, homologous, or derived from an antibody or antibodies of another species or belonging to another class or subclass of antibodies. Fragments of such antibodies exhibiting the desired biological activity are also included. In the context of the present invention, the variable regions of a chimeric antibody can be used in the contemplated bispecific binding construct formats. The invention provides bispecific binding constructs comprising the HHLL format and further comprising linkers comprising protease cleavage sites. In the most general sense, a bispecific binding construct as described herein comprises several polypeptide chains having different amino acid sequences which, when linked together, can bind to two different antigens. With the inclusion of a protease cleavage site in particular linkers (see, for example, Figures 1 and 2), the uncleaved binding construct has reduced or no binding to a desired target. Upon exposure to protease, the linkers are cleaved, and the binding construct is then able to bind to a desired target. Optionally, the HHLL molecules further comprise a half-life-extending moiety. In some embodiments, the half-life-extending moiety is an Fe polypeptide chain.In other embodiments, the half-life-prolonging residue is a single-stranded Fe. In yet another embodiment, the half-life-prolonging residue is a hetero-Fc. In still other embodiments, the half-life-prolonging residue is human albumin. LINKING MECHANISM Between the variable regions of immunoglobulin is a peptide linker, which can be the same linker or different linkers of varying lengths. These linkers can play a role in the structure of the bispecific binding construct. If the linker is too short, it will not allow sufficient flexibility for the appropriate variable regions on a single polypeptide chain to interact and form an antigen-binding site. If the linker is of the appropriate length, it will allow one variable region to interact with another variable region on the same polypeptide chain to form an antigen-binding site. In certain embodiments, the HHLL format comprises disulfide bonds, both within the domain (within H1 and L1) and between domains (between H1 and L1).To achieve the appropriate expression and conformation of the bispecific linkage constructs of the invention, specific linkers are used between the various immunoglobulin regions in certain embodiments (see, for example, Figure 1 herein). Example linkers are provided in Table 1 herein. In certain embodiments, increasing the linker length could result in increased protein truncation, an undesirable property. Therefore, it is desirable to achieve an appropriate balance between linker length to allow for the appropriate polypeptide structure and activity without increasing truncation. A linker, as understood herein, is a peptide that joins two polypeptides. In one embodiment, a linker can join two variable regions of immunoglobulin in the context of a bispecific linkage construct. A linker can be 2–30 amino acids in length. In some embodiments, a linker can be 2–25, 2–20, or 3–18 amino acids in length. In some embodiments, a linker can have a peptide no more than 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids in length. In other embodiments, a linker can be 5–25, 5–15, 4–11, 10–20, or 20–30 amino acids in length. In other embodiments, a linker may be approximately 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 in length.Example linkers include, for example, the amino acid sequences GGGGS (SEQ ID NO: 1), GGGGSGGGGGS (SEQ ID NO: 2), GGGGSGGGGGSGGGGGS (SEQ ID NO: 3), GGGGSGGGGGSGGGGGSGGGGS (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 (SEQ ID NO: 14), among others, including repetitions of the amino acid sequences or subunits of amino acid sequences mentioned above (e.g., repetitions of GGGGS (SEQ ID NO: 1) or GGGGQ (SEQ ID NO: 6)). IVIA / a / ZUZ II In certain embodiments, in the context of the HHLL molecules of the invention, the linking sequence of Linker 1 has at least 10 amino acids. In other embodiments, Linker 1 has at least 15 amino acids. In other embodiments, Linker 1 has at least 20 amino acids. In other embodiments, Linker 1 has at least 25 amino acids. In other embodiments, Linker 1 has at least 30 amino acids. In other embodiments, Linker 1 has 10-30 amino acids. In other embodiments, Linker 1 has 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 addition to other realizations, Linker 1 has more than 30 amino acids. In certain embodiments, in the context of the HHLL molecules of the invention, the linking sequence of Linker 2 has at least 15 amino acids. In other embodiments, Linker 2 has at least 20 amino acids. In other embodiments, Linker 2 has at least 25 amino acids. In other embodiments, Linker 2 has at least 30 amino acids. In other embodiments, Linker 2 has 15-30 amino acids. In other embodiments, Linker 2 has 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In still other embodiments, Linker 2 has more than 30 amino acids. In certain embodiments, in the context of the HHLL molecules of the invention, the linking sequence of Linker 3 has at least 15 amino acids. In other embodiments, Linker 3 has at least 20 amino acids. In other embodiments, Linker 3 has at least 25 amino acids. In other embodiments, Linker 3 has at least 30 amino acids. In other embodiments, Linker 3 has 15-30 amino acids. In other embodiments, Linker 3 has 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In still other embodiments, Linker 3 has more than 30 amino acids. In certain embodiments, in the context of the HHLL molecules of the invention, the linking sequence of Linker 4 has at least 5 amino acids. In other embodiments, Linker 4 has at least 10 amino acids. In other embodiments, Linker 4 has at least 15 amino acids. In other embodiments, Linker 4 has at least 20 amino acids. In other embodiments, Linker 4 has at least 25 amino acids. In other embodiments, Linker 4 has at least 30 amino acids. In other embodiments, Linker 4 has 530 amino acids. In other embodiments, Linker 4 has 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 still other embodiments, Linker 4 has more than 30 amino acids. In certain embodiments, in the context of the HHLL molecules of the invention, the linker sequences and positions are set out in the following Table 1, with linker positions corresponding to those set out in Figure 1, and Linker 4 being optionally used if an Fe region is also attached to the HHLL molecule. TABLE 1 Linkers Linker 1 SEQ ID NO: Linker 2 SEQ ID NO: Linker 3 SEQ ID NO: Linker 4 SEQ ID NO: (GGGGS)2 2 (GGGGS)3 3 (GGGGS)3 3 GGGG 100 (GGGGS)4 4 (GGGGS)4 4 (GGGGS)4 4 GGGG 100 (GGGGS)5 5 (GGGGS)5 5 (GGGGS)5 5 GGGG 100 (GGGGS)3 3 (GGGGS)5 5 (GGGGS)5 5 GGGG 100 (GGGGS)3 3 (GGGGS)3 3 (GGGGS)2 2 GGGG 100 (GGGGS)2. 10 96 (GGGGS)3. 10 98 (GGGGS)3- 10 98 (GGGG)i- 10 101 (GGGGQ)2 7 (GGGGQ)3 8 (GGGGQ)3 8 GGGG 100 (GGGGQ)4 9 (GGGGQ)4 9 (GGGGQ)4 9 GGGG 100 (GGGGQ)5 10 (GGGGQ)s 10 (GGGGQ)s 10 GGGG 100 (GGGGQ)3 8 (GGGGQ)s 10 (GGGGQ)s 10 GGGG 100 (GGGGQ)2 10 97 (GGGGQ)3 10 99 (GGGGQ)3_ 10 99 (GGGG)i- 10 101 *The numeric subscript indicates the number of repetitions, for example, (GGGGS)2 = GGGGSGGGGG (SEQ ID NO: 2) It should be noted that the 3-3-2 linker was intentionally designed with non-optimal lengths to serve as a negative control. PROTEASE CLEFISATION SITES In certain therapeutic applications, it can be advantageous to design the bispecific binding construct so that it is only active in the vicinity of target cells or their local microenvironment. For example, in certain cancers, inflammatory diseases, fibrotic diseases, and neurodegenerative diseases that produce proteases in the microenvironment, the bispecific binding construct is activated once it is present in the microenvironment of diseased cells. See, for example, Broder and Becker-Pauly (2013), Biochem. J. 450: pp. 253-264. See also, for example, Metz et al. (2012), Protein Engineering, Design and Selection, Vol. 25, issue 10, pp. 571-580. In this type of pathology, the bispecific binding construct can be activated in the presence of proteases produced by diseased cells, but not in their absence.Therefore, a bispecific junction construct, as described herein, can be specifically activated in a disease microenvironment and be less active or inactive in other areas of the body, which may result in fewer negative side effects experienced by the patient receiving the therapy. Accordingly, in certain embodiments, bispecific binding constructs comprise a protease cleavage site within linkers that bind to certain domains, wherein this protease cleavage site can be cleaved by a protease produced by target cells, e.g., cancer cells or infected cells or pathogens, and wherein this cleavage activates the molecule. A protease cleavage site, as defined herein, comprises an amino acid sequence that can be cleaved by a protease, such as, for example, a metalloproteinase (e.g., a matrix metalloproteinase (MMP) such as MMP2, MMP9, MMP11, or others), a serine protease, a cysteine ​​protease, plasmin or a plasminogen activator (such as urokinase-like plasminogen activator (u-PA) or tissue plasminogen activator (tPA)), fibroblast activation protein α (FAPα), or a furin, among others. Representative locations of protease cleavage sites within linkers are outlined in Figures 1 and 2 herein. Non-limiting examples of amino acid sequences comprised by such protease cleavage sites include those listed in Table 2 herein. In some embodiments, protease cleavage sites may include, for example, plasmin-cleaved sites. The proenzyme plasminogen is activated by proteolytic cleavage by u-PA, leading to its conversion into the active enzyme, plasmin. Plasmin, a serine protease, may play a role in metastasis due to its degradation of the extracellular matrix and its activation of other enzymes, such as type IV collagenase. See, for example, Kaneko et al. (2003), Cancer Sel. 94(1): 43-39. Matrix metalloproteinases (MMPs) MMP-2 and MMP-9 are overexpressed in a variety of human tumors, including ovarian, breast, and prostate tumors, as well as melanoma. Furthermore, an association between aggressive tumor growth and high levels of MMP-2 and / or MMP-9 has been observed in both clinical and experimental studies. See, for example, Roomi et al. (2009), One. Rep. 21: 1323-1333. An MMP-2 or MMP-9 cleavage site can be represented as P4-P3-P2-P1P1'-P2'-P3'-P4', where P1-P4 and P1-P4' are amino acids and the vertical line represents the cleavage site. Some generalizations can be made about an MMP-2 cleavage site. P1 is most likely to be glycine or proline. P2 is most likely to be proline, and somewhat less likely to be alanine, valine, or isoleucine. P3 is most likely to be alanine, serine, or arginine. P4 is most likely to be alanine, glycine, asparagine, or serine.PT is more likely to be leucine, while isoleucine, phenylalanine, or tyrosine are somewhat less likely. P2' is more likely to be lysine, while alanine, valine, isoleucine, or tyrosine are somewhat less likely. P3' is more likely to be alanine, serine, or glycine. P4' is more likely to be alanine, lysine, or aspartic acid. There are somewhat clearer preferences for the MMP-9 cleavage sites. P4 is more likely to be glycine. P3 is more likely to be proline. P2 is more likely to be lysine. P1 is more likely to be glycine or proline. PT is more likely to be leucine, while isoleucine is somewhat less likely. P2' is more likely to be lysine. P3' is more likely to be glycine or alanine. P4' is more likely to be alanine, proline, or tyrosine.Any MMP-2 or MMP-9 cleavage site may be located within the bispecific binding constructs (e.g., in linkers) described herein, including those described in Table 2 or in, e.g., Metz et al. (2012), Protein Engineering, Design and Selection, Vol. 25, Issue 10, pp. 571-580 or, e.g., Prudova et al. (2010), Mol. CelL Proteomics 9(5): 894-911. In some embodiments, the protease cleavage sites used in the linkers also include, for example, cleavage sites for the metalloproteinases meprin a and meprin β, which may be implicated in diseases such as certain cancers, inflammatory bowel diseases, cystic fibrosis, kidney diseases, diabetic nephropathy, and fibrotic dermal tumors. The meprin a and β cleavage sites are not limited to a single defined sequence for each of these proteases. However, at certain amino acid positions relative to the cleavage site, there is a strong preference for one or a series of specific amino acids. See, for example, Becker-Pauly et al. (2011), Molecular and Cellular Proteomics 10(9):M111.009233. DOI:10.1074 / mcp.M111.009233, parts of which, describing a particular cleavage site, including supplementary material, are incorporated herein by reference.Table 2 of this document provides a small selection of known cleavage sites for various proteases, including meprin a and meprin β. Higher than normal levels of u-PA are known to be associated with various cancers, including, for example, colorectal cancer, breast cancer, monocytic and myelogenous leukemias, bladder cancer, thyroid cancer, liver cancer, gastric cancer, and cancers of the pleura, lung, pancreas, ovaries, and head and neck. See, for example, Skelly et al. (1997), Clin. Can. Res. 3: 1837-1840; Han et al. (2005), Oncol. Rep. 14(1): 105-112; Kaneko et al. (2003), Cancer Sci. 94(1): 43-49; Liu et al. (2001), J. Biol. Chem. 276(21): 17976-17984. Table 2 of this document reports a small sample of sites that can be cleaved by u-PA. Therefore, the bispecific binding constructs described herein may comprise a cleavage site for any serine protease, including u-PA and tissue plasminogen activator (tPA), and include any of the cleavage sites listed in Table 2. Some cysteine ​​proteases, such as cathepsin B, have been found to be overexpressed in tumor tissue and probably play a causal role in some cancers. See, for example, Emmert-Buck et al. (1994), Am. J. Pathol. 145(6): 1285-1290; Biniosseek et al. (2011), J. Proteome Res. 10: 5363-5373. As with the cleavage sites for meprin α and meprin β, there is much heterogeneity in the cleavage sites of cathepsin B. A cleavage site for cathepsin B (as well as other proteases) can be represented as P3-P2-P1 IP1'-P2'-P3', where P1-P3 and PT-P3' are all amino acids and the vertical line represents the cleavage site. Some generalizations apply to cathepsin β cleavage sites. P3 is usually G, F, L, or P (using a one-letter code for amino acids). P2 is usually A, V, Y, F, or I. P1 is usually G, A, M, Q, or T. P1' is usually F, G, I, V, or L. P2' is usually V, I, G, T, or A. P3' is usually G. In addition, there are some cooperative subsites. For example, if P2 is F, then P3 is more likely to be G and less likely to be L, and P3 is more likely to be F and less likely to be L.This and other examples of subsite cooperativity are described in detail in Biniossek et al. (2011), J. Proteome Res. 10: 5363-5373. Accordingly, all cathepsin B cleavage sites, including, without limitation, those in Table 2 herein, may be comprised of the bispecific binding constructs described herein. In some embodiments, the bispecific junction constructs comprise the protease cleavage sites Gly-Gly-Pro-Leu-Gly-Met-Leu-Ser-GIn-Ser (SEQ ID NO: 45), Gly-ProLeu-Gly-Ile-Ala-Gly-GIn (SEQ ID NO: 44), or Ala-Val-Arg-Trp-Leu-Leu-Thr-Ala (SEQ ID NO: 102), which can be cleaved by metalloproteinases. Other examples of protease cleavage sites include Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 54), which is cleaved by a furin. Cleavage at the protease cleavage site can be assessed using various known assays, such as SDS-PAGE and / or Western blot. In certain embodiments, binding constructs bind to a target more effectively when the protease cleavage sites are essentially completely cleaved, which can be assessed, for example, by SDS-PAGE and / or Western blot. TABLE 2: EXAMPLES OF PROTEASE CLEAVAGE SITES Protease Cleavage site sequence* meprin to meprin β APMAIEGGG (SEQ ID NO: 17) EAQGIDKII (SEQ ID NO: 18) LAFSlDAGP (SEQ ID NO: 19) YVAIDAPK (SEQ ID NO: 20) u-PA SGRlSA (SEQ ID NO: 21) GSGRISA (SEQ ID NO: 22) SGKISA (SEQ ID NO: 23) Protease Cleavage site sequence* u-PA SGRlSS (SEQ ID NO: 24) SGRIRA (SEQ ID NO: 25) SGRINA (SEQ ID NO: 26) SGRlKA (SEQ ID NO: 27) tPA QRGRlSA (SEQ ID NO: 28) cathepsin B TQGlAAA(SEQ ID NO: 29) GAAlAAA (SEQ 30) GAGIAAG (SEQ ID NO: 31) AAAIAAG (SEQ ID NO: 32) LCGIAAI (SEQ ID NO: 33) FAQlALG (SEQ ID NO: 34) LAAIANP (SEQ ID NO: 35) LLQlANP (SEQ ID NO: 36) LAAIANP (SEQ ID NO: 37) LYGIAQF (SEQ ID NO: 37). NO: 38) LSQIAQG (SEQ ID NO: 39) ASAIASG (SEQ ID NO: 40) FLGlASL (SEQ ID NO: 41) AYGlATG (SEQ ID NO: 42) LAQIATG (SEQ ID NO: 43) MMP-2 GPLGIIAGQ (SEQ ID NO: 44) GGPLGIMLSQS (SEQ ID NO: 44) 45)** PLAGUE (SEQ ID NO: 46) MMP-11 AANILRN (SEQ ID NO: 47) AQAIYVK (SEQ ID NO: 48) AANlYMR (SEQ ID NO: 49) AAAILTR (SEQ ID NO: 50) AQNILMR (SEQ ID NO: 51) AANIYTK (SEQ ID NO: 52) Furin RRRRR (SEQ ID NO: 53) RRRRRR (SEQ ID NO: 54) GQSSRHRRAL (SEQ ID NO: 55) The 7 vertical lines, when present, represent the intended split site. **Note that this sequence is also split by MMP-9 IVIA / a / ¿U¿ II CYSTEINE CLAMPS A cysteine ​​clamp involves the introduction of a cysteine ​​into a polypeptide domain at a specific location, typically by replacing an existing amino acid at the specific location, so that when in proximity to another polypeptide domain, also with a cysteine ​​introduced at a specific location, a disulfide bond (a cysteine ​​clamp) can form between the two domains. In some embodiments, a linker sequence comprising a protease cleavage site may result in a molecule that, after cleavage of the protease cleavage site, does not produce the desired molecular structure due to the lack of a covalent bond between the appropriate polypeptide domains. Accordingly, in certain embodiments, the covalent bond is provided by one or more genome-manipulated disulfide bonds introduced at specified locations (a cysteine ​​clamp). Non-limiting examples of such cysteine ​​clamps can be found in U.S. Patent Solution Pub. No. 2016 / 0193295A1, U.S. Patent Solution Pub. No. 2017 / 0306033A1, and U.S. Patent Solution Pub. No. 2018 / 0079790A1. In certain embodiments, an antibody Fe domain may comprise the cysteine ​​clamp(s), such as the CH2 and / or CH3 domains. See, for example, U.S. Patent Sol. Pub. No. 2016 / 0193295A1. In one specific embodiment, an scFc comprises at least one cysteine ​​clamp resulting in a disulfide bond across both CH2 domains. In a further specific embodiment, an scFc comprises at least two cysteine ​​clamps resulting in a disulfide bond across both CH2 domains. In certain embodiments, the amino acid residues where the CH2 sequence has been altered to create the cysteine ​​clamp(s) can be selected from the following, where one or more amino acids are substituted by cysteine: R72C, V82C, R329C, R339C In certain embodiments, specific pairs of residues are substituted so that they preferentially form a disulfide bond with each other, thereby limiting or preventing alteration of the disulfide bond. Non-limiting examples of these specific pairs include, but are not limited to, 72C-82C and 329C-339C. In other embodiments, the VH and VL domains of a binding construct may comprise the cysteine ​​clamp(s) to result in the formation of disulfide bonds between the VH and VL domains. These cysteine ​​clamps will stabilize the VH and VL domains in an antigen-binding configuration. See, for example, U.S. Pat. Sol. Pub. No. 2017 / 0306033A1. In certain embodiments, the amino acid residues where the VH and VL sequence has been altered to create the cysteine ​​clamp(s) can be selected from the following, where one or more amino acids are substituted by cysteine: Kabat VH44 VL100 for anti-MSLN and VH103 VL43 for anti-CD3. In certain embodiments, specific residue pairs are substituted so that they preferentially form a disulfide bond with each other, thereby limiting or preventing alteration of the disulfide bond. Non-limiting examples of such specific pairs include, but are not limited to, MSLN VH44-VL100 and anti-CD3 VH103-VL43. AMINO ACID SEQUENCES OF THE JUNCTION REGIONS In the example embodiments described herein, the bispecific binding constructs maintain the desired binding to the various target molecules by assuming the appropriate conformation to allow this binding. The immunoglobulin variable region comprises a VH and VL domain, which associate to form the variable domain that binds to the desired target. Variable domains can be obtained from any immunoglobulin with the desired characteristics, and the methods for achieving this are further described herein. In one embodiment, VH1 and VL1 associate and bind to CD3e, and VH2 and VL2 associate and bind to a different target. In another embodiment, VH2 and VL2 bind to CD3c, and VH1 and VL1 bind to a different target. In another embodiment, the light chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the light chain variable domain sequence listed herein. In another embodiment, the light chain variable domain comprises an amino acid sequence 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 listed herein. In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately rigorous conditions with the complement of a polynucleotide encoding a light chain variable domain selected from the sequences listed herein.In another embodiment, the light chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under rigorous conditions with the complement of a polynucleotide encoding a light chain variable domain selected from the group consisting of the sequences listed herein. In another embodiment, the heavy chain variable domain comprises an amino acid sequence 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 listed herein. In another embodiment, the heavy chain variable domain comprises an amino acid sequence 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 encoding a heavy chain variable domain selected from the sequences listed herein.In another embodiment, the heavy chain variable domain comprises an amino acid sequence encoded by a polynucleotide that hybridizes under moderately rigorous conditions with the complement of a polynucleotide encoding a heavy chain variable domain selected from the sequences listed herein. REPLACEMENTS It will be appreciated that a bispecific binding construct of the present invention may have at least one amino acid substitution, provided 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 included 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 as 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 to the sequences set forth herein. These may include amino acid substitutions, which may be conservative or non-conservative and do not destroy the desired binding capacity of a bonding construct. Conservative amino acid substitutions may encompass amino acid residues of non-natural origin, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other inverted or reversed forms of amino acid residues.A conservative amino acid substitution can also involve replacing a native amino acid residue with a normative residue so that there is little or no effect on the polarity or charge of the amino acid residue at that position. Non-conservative substitutions can involve the exchange of a member of one class of amino acids or amino acid mimetics for a member of another class with different physical properties (e.g., size, polarity, hydrophobicity, charge). In certain embodiments, such substituted residues can be introduced into regions of a human antibody that are homologous to non-human antibodies, or into non-homologiverse regions of the molecule. Furthermore, a skilled worker can generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using activity assays known to skilled workers. Such variants could be used to gather information on suitable variants. For example, if a change at a particular amino acid residue is found to result in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from these routine experiments, a skilled worker can readily determine which amino acids should be avoided for further substitutions, either alone or in combination with other mutations. A person skilled in the art may determine suitable variants of the bispecific linkage construct as described herein using well-known techniques. In certain embodiments, a person skilled in the art may identify suitable areas of the molecule that can be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, residues and portions of molecules that are conserved among similar polypeptides as described herein may be identified. In certain embodiments, even areas that may be important for biological activity or structure may be subject to conservative amino acid substitutions without destroying biological activity or adversely affecting the polypeptide structure. Furthermore, a skilled worker can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. Based on such a comparison, the importance of amino acid residues in a protein can be predicted, specifically those that correspond to amino acid residues important for activity or structure in similar proteins. A skilled worker can then select chemically similar amino acid substitutions for these predicted important amino acid residues. In some embodiments, a skilled practitioner can identify residues that can be changed to achieve desired enhanced properties. For example, an amino acid substitution (conservative or non-conservative) can result in increased binding affinity to a desired target. A skilled worker can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. Based on this information, a skilled worker can predict the alignment of an antibody's amino acid residues with respect to its three-dimensional structure. In certain embodiments, a skilled worker may choose not to make radical changes to amino acid residues predicted to be on the protein surface, as these residues may be involved in important interactions with other molecules. Several scientific publications have been devoted to predicting secondary structure. See Moult J., Curr. Op. en 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). In addition, computer programs are currently available to help predict secondary structure. One method for predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins that have sequence identity greater than 30% or similarity greater than 40% often have similar structural topologies. The growth of the Protein Structural Database (PDB) has provided improved predictive capabilities for secondary structure, including the potential number of folds within the structure of a polypeptide or protein. See Holm et al., Nuci. Acid. Res., 27(1):244-247 (1999). Additional methods for 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 union (see Holm, above (1999), and Brenner, above (1997)). In certain embodiments, variants of the bispecific linkage construct include glycosylation variants in which the number and / or type of glycosylation site has been altered compared to the amino acid sequences of an original polypeptide. In certain embodiments, the variants comprise a greater or lesser number of N-linked glycosylation sites than the native protein. Alternatively, substitutions that delete this sequence will remove an existing N-linked carbohydrate chain. A rearrangement of the existing N-linked carbohydrate chains is also provided in which one or more N-linked glycosylation sites (typically those of natural origin) are deleted and one or more new N-linked sites are created.Additional antibody variants include cysteine ​​variants, in which one or more cysteine ​​residues are deleted or replaced with another amino acid (e.g., serine) compared to the original amino acid sequence. Cysteine ​​variants can be useful when antibodies or bispecific / U1I binding constructs need to 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. Skilled practitioners can determine the desired amino acid substitutions (conservative or non-conservative) at the time such substitutions are required. In certain embodiments, amino acid substitutions can be used to identify important residues of antibodies or bispecific binding constructs with respect to the target of interest, or to increase or decrease the affinity of the antibodies or bispecific binding constructs with respect to the target of interest described herein. According to certain embodiments, the desired amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter the binding affinity for forming protein complexes, (4) alter binding affinities, and / or (5) confer or modify other physicochemical or functional properties in said polypeptides. According to certain embodiments, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domains that form intermolecular contacts).In certain embodiments, a conservative amino acid substitution should typically not substantially change the structural characteristics of the original sequence (e.g., an amino acid substitution should not tend to break the helix present in the original sequence, nor to disrupt other types of secondary structures that characterize the original sequence). Examples of recognized secondary and tertiary polypeptide 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, NY (1991)); and Thornton et al. Nature 354:105 (1991), each of which is incorporated herein by reference. PROLONGATION OF HALF-LIFE AND REGIONS Fe In certain embodiments, it is desirable to prolong the in vivo half-life of the bispecific binding constructs of the invention. This can be achieved by including a half-life-prolonging moiety as part of the bispecific binding construct. Non-limiting examples of half-life-prolonging moietyes include an Fe polypeptide, albumin, an albumin fragment, a moiety that binds to albumin or the neonatal Fe receptor (FcRn), a fibronectin derivative that has been genome-manipulated to bind to albumin or a fragment thereof, a peptide, a single-domain protein fragment, or another polypeptide that can increase serum half-life. In alternative embodiments, a half-life-prolonging moiety may be a non-polypeptide molecule such as, for example, polyethylene glycol (PEG). The term "Fe polypeptide," as used herein, includes native and mutein forms of polypeptides derived from the Fe region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. In addition to other properties described herein, polypeptides comprising Fe moieties offer the advantage of purification by affinity chromatography over, for example, protein A or protein G columns. In certain embodiments, the half-life-extending component is an Fe region of an antibody. In some embodiments, the Fe region is located at the N-terminus of the HHLL bispecific binding construct. In other embodiments, the Fe region is located at the C-terminus of the HHLL bispecific binding construct. In still other embodiments, the Fe region may be located between the VH and VL subunits, as shown in Figure 2 herein. A linker may be present between the HHLL bispecific binding construct and the Fe region, but is not required. As explained herein, an Fe polypeptide chain may comprise all or part of a hinge region followed by a CH2 and CH3 region. The Fe polypeptide chain may be of mammalian (e.g., human, mouse, rat, rabbit, dromedary, or platyrrhine or cercopithecine monkey), avian, or shark origin.Furthermore, as explained in this document, an Fe polypeptide chain may include a limited number of alterations. For example, an Fe polypeptide chain may comprise one or more heterodimerizing alterations, one or more alterations that inhibit or enhance FcyR binding, or one or more alterations that increase FcRn binding. In one specific embodiment, the Fe used for half-life extension is a single-chain Fe (scFc). In some embodiments, the amino acid sequences of the Fe polypeptides can be mammalian amino acid sequences, for example, from a human. The isotype of the Fe polypeptide can be IgG, such as lgG1, lgG2, lgG3, or lgG4, IgA, IgD, IgE, or IgM. Table 3 below shows an alignment of the amino acid sequences of the Fe polypeptide chains of human lgG1, lgG2, lgG3, and lgG4. The Fe polypeptide sequences of human IgG1, IgG2, IgG3, and IgG4 that could be used are provided in SEQ IDs 56-59. Also considered are variants of these sequences containing one or more heterodimerizing alterations, one or more Fe alterations that prolong the half-life, one or more alterations that enhance ADCC, and / or one or more alterations that inhibit Fe gamma receptor (FcγR) binding, as well as other close variants containing no more than 10 single-amino-acid deletions, insertions, or substitutions per 100 amino acids of sequence. TABLE 3: AMINO ACID SEQUENCES OF HUMAN IqG Fe POLYPEPTIDE CHAINS IgG1 ----------------------------------------------IgG2 ----------------------------------------------IgG3 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP IgG4 ----------------------------------------------225 235 245 255 265 275 ***★**· IgGl EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF IgG2 ERKCCVE---CPPCPAPPVA-GPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQF IgG3 EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQF IgG4 ESKYG---PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQF 285 295 305 315 325 335 IgGl NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG2 NWYVDGMEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKT IgG3 KWYVDGVEVHNAKTKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG4 NWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT 345 355 365 375 385 395 IgGl ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG2 ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG3 ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTP IgG4 ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP 405 415 425 435 445 ★ ★ ★ ★★ IgGl PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQID NO:56) IgG2 PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQID NO:57) IgG3 PMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQID NO:58) IgG4 PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQID NO:59) The numbering shown in Table 3 is according to the EU numbering system, which is based on the sequential numbering of the constant region of an lgG1 antibody. Edelman et al. (1969), Proc. Nati. Acad. Sci. 63: 78-85. Therefore, it does not fit well with the additional length of the lgG3 hinge. Nevertheless, it is used here to designate positions in an Fe region because it is still commonly used in the technique to refer to positions in Fe regions. The Fe polypeptide hinge regions of lgG1, lgG2, and lgG4 extend from approximately position 216 to approximately 230. It is evident from the alignment that the hinge regions of lgG2 and lgG4 are each three amino acids shorter than the lgG1 hinge. The lgG3 hinge is much longer and extends an additional 47 amino acids up the chain. The CH2 region extends from approximately position 231 to 340, and the CH3 region extends from approximately position 341 to 447. The naturally occurring amino acid sequences of Fe polypeptides may vary slightly. Such variations may include no more than 10 insertions, deletions, or single-amino-acid substitutions per 100 amino acids in a naturally occurring Fe polypeptide chain. If substitutions occur, they may be conservative amino acid substitutions, as defined herein. The Fe polypeptides of the first and second polypeptide chains may differ in amino acid sequence. In some embodiments, they may include heterodimerizing alterations, such as charge-pair substitutions, as defined herein, which facilitate heterodimer formation. In addition, the Fe polypeptide portions of PABP may also contain alterations that inhibit or enhance FcεR binding. Such mutations are described herein and in Xu et al. (2000), Cell Immunol.200(1): 16-26, relevant portions of which are incorporated herein by reference. Fe polypeptide portions may also include a half-life-prolonging Fe alteration, as described herein, including those described, for example, in U.S. Patents 7,037,784, 7,670,600, and 7,371,827, U.S. Patent Application Publication 2010 / 0234575, and International Application PCT / US2012 / 070146, relevant portions of which are incorporated herein by reference. Furthermore, an Fe polypeptide may comprise ADCC-enhancing alterations, as defined herein. Another suitable Fe polypeptide, described in PCT application WO 93 / 10151 (which is hereby incorporated by reference), is a single-stranded polypeptide extending from the N-terminus hinge region to the native C-terminus of the Fe region of a human IgG1 antibody. Another useful Fe polypeptide is the Fe 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 Fe 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 Fe receptors. MA / a / ZUZI / U1I The effector function of an antibody or binding construct can be increased or decreased by introducing one or more mutations in the Fe. Embodiments of the invention include IL-2 mutein Fe fusion proteins having a genome-manipulated Fe to increase effector function (US patent 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both incorporated herein by reference in their entirety). For certain therapeutic indications, increasing effector function may be desirable. For other therapeutic indications, decreasing effector function may be desirable. Example lgG1 Fe molecules that have an enhanced effector function include those with 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 Another method for enhancing the effector function of Fe-containing IgG proteins is by reducing Fe fucosylation. Deletion of the core fucose from Fe-bound, two-strand complex oligosaccharides greatly increased the effector function of ADCC without altering antigen-binding or effector function of CDC. Several methods are known for reducing or suppressing fucosylation of Fe-containing molecules, such as antibodies. These include recombinant expression in specific mammalian cell lines, including a FUT8-attenuated cell line, a Lec13 variant CHO cell line, a YB2 / 0 rat hybridoma cell line, a cell line comprising a small interfering RNA specifically targeting the FUT8 gene, and a cell line co-expressing α-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II.Alternatively, the Fe-containing molecule can be expressed in a non-mammalian cell such as a plant cell, yeast, or prokaryotic cell, e.g., E. coli. In certain embodiments of the invention, the bispecific bonding constructs comprise a genome-manipulated Fe molecule to decrease its effector function. Example Fe molecules having decreased effector function include those with the following substitutions: N297A or N297Q (lgG1) L234A / L235A (lgG1) V234A / G237A (lgG2) L235A / G237A / E318A (lgG4) H268Q / V309L / A330S / A331S (lgG2) C220S / C226S / C229S / P238S (lgG1) C226S / C229S / E233P / L234V / L235A (lgG1) L234F / L235E / P331S (lgG1) S267E / L328F (lgG1) Human IgG1 is known to have a glycosylation site at N297 (EU numbering system), and glycosylation contributes to the effector function of IgG1 antibodies. An example IgG1 sequence is provided in SEQ ID NO: 36. N297 can be mutated to produce glycosylated antibodies. For example, mutations can substitute N297 with amino acids that resemble asparagine in physicochemical nature, such as glutamine (N297Q), or with alanine (N297A), which mimics asparagines without polar groups. In certain embodiments, the mutation of the amino acid N297 of human IgG1 to glycine, i.e., N297G, provides purification efficiency and biophysical properties far superior to other amino acid substitutions at that residue. See, for example, U.S. Patent Nos. 9,546,203 and 10,093,711. In one specific embodiment, the bispecific binding constructs of the invention comprise a human IgG1 Fe having an N297G substitution. A bispecific junction construct of the invention comprising a human IgG1 Fe having the N297G mutation may also comprise additional insertions, deletions, and substitutions. In certain embodiments, the human IgG1 Fe 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: 36. In a particularly preferred embodiment, the C-terminus lysine residue is substituted or deleted. In certain cases, aglycosylated IgG1 Fe-containing molecules may be less stable than glycosylated IgG1 Fe-containing molecules. Therefore, the Fe region can be further manipulated to increase the stability of the aglycosylated molecule. In some embodiments, one or more amino acids are substituted with cysteine ​​to form disulfide bonds in the dimeric state. In specific embodiments, residues V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence shown in SEQ ID NO: 56-59 may be substituted with cysteine. In other embodiments, specific pairs of residues are substituted so that they preferentially form a disulfide bond with each other, thereby limiting or preventing disruption of the disulfide bond. In specific embodiments, the pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. As discussed in the Linkers section of this document, in certain embodiments, the bispecific linkage constructs of the invention comprise a linker between the Fe and the bispecific linkage construct of HHLL, specifically, linking the Fe to VL2. 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) are located between the Fe and the HHLL polypeptide. In some embodiments, the polypeptide region between the Fe region and the HHLL 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 GGNGT (SEQ ID NO: 15) or YGNGT (SEQ ID NO: 16) linkers are glycosylated when expressed in the appropriate cells, and such glycosylation can help stabilize the protein in solution and / or when administered in vivo.Accordingly, in certain embodiments, a bispecific linkage construction of the invention comprises a glycosylated linker between the Fe region and the HHLL polypeptide. NUCLEIC ACIDS THAT ENCODE BIESPECIFIC JUNCTION CONSTRUCTS In another embodiment, the present invention provides isolated nucleic acid molecules encoding the bispecific junction constructs of the present invention. In addition, vectors comprising the nucleic acids, cells comprising the nucleic acids, and methods for manufacturing the bispecific junction constructs of the invention are provided. The nucleic acids comprise, for example, polynucleotides encoding all or part of the bispecific junction construct, or a fragment, derivative, mutain, or variant thereof; sufficient polynucleotides for use as hybridization probes, PCR primers, or sequencing primers for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide; non-coding nucleic acids for inhibiting the expression of a polynucleotide; and complementary sequences thereof.Nucleic acids can be of any length as appropriate for their function or intended use, and may comprise one or more additional sequences, such as regulatory sequences, and / or be part of a larger nucleic acid, such as a vector. Nucleic acids can be single-stranded or double-stranded and may comprise RNA and / or DNA nucleotides, and artificial variants thereof (e.g., peptidonucleotic acids). Nucleic acids encoding polypeptides (e.g., heavy or light chain, variable domain only, or full length) can be isolated from B lymphocytes of mice immunized with antigen. The nucleic acid can be isolated using conventional procedures such as polymerase chain reaction (PCR). This document includes nucleic acid sequences that encode the variable regions of the heavy and light chains. Those skilled in the art 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 that encodes each bispecific junction construct of the invention. The invention further provides nucleic acids that hybridize with other nucleic acids under particular hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. As defined herein, for example, a moderately rigorous hybridization condition uses a prewash solution containing 5X sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of approximately 50% formamide, 6X SSC, and a hybridization temperature of 55 °C (or other similar hybridization solutions, such as one containing approximately 50% formamide, with a hybridization temperature of 42 °C), and wash conditions of 60 °C, in 0.5X SSC, 0.1% SDS. A rigorous 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, a skilled technician can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization so 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. Basic parameters affecting the choice of hybridization conditions and guidelines for establishing appropriate conditions are set out, for example, in Sambrook, Fritsch, and Maniatis (1989). Molecular Cloning: A Laboratory Manual, Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY, 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 skilled in the technique based on, for example, the length and / or base composition of the DNA. Changes can be introduced by mutation in a nucleic acid, leading to changes in the amino acid sequence of a polypeptide (for example, a bispecific junction construct) that it encodes. Mutations can be introduced using any technique known in the field. In one embodiment, one or more particular amino acid residues are changed using, for example, a directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed, for example, using a random mutagenesis protocol.However, if done, a mutating polypeptide can be expressed and screened for a desired property. Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made that produce amino acid substitutions at non-essential amino acid residues. In one embodiment, a nucleotide sequence provided herein for the junction constructs of the present invention, or a desired fragment, variant, or derivative thereof, is mutated so as to encode an amino acid sequence comprising one or more deletions or substitutions of amino acid residues shown herein for the light chains of the junction constructs of the present invention or the heavy chains of the junction constructs of the present invention, such that the residues differ at two or more sequences.In another embodiment, mutagenesis inserts an amino acid adjacent to one or more amino acid residues shown herein for the light chains of the linking constructs of the present invention or the heavy chains of the linking constructs of the present invention, so that they are residues where two or more sequences differ. Alternatively, one or more mutations may be introduced into a nucleic acid that selectively change the biological activity of a polypeptide it encodes. 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. The recombinant expression vectors of the invention may comprise a nucleic acid of the invention in a form suitable for nucleic acid expression in a host cell. The recombinant expression vectors include one or more regulatory sequences, selected based on the host cells to be used for expression, which are operatively linked to the nucleic acid sequence to be expressed. The regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many host cell types (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter, and cytomegalovirus promoter), and those that direct the 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 herein by reference in their entirety), and those that direct the inducible expression of a nucleotide sequence in response to a particular treatment or condition (e.g., the metallothionein promoter in mammalian cells and the tet-sensitive and / or streptomycin-sensitive promoter in prokaryotic and eukaryotic systems (see id.). It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, 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. In another embodiment, the present invention provides host cells into which a recombinant expression vector of the invention has been introduced. A host cell may be any prokaryotic 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 grown in serum-free media (see Rasmussen et al., 1998, Cytotechnology 28:31) or the CHO strain DXB-11, which is DHFR-deficient (see Urlaub et al., 1980, Proc. Nati. Acad. Sci. USA 77:4216-20). Additional CHO cell lines include CHO-K1 (ATCC No. CCL-61), EM9 (ATCC No. CRL-1861) and UV20 (ATCC No. CRL-1862).Additional host cells include the COS-7 monkey kidney cell line (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L lymphocytes, C127 cells, 3T3 cells (ATCC CCL 163), AM-1 / D cells (described in U.S. Patent No. 6,210,924), HeLa cells, BHK cell lines (ATCC CRL 10), the CV1 / EBNA cell line derived from the CV1 African green monkey kidney cell line (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, and other primate cell lines transformed, normal diploid cells, cell strains obtained from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Vectors of. MA / a / zuzi / un i cloning and expression appropriate for use with bacterial, fungal, yeast, and mammalian cell hosts by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). The vector DNA can be introduced into prokaryotic or eukaryotic cells using conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that, depending on the expression vector and the transfection technique used, only a small fraction of the cells will integrate the foreign DNA into their genome. In order to identify and select these integrators, a gene encoding a selectable marker (e.g., for antibiotic resistance) is typically introduced into the host cells along with the gene of interest. Additional selectable markers include those that confer drug resistance, 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 other cells will die), among other methods. The transformed cells can be cultured under conditions that promote polypeptide expression, and the recovered polypeptide can be purified using conventional protein purification procedures. The polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian polypeptides substantially free of contaminating endogenous materials. Cells containing the nucleic acid encoding the bispecific junction constructs of the present invention also include hybridomas. The production and cultivation of hybridomas are discussed herein. 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. In some embodiments, a nucleic acid molecule is provided that encodes the bispecific junction constructs as described herein. In some embodiments, a pharmaceutical composition comprising at least one bispecific bonding construct described herein is provided. PRODUCTION METHODS The bispecific junction constructs of the invention can be produced by any method known in the art for protein synthesis (e.g., antibodies), in particular by chemical synthesis or preferably by recombinant expression techniques. The recombinant expression of bispecific junction constructs requires the construction of an expression vector containing a polynucleotide encoding the bispecific junction construct. Once a polynucleotide encoding the bispecific junction construct has been obtained, the vector for its production can be generated using recombinant DNA technology. An expression vector is constructed containing the bispecific junction construct that encodes the appropriate transcription and translation control sequences and signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The expression vector is transferred to a host cell using conventional techniques and then the transfected cells are cultured using conventional techniques to produce a bispecific junction construct of the invention. A variety of host expression vector systems can be used and readily adapted to express the bispecific binding constructs of the invention. Such host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but they also represent cells that can express a molecule of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequences. Bacterial cells such as E. coli and eukaryotic cells are commonly used for the expression of a recombinant antibody molecule, especially for the expression of a complete recombinant antibody molecule.For example, mammalian cells such as Chinese hamster ovary (CHO) cells, together with a vector such as the major early intermediate genetic promoter element of human cytomegalovirus, is an efficient expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio / Technology 8:2 (1990)). Furthermore, a host cell strain can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for protein function. 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 selected to ensure the correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells that possess the cellular machinery for the appropriate processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, COS, 293, 3T3, or myeloma cells. ML / a / ZUZ 1 I For long-term, high-throughput production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the molecule can be genome-manipulated. Instead of using expression vectors containing 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. After the introduction of the foreign DNA, the genome-manipulated cells can be allowed to grow for 1–2 days in an enriched medium and then transferred to a selective medium.The selectable marker on the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. This method can be advantageously used to genome-manipulate cell lines that express the molecule. Such genome-manipulated cell lines can be particularly useful in screening and evaluating compounds that interact directly or indirectly with the molecule. Several selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Nati. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase genes (Lowy et al., Cell 22:817 (1980)) in tk, hgprt, or aprt cells, respectively. In addition, antimetabolite resistance can be used as a basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Nati. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Nati. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Nati. 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)).The methods commonly known in recombinant DNA technology can 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 in their entirety herein. The expression levels of a molecule 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). ML / a / ZUZ 1 I Academic Press, New York, 1987). When a marker in the vector system expressing the molecule is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the molecule will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)). The host cell can be co-transfected with multiple expression vectors of the invention. The vectors can contain identical selectable markers that allow equal expression of the expressed polypeptides. Alternatively, a single vector encoding and capable of expressing, for example, the polypeptides of the invention can be used. The coding sequences can comprise cDNA or genomic DNA. Once a molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it can be purified by any method known in the art for the purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion-exchange, affinity, particularly antigen-specific affinity after protein A, and size-exclusion chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Furthermore, 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 can be varied, depending on whether an Fe region (e.g., an scFC) is bonded to the bispecific bonding constructs of the invention. In some embodiments, the present invention encompasses recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) linkage constructs to a polypeptide. The fused or conjugated linkage constructs of the present invention can be used to facilitate purification. See, for example, Harbor et al., above, and PCT Publication WO 93 / 21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent No. 5,474,981; Gillies et al., Proc. Nati. Acad. Sci. 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991). Furthermore, the junction constructs or fragments thereof of the present invention can be fused with marker sequences, such as a peptide, to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide (SEQ ID NO: 103), 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. Nati. Acad. Sci. USA 86:821-824 (1989), for example, hexahistidine (SEQ ID NO: 103) provides 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 obtained from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the flag tag. GENERATION OF BIESPECIFIC JOINT CONSTRUCTIONS The bispecific binding constructs of the invention, in a general sense, are constructed by selecting the VH and VL regions of the desired antibodies and linking them using polypeptide linkers as described herein to form the HHLL bispecific binding construct, optionally with an attached Fe region. More specifically, nucleic acids encoding VH, VL, and linkers, and optionally IA Fe, are combined to create the HHLL nucleic acid constructs that encode the bispecific binding constructs of the invention. Antibody generation In certain embodiments, prior to the generation of the bispecific binding constructs of the invention, monospecific antibodies with binding specificities to the desired targets are first generated. The antibodies used to generate the bispecific binding molecules of the invention can be prepared using techniques well known to those skilled in the art. For example, by immunizing an animal (e.g., a mouse, rat, or rabbit) and then immortalizing splenocytes collected from the animal after the completion of the immunization program. The splenocytes can be immortalized using any technique known in the art, for example, by fusing them with myeloma cells to produce hybridomas. (See, for example, Antibodies; Hariow and Lane, Coid Spring Harbor Laboratory Press, 1st edition, e.g., 1988, or 2nd edition, e.g., 2014). In one embodiment, a humanized monoclonal antibody to be used to generate the bispecific binding molecules of the invention comprises the variable domain of a murine antibody (or all or part of the antigen-binding site thereof) and a constant domain obtained 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) obtained from a human antibody. Processes for the production of genome-manipulated monoclonal antibodies 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 an antibody grafted with a CDR. Techniques for humanizing antibodies are being analyzed, for example, in the US Patents.N.° 5,869,619; 5,225,539; 5,821,337; 5,859,205; 6,881,557, Padlan etal., 1995, FASEB. ΜΛ / a / ZUZ 1 I 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(1):35-42, 2005; Dall'Acqua WF, et al., Methods 36(1):43-60, 2005; y Clark, M„ Immunology Today. 21(8):397-402, 2000. An antibody of the present invention may also be a fully human monoclonal antibody used to generate the bispecific binding molecules of the invention. Fully human monoclonal antibodies may be generated by various techniques familiar to those skilled in the art. 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 lymphocytes, fusion of splenocytes from immunized transgenic mice bearing inserted immunoglobulin genes, isolation of phage libraries from the V region of human immunoglobulin, or other procedures known in the art and based on disclosure herein. Procedures have been developed to generate human monoclonal antibodies in non-human animals. For example, mice have been prepared in which one or more endogenous immunoglobulin genes have been inactivated by various means. Human immunoglobulin genes have been introduced into mice to replace the inactivated mouse genes. In this technique, human heavy and light chain locus elements are introduced into mouse strains derived from embryonic stem cell lines containing targeted alterations of the endogenous heavy and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455-58 (1997)). For example, human immunoglobulin transgenes can be minigene constructs, or trans-acting loci on yeast artificial chromosomes, which undergo B-cell-specific DNA rearrangement and hypermutation in mouse lymphoid tissue. The 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. Examples of techniques for the production and use of transgenic animals for the production of human or partially human antibodies 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, Infecí Immun. 68:1820-26, Gallo et al., 2000, Eur J Immun. 30:534-40, Davis el 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. 42:41321, Mendez et al., 1997, Nat Genet. 15:146-56, Jakobovits, 1994, Curr Biol. 4:761-63, Arbones etal., 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 Nati Acad Sci USA. 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 etal., 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, Infernal Review of Immunology 13: 65-93, Neuberger, 1996, Nature Biotechnology 14: 826, TayloretaL, 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, y Tuaillon etal., 1994, Journal of Immunology 152: 2912-20.; Lonberg et aL, Nature 368:856, 1994; Taylor et al., Int. Immun. 6:579, 1994; Patente de EE.UU. N.° 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. Además, se describen protocolos que implican el XenoMouse® (Abgenix, ahora Amgen, Inc.), por ejemplo, en los documentos U.S. 05 / 0118643 and WO 05 / 694879, WO 98 / 24838, WO 00 / 76310 and U.S. Patent 7,064,244. Lymphoid cells from immunized transgenic mice are fused with myeloma cells, for example, to produce hybridomas. Preferably, the myeloma cells used in fusion procedures that produce hybridomas do not produce antibodies, have high fusion efficiency, and exhibit enzyme deficiencies that render them unable to grow in certain selective media that only favor the growth of the desired fused cells (hybridomas). Examples of cell lines suitable for use in such fusions include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XX0 Bul. Examples of cell lines 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, LICRLON-HMy2, and UC729-6. Lymphoid cells (e.g., from the spleen) and myeloma cells can 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 such selective medium is HAT (hypoxanthine, aminopterin, thymidine). After a sufficient time, usually about one to two weeks, cell colonies are observed. Individual colonies are isolated, and the antibodies produced by the cells can be assayed for binding activity against desired targets using any one of a variety of immunoassays known in the art and described herein.Hybridomas are cloned (e.g., by limited dilution cloning or by isolation on soft agar plates), and positive clones that produce an antibody specific for a desired target are selected and cultured. Monoclonal antibodies from hybridoma cultures can be isolated from the hybridoma culture supernatants. The present invention provides hybridomas comprising polynucleotides encoding the bispecific junction constructs of the invention on the cell chromosomes. These hybridomas can be cultured according to the methods described herein and known in the art. Another method for generating human antibodies to be used to generate the bispecific binding molecules of the invention includes immortalizing human peripheral blood cells by EBV transformation. See, for example, U.S. Patent No. 4,464,456. Such an immortalized B-lymphocyte line (or lymphoblastoid cell line) that produces a monoclonal antibody that binds specifically 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 antibody-producing lymphoblastoid cell line can 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, for example, Glasky et al., Hybridoma 8:377-89 (1989)).Another method for generating human monoclonal antibodies is in vitro immunization, which involves priming human splenic B lymphocytes with antigen, followed by fusion of primed B lymphocytes with a heterohybrid fusion partner. See, for example, Boerner et al., 1991 J. Immunol. 147:86-95. In certain embodiments, a B lymphocyte producing a desired antibody is selected, and the light-chain and heavy-chain variable regions are cloned from the B lymphocyte according to molecular biology techniques known in the art (WO 92 / 02551; U.S. Patent 5,627,052; Babcook et al., Proc. Nati. Acad. Sci. USA 93:7843-48 (1996)) and described herein. B lymphocytes from an immunized animal can be isolated from a spleen, lymph node, or peripheral blood sample by selecting a cell producing a desired antibody. B lymphocytes can also be isolated from humans, for example, from a peripheral blood sample.Methods for detecting individual B lymphocytes producing an antibody with the desired specificity are well known in the field, for example, by plaque formation, fluorescence-activated cell sorting, in vitro stimulation followed by detection of specific antibodies, and similar techniques. Methods for selecting specific antibody-producing B lymphocytes include, for example, preparing a suspension of individual B lymphocytes on soft agar containing antigen. The binding of the specific antibody produced by the B lymphocyte to the antigen results in the formation of a complex, which may be visible as an immunoprecipitate.After selecting the B lymphocytes that produce the desired antibody, the specific antibody genes can be cloned by isolating and amplifying DNA or mRNA according to methods known in the art and described herein and can be used to generate the bispecific binding molecules of the invention. An additional method for obtaining antibodies to be used to generate the bispecific binding molecules of the invention is phage presentation. See, for example, Winter et al., 1994 Annu. Rev. Immunol. 12:433-55; Burton et al., 1994 Adv. Immunol. 57:191-280. Combinatorial libraries of human or murine immunoglobulin variable region genes can be created in phage vectors that can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that bind specifically to the TGF-beta binding protein or a variant or fragment thereof. See, for example, U.S. Patent No. 5,223,409; Husee et al., 1989 Science 246:1275-81; Sastry et al., Proc. Nati. Academic Sci. USA 86:5728-32 (1989); Alting-Mees et al, Strategies in Molecular Biology 3:1-9 (1990); Kang et al., 1991 Proc. Nati. Academic Sci USA 88:4363-66; Hoogenboom et al., 1992 J. Molec. Biol. 227:381388; Schlebusch et al., 1997 Hybridoma 16:47-52 and references cited therein. For example, a library containing a plurality of polynucleotide sequences encoding fragments of the variable region of Ig can be inserted into the genome of a filamentous bacteriophage, such as M13 or a variant thereof, within the frame containing the sequence encoding a phage coat protein. A fusion protein can 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, Fab fragments of immunoglobulin can also be presented in a phage particle (see, for example, U.S. Patent No. 5,698,426). Immunoglobulin heavy and light chain cDNA expression libraries can also be prepared in lambda phages, for example, using the AlmmunoZap™(H) and AlmmunoZap™(L) vectors (Stratagene, La Jolla, California). Briefly, mRNA is isolated from a B-cell population and used to create immunoglobulin heavy and light chain cDNA expression libraries in the AlmmunoZap™(H) and AlmmunoZap™(L) vectors. These vectors can be screened individually or co-expressed to form fragments. Fab or antibodies (see Huse et al., above; see also Sastry et al., above). Positive plaques can subsequently be converted into a non-lytic plasmid that allows high-level expression of E. coli monoclonal antibody fragments. In one embodiment, in a hybridoma, the variable regions of a gene expressing a monoclonal antibody of interest are amplified using nucleotide primers, and these genes can be used to generate the bispecific binding molecules of the invention. These primers can be synthesized by a person skilled in the art or can be purchased from commercially available sources. (See, for example, Stratagene (La Jolla, California), which sells primers for mouse and human variable regions, including, but not limited to, primers for the VHa, VHb, VHc, VHd, CH1, VL, and CL regions.) These primers can be used to amplify heavy-chain or light-chain variable regions, which can then be inserted into vectors such as ImmunoZAPTMH or ImmunoZAPTML (Stratagene), respectively. These vectors can then be introduced into E. coli-based, yeast-based, or mammalian-based systems for expression.Large quantities of a single-stranded protein containing a fusion of the VH and VL domains can be produced using these methods (see Bird et al., Science 242:423-426, 1988). In certain embodiments, the antibodies used to generate the bispecific binding molecules of the invention are obtained from transgenic animals (e.g., mice) that produce only heavy-chain antibodies, or HCAbs. HCAbs are analogous to naturally occurring single-chain camel and llama VHH antibodies. See, for example, U.S. Patent Nos. 8,507,748 and 8,502,014, and U.S. Patent Application Publications Nos. US2009 / 0285805A1, US2009 / 0169548A1, US2009 / 0307787A1, US2011 / 0314563A1, US2012 / 0151610A1, WO2008 / 122886A2, and WO2009 / 013620A2. Once molecule-producing cells have been obtained according to the invention using any of the immunization and other techniques described above, the specific antibody genes can be cloned by isolating and amplifying DNA or mRNA from them according to standard procedures as described herein and then used to generate the bispecific binding constructs of the present invention. The antibodies produced therefrom can be sequenced, the CDRs can be identified, and the DNA encoding the CDRs can be manipulated as previously described to generate other bispecific binding constructs according to the invention. Molecular evolution of complementarity-determining regions (CDRs) in the center of the antibody-binding site has also been used to isolate antibodies with higher affinity, for example, those described by Schiere et al., 1996, J. Mol. Biol. 263:551. Consequently, such techniques are useful for preparing binding constructs of the invention. ivi i 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 obtained, for example, from any antibody-producing animal, such as a mouse, rat, rabbit, goat, donkey, or non-human primate (e.g., a monkey, such as a crab-eating macaque or rhesus monkey) or an ape (e.g., a chimpanzee).An antibody of a particular species can be produced, for example, by immunizing an animal of that species with the desired immunogen or by using an artificial system to generate antibodies of that species (for example, a bacteria-based system or phage presentation for generating antibodies of a particular species), or by converting an antibody of one species into an antibody of another species by replacing, for example, the constant region of the antibody with a constant region of 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 of the other species. In one embodiment, the antibody is a chimeric antibody comprising amino acid sequences obtained from antibodies of two or more different species. The desired binding region sequences can then be used to generate the bispecific binding constructs of the present invention. When it is desired to improve the affinity of the binding constructs according to the invention containing one or more of the aforementioned CDRs, it can be achieved by various affinity maturation protocols including maintenance of the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain rearrangement (Marks et al., Bio / Technology, 10, 779-783, 1992), use of mutation strains of E. coli. (Low et al., J. Mol. Biol., 250, 350-368, 1996), DNA rearrangement (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage presentation (Thompson et al., J. Mol. Biol., 256, 7-88, 1996), and additional PCR techniques (Crameri et al., Nature, 391, 288-291, 1998). All these affinity maturation methods are discussed by Vaughan et al. (Nature Biotechnology, 16, 535-539, 1998). In certain embodiments, to generate the bispecific HHLL binding constructs of the present invention, it may be desirable to first generate a more typical single-stranded antibody that can be formed by joining heavy- and light-chain variable domain fragments (Fv region) via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-stranded Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNA 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 (KorttetaL, 1997, Prot. Eng. 10:423; Korttet al., 2001, Biomol. Eng. 18:95-108).The techniques developed for the production of single-stranded antibodies include those described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Nati. 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-stranded antibodies are distinct from and differ from the bispecific binding constructs of the invention. Antigen-binding fragments derived from an antibody can also be obtained, for example, by proteolytic hydrolysis of the antibody, such as pepsin or papain digestion of whole antibodies according to conventional methods. For instance, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to yield a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol-reducing agent to produce monovalent Fab' 3.5S fragments. Optionally, the cleavage reaction can be carried out using a blocking group for the sulfhydryl groups resulting from the cleavage of disulfide bonds. Alternatively, enzymatic cleavage with papain directly yields two monovalent Fab fragments and one Fe fragment. 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, SM and Tifus, JA in Current Protocols in Immunology (Coligan JE, 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 can also be used to cleave antibodies, such as the separation of heavy chains to form monovalent heavy-light chain (Fd) fragments, further fragment cleavage, or other enzymatic, chemical, or genetic techniques, provided that the fragments bind to the antigen recognized by the intact antibody. In certain embodiments, 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 from 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.), p. 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.), p. 137 (Wiley-Liss, Inc. 1995)). The antibody fragment may further 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 to a desired target (e.g., human CD3) with an affinity at least equal to 10⁻⁷ M or less as described herein. The variable region can be any naturally occurring variable domain or a genome-manipulated version thereof. A genome-manipulated version is a variable region created using recombinant DNA genome manipulation techniques. Such genome-manipulated versions include those created, for example, from a specific antibody variable region by insertions, deletions, or changes in the amino acid sequences of the specific antibody. A person skilled in the technique can use any known method to identify amino acid residues suitable for genome manipulation. Additional examples include genome-manipulated 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.Genomipulated versions of antibody variable domains can be generated using any number of techniques familiar to those skilled in the art. Once these domains are generated, they can then be used to generate the bispecific binding molecules of the invention. The variable region can be covalently linked at one C-terminus amino acid to at least one other antibody domain or a fragment thereof. Thus, for example, a VH present in the variable region can be linked to an immunoglobulin CH1 domain. Similarly, a VL domain can be linked to a CK domain. In this way, for example, the construct can be a Fab fragment where the antigen-binding domain contains the associated VH and VL domains covalently linked at their C-terminus to a CH1 and CK domain, respectively. The CH1 domain can be extended with more 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 additional domains, such as antibody CH2 and CH3 domains. JOINT SPECIFICITY An antibody or bispecific binding construct binds specifically 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. Affinity can be determined using a variety of techniques known in the field, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent 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 kinetics-based assay mechanism (e.g., BIACORE® assay or Octet® assay (forteBIO)), and other methods such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), electrophoresis, and gel chromatography (e.g., gel filtration). These and other methods may use a marker in 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 markers.A detailed description of binding affinities and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. An example of a competitive binding assay is a radioimmunoassay involving the incubation of the labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding dissociation constants can be determined from the data by analyzing Scatchard plots. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with the antibody of interest conjugated to a labeled compound in the presence of increasing amounts of a second unlabeled antibody.These tests can be easily adapted to the bispecific joint constructions of the invention. Further embodiments of the invention provide bispecific binding constructs that bind to desired targets with an equilibrium dissociation constant or KD (kdissociation / kasociation) of less than 10-7 M, or less than 10-8 M, or less than 109 M, or less than 10-10 M, or less than 10-11 M, or less than 10-12 M, or less than 10-13 M, or less than 5x10-13 M (lower values ​​indicate a tighter binding affinity). Still further embodiments of the invention are bispecific junction constructs that junction to desired targets with an equilibrium dissociation constant or KD (kdissociation / kasociation) of less than approximately 10-7 M, or less than approximately 10-8 M, or less than approximately 10-9 M, or less than approximately 10-10 M, or less than approximately 10-11 M, or less than approximately 10-12 M, or less than approximately 10-13 M, or less than approximately 5x10-13 M. In yet another embodiment, the bispecific bonding constructs that bind to desired targets have an equilibrium dissociation constant or KD (kdissociation / kasociation) of between approximately 10⁻⁷ M and approximately 10⁻⁸ M, between approximately 10⁻⁸ M and approximately 10⁻⁹ M, between approximately 10⁻⁹ M and approximately 10⁻¹⁰ M, between approximately 10⁻¹⁰ M and approximately 10⁻¹¹ M, between approximately 10⁻¹¹ M and approximately 10⁻¹² M, and between approximately 10⁻¹² M and approximately 10⁻¹³ M. In yet another embodiment, a bonding construct of the invention has an equilibrium dissociation constant or KD (kdissociation / kasociation) 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, and between 10⁻¹⁰ M and 10-11 M, between 10-11 M and 10-12 M, between 10-12 M and 1013 M. MOLECULAR STABILITY Various aspects of molecular stability may be desirable, particularly in the context of a biopharmaceutical therapeutic molecule. For example, stability at various temperatures (thermostability) may be desired. In some embodiments, this may encompass stability within physiological temperature ranges, for example, at approximately 37 °C, or from 32 °C to 42 °C. In other embodiments, this may encompass stability at higher temperature ranges, for example, from 42 °C to 60 °C. In still other embodiments, this may encompass stability at colder temperature ranges, for example, from 20 °C to 32 °C. In yet other embodiments, this may encompass stability while in a frozen state, for example, at 0 °C or below. Tests for determining the thermostability of protein molecules are known in the art. For example, the fully automated UNcle platform (Unchained Labs) was used, which allowed for the simultaneous acquisition of intrinsic protein fluorescence and static light scattering (SLS) data during the thermal ramp and is further described in the Examples. In addition, the aggregation and thermal stability assays described in the Examples, such as differential scanning fluorimetry (DSF) and static light scattering (SLS), can also be used to measure both thermal melting (Tm) and thermal aggregation (Tagg), respectively. Alternatively, 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 time points, where lower levels of aggregation indicate better protein stability. Alternatively, the thermostability parameter can be determined in terms of the aggregation temperature of molecules as follows: A solution of molecules at a concentration of 250 pg / ml is transferred to 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 continuous acquisition of the measured radius. The increase in radius, which indicates protein melting and aggregation, is used to calculate the aggregation temperature of the molecule.Alternatively, melting temperature curves can be determined by differential scanning calorimetry (DSC) to assess the intrinsic biophysical stabilities of the binding construct proteins. These experiments are performed using a VP-DSC MicroCal LLC device (Northampton, MA, USA). The energy absorption 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, for example, in SEC migration buffer. To record the respective melting curve, the overall sample temperature is progressively increased. At each temperature T, the energy absorption of the sample and the formulation buffer reference is recorded.The difference in energy absorption Cp (kcal / mol / °C) between the sample and the reference is plotted against the respective temperature. The melting point is defined as the temperature at the first maximum of energy absorption. In a further embodiment, the bispecific bonding constructs according to the invention are stable at approximately physiological pH, i.e., approximately pH 7.4. In other embodiments, the bispecific bonding constructs are stable at a lower pH, e.g., down to pH 6.0. In other embodiments, the bispecific bonding constructs are stable at a higher pH, e.g., up to pH 9.0. In one embodiment, the bispecific bonding constructs are stable at a pH of 6.0 to 9.0. In another embodiment, the bispecific bonding constructs are stable at a pH of 6.0 to 8.0. In another embodiment, the bispecific bonding constructs are stable at a pH of 7.0 to 9.0. In certain embodiments, the more tolerant the bispecific binding construct is to non-physiological pH (e.g., pH 6.0), the greater the recovery of the eluted binding construct from an ion-exchange column relative to the total amount of loaded protein. In one embodiment, the recovery of the binding construct from an ion-exchange column (e.g., cationic) is >30%. In another embodiment, the recovery of the binding construct from an ion-exchange column (e.g., cationic) is >40%. In another embodiment, the recovery of the binding construct from an ion-exchange column (e.g., cationic) is >50%. In another embodiment, the recovery of the binding construct from an ion-exchange column (e.g., cationic) is >60%. In another embodiment, the recovery of the binding construct from an ion-exchange column (e.g., cationic) is >70%.In another embodiment, the recovery of the bonding construct of an ion-exchange column (e.g., cationic) is >80%. In another embodiment, the recovery of the bonding construct of an ion-exchange column (e.g., cationic) is >90%. In another embodiment, the recovery of the bonding construct of an ion-exchange column (e.g., cationic) is >95%. In another embodiment, the recovery of the bonding construct of an ion-exchange column (e.g., cationic) is >99%. In certain embodiments, it may be desirable to determine the chemical stability of the molecules. The determination of the chemical stability of the bispecific junction construct can be performed through isothermal chemical denaturation (ICD) by monitoring the fluorescence of the intrinsic protein, as further described in the Examples section of this document. ICD produces C1 / 2 and AG, which can be useful metrics for protein stability. C1 / 2 is the amount of chemical denaturant required to denature 50% of the protein and is used to obtain AG (or unfolding energy). Protein chain truncation is another critical product quality attribute that is carefully monitored and reported for biologic drugs. Typically, a longer and / or less structured linker is expected to result in increased truncation as a function of incubation time and temperature. Truncation is a critical issue for bispecific binding constructs, as fragments at linkers connecting T-cell or target docking domains have a terminal negative impact on drug potency and efficacy. Fragments at additional sites, including scFc, can affect pharmacodynamic / kinetic properties. Increased truncation is an attribute that must be avoided in a pharmaceutical product. Therefore, in certain embodiments, protein truncation can be assayed as described herein in the Examples. IMMUNE EFFECTOR CELLS AND EFFECTOR CELL PROTEINS A bispecific binding construct can bind to a molecule expressed on the surface of an immune effector cell (referred to as the effector cell protein herein) and another molecule expressed on the surface of a target cell (referred to as the target cell protein herein). The immune effector cell can be a T lymphocyte, 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 TCRα and TCRβ or TCRγ and TCRβ plus various CD3 chains from among the CD3 zeta chain (CD3γ), the CD3 epsilon chain (CD3ε), the CD3 gamma chain (CD3γ), and the CD3 delta chain (CD3δ). The CD3 receptor complex is a protein complex comprising four chains. In mammals, the complex contains one CD3y (gamma) chain, one CD3o (delta) chain, and two CD3s (epsilon) chains. These chains associate with the T-cell receptor (TCR). The ML / a / ZUZ 1 / U1I and the so-called ζ (zeta) chain combine to form the CD3 T-cell receptor complex and to generate an activation signal in T cells. The CD3γ (gamma), CD3δ (delta), and CD3e (epsilon) chains are closely related cell-surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The intracellular tails of CD3 molecules contain a unique conserved motif known as a tyrosine-dependent immunoreceptor activation motif, or ITAM for short, which is essential for TCR signaling capability. The CD3ε molecule is a polypeptide that in humans is encoded by the CD3E gene located on chromosome 11. The preferred epitope of CD3e is contained within amino acid residues 1-27 of the human CD3c extracellular domain.It is anticipated that bispecific binding constructs according to the present invention will typically and advantageously exhibit less nonspecific T-cell activation, which is undesirable in targeted immunotherapy. This translates into a lower risk of side effects. In some embodiments, the effector cell protein may be the human CD3 epsilon (CD3c) chain (whose mature amino acid sequence is disclosed in SEQ ID NO: 40), which may be part of a multimeric protein. Alternatively, the effector cell protein may be TCRα, TCRp, TCR5, TCRγ, the CD3β (CD3p) chain, the CD3γ (CD3y) chain, the CD3δ (CD35) chain, or the CD3ζ (δ3ζ) chain from humans and / or crab-eating macaques. Furthermore, in some embodiments, a bispecific binding construct can also bind to a CD3e chain from a non-human species, such as mouse, rat, rabbit, New World monkey, or vervet monkey species. Such species include, but are not limited to, the following mammalian species: Mus musculus; Rattus rattus; Rattus norvegicus; the crab-eating macaque, Macaca fascicularis; the hamadryas monkey, 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 Saimir sciureus. The mature amino acid sequence of the crab-eating macaque CD3e chain is provided in SEQ ID NO: 41.Having a therapeutic molecule that exhibits comparable activity in humans and species commonly used for preclinical testing, such as mice and monkeys, can simplify, accelerate, and ultimately yield better results in drug development. In the lengthy and costly process of bringing a drug to market, these advantages can be critical. In certain embodiments, the bispecific binding construct can bind to an epitope within the first 27 amino acids of the CD3e chain (SEQ ID NO: 43), which can be a human CD3e chain or a CD3e chain from different species, particularly one of the mammalian species listed herein. The epitope can contain the amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ ID NO: 104). The advantages of a binding construct that binds to such an epitope are explained in detail in U.S. Patent Application Publication 2010 / 0183615A1, relevant portions of which are incorporated herein by reference. The epitope to which an antibody or bispecific binding construct binds can be determined by alanine scanning, which is described, for example, in U.S. Patent Application Publication 2010 / 0183615A1, relevant parts of which are incorporated herein by reference.In other embodiments, the bispecific binding construct can bind to an epitope within the extracellular domain of CD3e (SEQ ID NO: 42). In embodiments where a T lymphocyte is the immune effector cell, effector cell proteins to which a bispecific binding construct can bind include, but are not limited to, the CD3e chain, the CD3y chain, the CD3o chain, the CD3( chain, TCR3, TCR3, TCRy, and TCRo. In embodiments where an NK cell or a cytotoxic T lymphocyte is an immune effector cell, NKG2D, CD352, NKp46, or CD16a can be, for example, an effector cell protein. In embodiments where a CD8+ T lymphocyte 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 lymphocytes, NK cells, macrophages, or neutrophils. TARGET CELLS AND TARGET CELL PROTEINS EXPRESSED IN TARGET CELLS As explained herein, a bispecific binding construct can bind to both an effector cell protein and a target cell protein. The target cell protein can be expressed, for example, on the surface of a cancer cell, a cell infected with a pathogen, or a cell that mediates a disease, such as 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. When the target cell is a cancer cell, a bispecific binding construct as described herein can bind to a cancer cell antigen as described herein. A cancer cell antigen can be a human protein or a protein from another species. For example, a bispecific binding construct can bind to a target cell protein from a species of mouse, rat, rabbit, platyrrhine monkey, or cercopithecid monkey, among many others. Such species include, but are not limited to, the following: Mus musculus; Rattus rattus; Rattus norvegicus; the crab-eating macaque, Macaca fascicularis; the hamadryas baboon, Papio hamadryas; the Guinea baboon, Papio papio; the olive baboon, Papio anubis; the yellow baboon, Papio cynocephalus; and the chacma baboon. Papio ursinus; Callithrix jacchus; Saguinus Oedipus; and Saimirí sciureus. In some examples, the target cell protein may be a protein selectively expressed in an infected cell. For example, in the case of HBV or HCV infection, the target cell protein may be an HBV or HCV envelope protein expressed on the surface of an infected cell. In other embodiments, the target cell protein may be encoded by gp120 by the human immunodeficiency virus (HIV) in HIV-infected cells. In other respects, 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 the EGF-like module containing the mucin-like hormone receptor 1 (EMR1), for example, could be a target cell protein. Alternatively, excess human B lymphocytes in a patient with systemic lupus erythematosus can be target cells, in which case CD19 or CD20, for example, could be a target cell protein. In other autoimmune conditions, excess human Th2 T lymphocytes can be target cells, in which case CCR4 could be, for example, 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 pulmonary fibrosis, including idiopathic pulmonary fibrosis and / or idiopathic pulmonary hypertension. For such fibrotic conditions, fibroblast activation protein alpha (FAP-alpha) can be, for example, a target cell protein. THERAPEUTIC METHODS AND COMPOSITIONS Bispecific junction 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. Another embodiment provides for the use of the joint construct of the invention (or of the joint construct produced according to the process of the invention) in the manufacture of a medicament for the prevention, treatment, or improvement of a disease. This document provides pharmaceutical compositions comprising bispecific bonding constructs. These pharmaceutical compositions comprise a therapeutically effective amount of a bispecific bonding construct and one or more additional components such as a physiologically acceptable carrier, excipient, or diluent. In some embodiments, these additional components may include buffers, carbohydrates, polyols, amino acids, chelating agents, stabilizers, and / or preservatives, among many other possibilities. In some embodiments, a bispecific junction construct can be used to treat proliferative cell diseases, including cancer, which involve unregulated and / or inappropriate cell proliferation, sometimes accompanied by destruction of adjacent tissue and growth of new blood vessels, which can allow cancer cells to invade new areas, i.e., metastasis. Conditions treatable with a bispecific junction construct include non-neoplastic conditions involving inappropriate cell growth, such as colorectal polyps, cerebral ischemia, macroscopic cystic disease, polycystic kidney disease, benign prostatic hyperplasia, and endometriosis. A bispecific junction construct can also be used to treat solid tumor or hematologic malignancies. More specifically, proliferative cell diseases that can be treated using a bispecific junction construct include, 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, Sézary syndrome, multiple myeloma and cancer of the lung, non-small cell lung, small cell lung, larynx, breast, head and neck, bladder, ovary, skin, prostate, cervical, vaginal, gastric, renal cell, kidney, pancreas, colorectal, endometrial and esophagus, hepatobiliary, bone, cutaneous and hematologic, as well as cancers of the nasal cavity and paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypolarynx, salivary glands, mediastinum, stomach, small intestine, colon, rectum and anal region, ureter, urethra, penis, testicles, vulva, system endocrine system, central nervous system, and plasma cells. Among the texts that provide guidance for cancer therapy is Cancer: Principles and Practice of Oncology, 4th edition, DeVita et al., Eds. JB Lippincott Co., Philadelphia, PA (1993). An appropriate therapeutic approach is chosen according to the particular type of cancer and other factors, such as the patient's overall condition, as recognized in the relevant field. A bispecific binding construct may be added to a therapy regimen using other antineoplastic agents in the treatment of a cancer patient. In some embodiments, a bispecific junction construct can be administered concurrently with, before, or after a variety of drugs and treatments widely used in cancer treatment, such as chemotherapeutic and non-chemotherapeutic agents, antineoplastic agents, and / or radiation. For example, chemotherapy and / or radiation may be administered before, during, and / or after any of the treatments described herein.Examples of chemotherapeutic agents discussed herein 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), antineoplastic antibiotics such as doxorubicin and daunomycin, and all chemotherapeutic agents mentioned herein. A bispecific junction construct can also be used to treat infectious diseases, for example, chronic hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, human immunodeficiency virus (HIV) infection, Epstein-Barr virus (EBV) infection, or cytomegalovirus (CMV) infection, among many others. A bispecific junction construct may find additional use in other types of conditions where it is beneficial to deplete certain cell types. For example, it may be beneficial to deplete human eosinophils in asthma, excess human B lymphocytes in systemic lupus erythematosus, excess human Th2 T lymphocytes in autoimmune conditions, or pathogen-infected cells in infectious diseases. In a fibrotic condition, it may be useful to deplete the cells that form fibrotic tissue. Therapeutically effective doses of a bispecific bonding construct can be administered. The amount of bispecific bonding construct that constitutes a therapeutic dose may vary depending on the condition being treated, the patient's weight, and the patient's calculated skin surface area. The dosage of a bispecific bonding construct can be adjusted to achieve the desired effects. In many cases, repeated doses may be necessary. A bispecific binding construct, or a pharmaceutical composition containing such a molecule, can be administered by any feasible method. Protein therapeutic agents are normally administered parenterally, for example, by injection, since oral administration, in the absence of some special formulation or circumstance, would lead to protein hydrolysis in the acidic environment of the stomach. Rapid subcutaneous, intramuscular, intravenous, intra-arterial, intralesional, or peritoneal injection are possible routes of administration. A bispecific binding construct can also be administered by infusion, for example, intravenous or subcutaneous infusion. Topical administration is also possible, especially for diseases affecting the skin.Alternatively, a bispecific binding construct can be administered via contact with a mucous membrane, for example, by intranasal, sublingual, vaginal, or rectal administration, or as an inhalant. Alternatively, certain suitable pharmaceutical compositions comprising a bispecific binding construct can be administered orally. The term "treatment" encompasses the relief of at least one symptom or other manifestation of a disorder, or the reduction of the severity of a disease, and the like. A bispecific junction construct according to the present invention need not effect a complete cure, nor eradicate every symptom or manifestation of a disease, to constitute a viable therapeutic agent. As recognized in the relevant field, drugs employed as therapeutic agents may reduce the severity of a given pathology, but they need not abolish all manifestations of the disease to be considered useful therapeutic agents. It is sufficient to reduce the impact of a disease (for example, by reducing the number or severity of symptoms, or by increasing the efficacy of another treatment, or by producing another beneficial effect), or to reduce the likelihood of the disease appearing or worsening in a subject.One embodiment of the invention is directed to a method comprising administering to a patient a bispecific junction construct of the invention in a quantity and for a time sufficient to induce a sustained improvement over the baseline value of an indicator reflecting the severity of the particular disorder. The term prevention encompasses the prevention of at least one symptom or other manifestation of a disorder or the like. A prophylactically administered treatment incorporating a bispecific junction construct according to the present invention need not be completely effective in preventing the occurrence of a condition to constitute a viable prophylactic agent. It is sufficient to reduce the probability of the disease occurring or worsening in a subject. As understood in the relevant field, pharmaceutical compositions comprising the bispecific bonding construct are administered to a subject in a manner appropriate to the indication and composition. Pharmaceutical compositions may be administered by any suitable technique, including, but not limited to, parenteral, topical, or inhalation routes. If injected, the pharmaceutical composition may be administered, for example, intra-articularly, intravenously, intramuscularly, intralesionally, intraperitoneally, or subcutaneously, by rapid injection or continuous infusion. Inhalation delivery includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the bispecific bonding construct in aerosol form, and similar methods. Other alternatives include oral preparations such as pills, syrups, or lozenges. Bispecific binding constructs may be administered as a composition comprising one or more additional components such as a physiologically acceptable carrier, excipient, or diluent. Optionally, the composition further 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. Kits are provided for use by medical professionals that include one or more bispecific junction constructs and a label or other instructions for use in the treatment of any of the conditions described herein. In one embodiment, the kit includes a sterile preparation of one or more bispecific junction constructs, which may be in the form of a composition as disclosed herein and may be in one or more vials. Dosage and frequency of administration may vary according to factors such as the route of administration, the particular bispecific bond construction 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. Having described the invention in general terms above, the following examples are offered by way of illustration and not as a limitation. EXAMPLES EXAMPLE 1 GENERATION AND EXPRESSION OF BIESPECIFIC HHLL JUNCTION CONSTRUCTS WITH PROTEASE CLEAVAGE SITES The open reading frames of the different formats (Figure 1-3) were sequenced as gene syntheses and subcloned into a mammalian expression vector containing an IgG-derived signal peptide for secreted expression in the cell culture supernatant. The sequence-verified plasmid clones were transiently transfected into 293 HEK cells or stably transfected into CHO cells. The cell culture supernatant was collected after 3 days for transient expression or 6 days for stable transfects. The cell culture supernatant was stored at -80 °C until protein purification. Figures 1-3 show the single-stranded pro-bispecific binding construct formats (i.e., without protease cleavage) in the absence of MMP2 / 9 and the resulting fragments in the presence of MMP2 / 9. Format A contains the following domains from N-terminus to C-terminus: CD3e peptide (aa 1-6 or aa 1-27)-L0-VH, Anti-CD3-L1-VH, Anti-MSLN2-VL, Anti-CD3-L3-VL, Anti-MSLN-L4-HLE domain 1, and Anti-MSLN2 domain 5, wherein the variable anti-CD3 and anti-MSLN domains contain a genome-manipulated disulfide bridge that constructs a covalent bond between the specific VH and VL domains. In this format, L0, L1, L3, and L4 contain an MMP2 / 9 restriction site (SEQ ID NO: 45). Format B contains a CD3c peptide (aa 1-6 or aa1-27)-L0-VH Anti CD3-L1-HLE domain1-L2-VH Anti MSLN-L3-VL Anti CD3-L4-HLE domain2-L5-VL N-terminus anti MSLN, wherein the variable anti CD3 and anti MSLN domains contain a genome-manipulated disulfide bridge that forms a covalent link between the specific VH and VL domains. In this format, LO, L1, L2, L4, and L5 contain an MMP2 / 9 restriction site. The length of the L3 linker was varied between constructs V1E (G4S)3, B1U (G4S)6, and Z9P (G4S)12. Format C contains the following domains: VH anti CD3-L1-VH Anti MSLN-L2-VL Anti CD3-L3-VL Anti MSLN-L4-HLE domain1-L5-HLE domain2. N-terminus IVIA / a / ZUZ II, in which the anti-CD3 and anti-MSLN variable domains contain a genome-manipulated disulfide bridge that forms a covalent link between the specific VH and VL domains. In this format, L3 contains an MMP2 / 9 restriction site. Format D contains an N-terminus CD3s-L0-Human Serum Albumin-L1-VH anti-CD3-L2-VH anti-MSLN-L3-VL anti-CD3-L4-VL anti-MSLN-L5-HLE domain1-L6-HLE domain2 peptide. The CD3s peptide was used in two different lengths (G2P AA1-6, W9A AA1-27), where LO is an SG linker and L5 is a G4 linker. In this format, L1, L2, L4, and L5 contain an MMP2 / 9 restriction site. A second construct of this format was generated by omitting the N-terminus CD3 peptide (O7H). Format E contains an N-terminus CD3 peptide (AA16 or AA1-27)-L0-HLE domain1-L1-HLE domain2-L2-VH anti-CD3-L3-VH anti-MSLN-L4-VL anti-CD3-L5-VL anti-MSLN. In this format, L2, L3, and L5 contain an MMP2 / 9 restriction site.A second construct of this format was generated by omitting the N-terminus CD3 peptide (T7U). EXAMPLE 2 CHROMATOGRAPHY ANALYSIS Protein purification was performed using protein A affinity chromatography followed by size exclusion chromatography (Figures 3-12). Peaks were combined according to the OD280 nm signal (blue), and molecular weight was analyzed by SDS-PAGE. Protein monomer peaks were formulated in 10 mM citrate, 75 mM lysine, and 4% trehalose for storage in aliquots at -80 °C. The results for the following constructs are shown in Figures 4-13, respectively: N4J, N7A, V1E, B1U, Z9P, O7H, W9A, B2P, T7U, and L2G, with expression indications for the various constructs. EXAMPLE 3 GEL SIZE / TRANSFER ANALYSIS TO DETERMINE IF THE EXCISION SITES ARE FUNCTIONAL IN VITRO To determine the in vitro cleavage of bispecific binding constructs, purified bispecific binding constructs were incubated with recombinant MMP-9 at a 1:1 molar ratio for 18 h at 37 °C (or PBS as a control). The samples were then denatured at 95 °C for 5 min and subjected to non-reducing SDS-PAGE (Figures 13–14). The expected molecular weight of the bispecific binding construct is shown in its pro conformation (i.e., without protease cleavage) in the absence of MMP9 (-MMP9) and in its active form (i.e., with protease cleavage) in the presence of MMP9 (+MMP9). Samples incubated with MMP9 were not subsequently purified, as indicated by additional MMP9-specific bands (67, 82 kDa). V1E (-MMP9) showed a lower than expected PM and no difference in its activated conformation (+MMP9). The results of this are shown in Figures 14A and 14B. wiA / ai¿v¿i iv i ^yji EXAMPLE 4 IN VITRO FACS BONDING ANALYSIS Purified bispecific binding constructs were flow cytometrically analyzed to determine binding to CHO cells transfected with a target antigen (MSLN+ CHO) or a human CD3-positive T lymphocyte line (HPB-ALL). Undigested and MMP-9-digested bispecific binding constructs and a B¡TE®-HLE (W2K) construct were compared to determine binding signals under both conditions (Figures 15-19). The N4J bispecific binding construct was pre-incubated at a 1:1 molar ratio with humMMP-9 or PBS for 20 h at 37 °C. In Figures 15-17, the bispecific molecules were stained using mouse anti-CD3 (scFv) 3E5A5 (5 pg / ml) and mouse anti-IgG (1:200). The assay was performed on bispecific binding constructs 100 / 10 / 1 / 0.1 nM for 30 minutes at 4 °C. Staining was done with reference to cells stained only by the mouse PE-conjugated polyclonal secondary Ab specific anti-Fe.In Figures 18 and 19, bispecific junction constructs were pre-incubated in a 1:1 molar ratio with huMMP-9 or PBS for 18 hours at 37 °C and the assay was performed on 50 / 4.2 / 1 / 0.35 nM bispecific junction constructs for 30 minutes at 4 °C. EXAMPLE 5 FACS-BASED IN VITRO CYTOTOXICITY ASSAYS Bispecific binding constructs were applied to in vitro TDCC assays to determine the difference in activity between undigested and MMP-9-digested bispecific binding constructs (Figures 20–25). The bispecific binding constructs were incubated with recombinant MMP-9 at a 1:1 molar ratio for 18 h at 37 °C (or PBS as a control). CHO cells transfected with the target antigen (target cells) were labeled using Vybrant DiO prior to assay setup, and human pan-T lymphocytes (effector cells) were isolated using a Pan-T lymphocyte isolation kit (Miltenyi) from human PBMCs donated by healthy volunteer donors. Signals of bispecific junction construct dilutions were incubated in combination with target and effector cell populations at an effector:target ratio of 10:1 and incubated for 48 hours at 37 °C, 5% CO2, 95% humidity.After 48 hours, the cells were centrifuged, stained with propidium iodide (Pl1), and subjected to flow cytometry. The percentage of cells positive for Vybrant DiO and propidium iodide (Pl1) was plotted against the corresponding concentration of bispecific binding construct to determine the EC50 value from the dose-response curves for activity comparison. The EC50 values ​​and the ΔU1 factor (potency difference factor) were calculated by dividing the EC50 of the bispecific binding construct incubated with MMP9 by the EC50 of the bispecific binding construct incubated with PBS. The range of EC50 values, number of assays, and ΔU1 factors (difference factor between the bispecific binding constructs incubated with PBS and MMP9) is shown in Figure 26. An MMP9 non-cleaving bispecific binding construct (W2K) was used as a reference. Each and every reference cited in this document is incorporated herein by reference in its entirety for all purposes. The scope of the present invention should not be limited to the specific embodiments described herein, which are intended as unique illustrations of individual embodiments of the invention and functionally equivalent methods and components of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. It is intended that such modifications fall within the scope of the claims. SEQUENCES EXAMPLE 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 (SEQ ID NO: 14) GGNGT (SEQ ID NO: 15) YGNGT (SEQ ID NO: 16) FE REGIONS (SEQ ID NO: 56-59) IgGl ----------------------------------------------IgG2 ----------------------------------------------IgG3 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP IgG4 -------------------------------------------225 235 245 255 265 275 IgGl EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF IgG2 ERKCCVE---CPPCPAPPVA-GPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQF IgG3 EPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQF IgG4 ESKYG---PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQF 285 295 305 315 325 335 IgGl NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG2 NWYVDGMEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKT IgG3 KWYVDGVEVHNAKTKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT IgG4 NWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT 345 355 365 375 385 395 ★ ★★★★★ IgGl ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG2 ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP IgG3 ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTP IgG4 ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP 405 415 425 435 445 ★ * ★ ★ ★ IgGl PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:56) IgG2 PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID) NO:57) IgG3 PMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID) NO:58) IgG4 PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID) NO:59) SEQ ID NQ:60 Secuencia de aminoácidos de CD3e humano maduro QDGNEEMGG ITQTPYKVSI SGTTVILTCP QYPGSEILWQ HNDKNIGGDE DDKNIGSDED HLSLKEFSEL EQSGYYVCYP RGSKPEDANF YLYLRARVCE NCMEMDVMSV ATIVIVDICI TGGLLLLVYY WSKNRKAKAK PVTRGAGAGG RQRGQNKERP PPVPNPDYEP IRKGQRDLYS GLNQRRI SEQ ID NO:61 Secuencia de aminoácidos del CD3e maduro de mono cangrejero QDGNEEMGS ITQTPYQVSI SGTTVILTCS QHLGSEAQWQ HNGKNKGDSG DQLFLPEFSE MEQSGYYVCY PRGSNPEDAS HHLYLKARVC ENCMEMDVMA VATIVIVDIC ITLGLLLLVY YWSKNRKAKA KPVTRGAGAG GRQRGQNKER PPPVPNPDYE PIRKGQQDLY SGLNQRRI SEQ ID NO:62 Secuencia de aminoácidos del extracellularado de CD3e humano QDGNEEMGG ITQTPYKVSI SGTTVILTCP QYPGSEILWQ HNDKNIGGDE DDKNIGSDED HLSLKEFSEL EQSGYYVCYP RGSKPEDANF YLYLRARVCE NCMEMDVMS SEQ ID NO:63 Aminoácidos 1-27 de CD3e humano QDGNEEMGG ITQTPYKVSI SGTTVILT SEQ ID NQ: 64 Aminoácidos 1-6 de CD3£ humano QDGNEE VH anti CD3 (SEQ ID NO: 65) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTV SS VL anti CD3 (SEQ ID NO: 66) QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPA RFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL VH anti-CD3 included in the package W103C (Kabat) (SEQ ID NO: 67) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYCGQGTLVTV SS VL anti-CD3 that includes the ring A43C (Kabat) (SEQ ID NO: 68) QTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQCPRGLIGGTKFLAPGTP ARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL CDR1 VH anti CD3 (SEQ ID NO: 69) KYAMN CDR2 VH anti CD3 (SEQ ID NO: 70) RIRSKYNNYATYYADSVKD CDR3 VH anti CD3 (SEQ ID NO: 71) HGNFGNSYISYWAY CDR1 VL anti-CD3 (SEQ ID NO: 72) GSSTGAVTSGNYPN CDR2 VL anti CD3 (SEQ ID NO: 73) GTKFLAP CDR3 VL anti CD3 (SEQ ID NO: 74) VLWYSNRWV VH anti MSLN (SEQ ID NO: 75) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWLSYISSSSGSTIYYAD SVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDRNSHFDYWGQGTLVTVSS VL anti MSLN (SEQ ID NO: 76) DIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRTFGQGTKVEIK ΜΛ / a / ZUZ 1 I VH anti MSLN that includes the pinza G44C (Kabat) (SEQ ID NO: 77) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKCLEWLSYISSSGSTIYYAD SVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDRNSHFDYWGQGTLVTVSS VL anti-MSLN that includes the pinza Q100C (Kabat) (SEQ ID NO: 78) DIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRTFGCGTKVEIK CDR1 VH anti MSLN (SEQ ID NO: 79) DYYMT CDR2 VH anti MSLN (SEQ ID NO: 80) YISSSGSTIYYADSVKG CDR3 VH anti MSLN (SEQ ID NO: 81) DRNSHFDY CDR1 VL anti MSLN (SEQ ID NO: 82) RASQGINTWLA CDR2 VL anti MSLN (SEQ ID NO: 83) GASGLQS CDR3 VL anti MSLN (SEQ ID NO: 84) QQAKSFPRT scFc (SEQ ID NO: 85) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK subdominiol de scFc (SEQ ID NO: 86) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK subdominio2 de scFc (SEQ ID NO: 87) DKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPCEEKYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL ινΐΛ / a / zuz i / un YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK W2K (SEQ ID NO: 88) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLEWLSYISSSGSTIYYAD SVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDRNSHFDYWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGAS GLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRTFGQGTKVEIKSGGGGSE VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYY ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK N4J (SEQ ID NO: 89) EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTV SSGGGGSGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWI RQAPGKCLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDR NSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTWTQEPSLTVSPGGTVTLTC GSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGSGGPLGMLSQSGGGGSDIQMTQSPSSVSAS VGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQAKSFPRTFGCGTKVEIKLTVLGGGGDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPCEE QYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ML / a / zuzi / un i Ν7Α (SEQ ID NO: 90) NSYISYWAYCGQGTLVTVSSGGPLGMLSQSGQVQLVESGGGLVKPGGSLRLSCAASGFTFS DYYMTWIRQAPGKCLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAV YYCARDRNSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQTWTQEPSLTVSPGGTVTLT CGSSTGAVTSGNYPNWVQQKPGQCPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCVLWYSNRWVFGGGTKLTVLSGGGPLGMLSQSGGGDIQMTQSPSSVSASVGDR VTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQAKSFPRTFGCGTKVEIKSGPLGMLSQSGDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPCEE QYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK B1U (SEQ ID NO: 91) QDGNEESGGPLGMLSQSGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPG KGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNF GNSYISYWAYCGQGTLVTVSSSGGPLGMLSQSGDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKSGGPLGMLSQSGQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWI RQAPGKCLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDR NSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQTWTQEPSLT VSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQCPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSGGPLGMLSQSGDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPCEE QYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKSGGPLGMLSQSGDIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQAKS FPRTFGCGTKVEIK ΜΛ / a / ZUZ 1 I Ζ9Ρ (SEQ ID NO: 92) QDGNEESGGPLGMLSQSGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPG KGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNF GNSYISYWAYCGQGTLVTVSSSGGPLGMLSQSGDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKSGGPLGMLSQSGQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWI RQAPGKCLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDR NSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQCPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVLSGGPLGMLSQSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKS GGPLGMLSQSGDIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRTFGCGTKVEIK V1E (SEQ ID NO: 93) QDGNEESGGPLGMLSQSGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPG KGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNF GNSYISYWAYCGQGTLVTVSSSGGPLGMLSQSGDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKSGGPLGMLSQSGQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWI RQAPGKCLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDR NSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGA VTSGNYPNWVQQKPGQCPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYY CVLWYSNRWVFGGGTKLTVLSGGPLGMLSQSGDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKSGGPLGMLSQSGDIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRTFGC GTKVEIK ΜΛ / a / ZUZ 1 I Β2Ρ (SEQ ID NO: 94) QDGNEESGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCV ADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLL PKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKV HTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSL AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHE CYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLG KVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEV DETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEK CCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGPLGMLSQSGEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVA RIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYW AYCGQGTLVTVSSGGPLGMLSQSGQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWI RQAPGKCLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDR NSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQCPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYY CVLWYSNRWVFGGGTKLTVLSGGGPLGMLSQSGGGDIQMTQSPSSVSASVGDRVTITCRAS QGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAKSFPRTFGCGTKVEIKSGPLGMLSQSGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK W9A (SEQ ID NO: 95) QDGNEEMGGITQTPYKVSISGTTVILTSGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCP FEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLERTYGEMADCCAKQEPE RNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQR FPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEK SHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRPDYSWLLLRL AKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYT KKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVT KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPK ATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGPLGMLSQSGEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTED TAVYYCVRHGNFGNSYISYWAYCGQGTLVTVSSGGPLGMLSQSGQVQLVESGGGLVKPGG SLRLSCAASGFTFSDYYMTWIRQAPGKCLEWLSYISSSGSTIYYADSVKGRFTISRDNAKNSLF LQMNSLRAEDTAVYYCARDRNSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQE PSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQCPRGLIGGTKFLAPGTPARFSGSLL GGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSGGGPLGMLSQSGGGDIQMT QSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQAKSFPRTFGCGTKVEIKSGPLGMLSQSGDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPCEE QYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK L2G (SEQ ID NO: 96) QDGNEESGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGPLGMLSQSGEVQLVESGGGLVQPGG SLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK NTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYCGQGTLVTVSSGGPLGMLSQSGQVQ LVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKCLEWLSYISSSGSTIYYADSVKG RFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDRNSHFDYWGQGTLVTVSSGGGGSGGGGS GGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQCPRGLIGGTKFL APGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSGGGPLGM LSQSGGGDIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRTFGCGTKVEIK T7U (SEQ ID NO: 97) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSGGPLGMLSQSGEVQLVESGGGLVQPGGSLKLSCAA SGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQM NNLKTEDTAVYYCVRHGNFGNSYISYWAYCGQGTLVTVSSGGPLGMLSQSGQVQLVESGGG LVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKCLEWLSYISSSGSTIYYADSVKGRFTISRD NAKNSLFLQMNSLRAEDTAVYYCARDRNSHFDYWGQGTLVTVSSGGGGSGGGGSGGGGS QTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQCPRGLIGGTKFLAPGTP ARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSGGGPLGMLSQSG GGDIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPRTFGCGTKVEIK O7H (SEQ ID NO: 98) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCD KSLHTLFGDKLCTVATLETRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCT AFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEG KASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLL ECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDV CKNYAEAKDVFLGMFLYEYARRPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFK PLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHP EAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFN AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETC FAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSGGGGSGGGGSGGPLGMLSQS GEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYAT YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYCVRHGNFGNSYISYWAYCGQGTLVT VSSGGPLGMLSQSGQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKCLE WLSYISSSGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDRNSHFDYWG QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPN WVQQKPGQCPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNR WVFGGGTKLTVLSGGGPLGMLSQSGGGDIQMTQSPSSVSASVGDRVTITCRASQGINTWLA WYQQKPGKAPKLLIYGASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAKSFPR TFGCGTKVEIKSGPLGMLSQSDGTHTCPPPAPELLGGPSVLFPPKPKDTLMISRTPEVTC WVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK ΜΛ / a / ZUZ 1 I

Claims

NOVELTY OF THE INVENTION Having described the present invention as above, the following claims are considered novel and are therefore claimed as property: CLAIMS 1. A bispecific binding construct comprising a polypeptide chain comprising an amino acid sequence having the formula VH1-L1-VH2L2-VL1-L3-VL2, wherein VH1 and VH2 comprise immunoglobulin heavy chain variable regions, VL1 and VL2 comprise immunoglobulin light chain variable regions, and L1, L2, and L3 are linkers, wherein L1 has at least 10 amino acids, L2 has at least 15 amino acids, and L3 has at least 10 amino acids, wherein L1 or L3 comprises a protease cleavage site, and wherein the bispecific binding construct can bind to an immune effector cell and a target cell.

2. A bispecific linker construct comprising a polypeptide chain comprising an amino acid sequence having the formula VH1-L1scFcsubdomain1-L2-VH2-L3-VL1-L4-scFcsubdomain2-L5-VL2, wherein VH1 and VH2 comprise immunoglobulin heavy chain variable regions, VL1 and VL2 comprise immunoglobulin light chain variable regions, scFc comprises either subdomain 1 or subdomain 2 of an immunoglobulin heavy chain constant domain 2 and an immunoglobulin heavy chain constant domain 3, and L1, L2, L3, L4, and L5 are linkers, wherein L1 has at least 10 amino acids, L2 has at least 10 amino acids, L3 has at least 15 amino acids, L4 has at least 10 amino acids, and L5 has at least 10 amino acids, and wherein L1, L2, L4 and L5 further comprise a protease cleavage site of at least 5 amino acids, and where the bispecific junction construct can bind to an immune effector cell and a target cell.

3. The bispecific junction construct of claim 1, wherein the protease cleavage site is present in both L1 and L3.

4. The bispecific joint construction of claim 1, further comprising at least one cysteine ​​clamp.

5. The bispecific junction construction of claim 4, wherein the cysteine ​​clamp is positioned to facilitate junction between the VH1 and VL1 subunits, the VH2 and VL2 subunits, or the scFc subunits.

6. The bispecific joint construction of claim 2, further comprising at least one cysteine ​​clamp.

7. The bispecific junction construction of claim 6, wherein the cysteine ​​clamp is positioned to facilitate junction between the VH1 and VL1 subunits, the VH2 and VL2 subunits, or the scFc subunits.

8. The bispecific joint construction of claim 1, further comprising a residue that prolongs the half-life.

9. The bispecific binding construct of claim 8, wherein the half-life-prolonging remainder comprises an additional linker and a single-stranded immunoglobulin Fe (scFc) region encoding a human lgG1, lgG2, or lgG4 antibody.

10. The bispecific linkage construction of claim 9, wherein the additional linker comprises a protease cleavage site.

11. The bispecific binding construct of claim 10, wherein the scFc polypeptide chain comprises one or more alterations that inhibit Fe gamma receptor (FcyR) binding and / or one or more alterations that prolong the half-life.

12. The bispecific joint construction of claim 1 or 2, wherein VH1, VH2, VL1 and VL2 have different sequences.

13. The bispecific joint construction of claim 1 or 2, wherein a. sequence VH1 comprises SEQ ID NO: 65 or 67, and sequence VL1 comprises SEQ ID NO: 66 or 68, and sequence VH2 comprises SEQ ID NO: 75 or 77, and sequence VL2 comprises SEQ ID NO: 76 or 78, or b. sequence VH1 comprises SEQ ID NO: 75 or 77, and sequence VL1 comprises SEQ ID NO: 76 or 78, and sequence VH2 comprises SEQ ID NO: 65 or 67, and sequence VL2 comprises SEQ ID NO: 66 or 68.

14. The bispecific linkage construction of claim 1 or 2, further comprising an additional residue linked to VH1 with an additional linker (LO), wherein LO is at least 5 amino acids in length.

15. The bispecific bonding construct of claim 14, wherein the additional residue is a CD3e, or a human serum albumin-CD3 linker (aa 1-6), or a human serum albumin-CD3 linker (aa 1.27), or an scFc-CD3e linker.

16. The bispecific junction construction of claims 14 or 15, wherein LO further comprises a protease site.

17. The bispecific joint construction of claim 1 or 2, wherein the linkers have different lengths.

18. The bispecific joint construction of claim 1 or 2, wherein the linkers have the same length.

19. The bispecific joint construction of claim 1, wherein L1 and L2 have the same length.

20. The bispecific joint construction of claim 1, wherein L1 and L3 have the same length.

21. The bispecific joint construction of claim 1, wherein L2 and L3 IVIA / a / ZUZ II have the same length.

22. The bispecific bonding construct of claim 1, wherein the amino acid sequence of L1 is at least 10 amino acids in length, the amino acid sequence of L2 is at least 15 amino acids in length, and the amino acid sequence of L3 is at least 15 amino acids in length.

23. The bispecific junction construct of claim 1 or 2, wherein the effector cell expresses an effector cell protein that is part of a human T lymphocyte receptor (TCR)-CD3 complex.

24. The bispecific binding construct of claim 1 or 2, wherein the effector cell protein is the CD3c chain 25. A nucleic acid encoding the bispecific junction construct of claim 1 or 2.

26. A vector comprising the nucleic acid of claim 25.

27. A host cell comprising the vector of claim 26.

28. A method for manufacturing the bispecific junction construct of claim 1 or 2, comprising (1) growing a host cell under conditions expressing the bispecific junction construct and (2) recovering the bispecific junction construct from the supernatant of the cell mass or cell culture, wherein the host cell comprises one or more nucleic acids encoding the bispecific junction construct of claim 1 or 2.

29. A method for treating a cancer patient, comprising administering to the patient a therapeutically effective amount of the bispecific bonding construct of claim 1 or 2.

30. The method of claim 29, wherein a chemotherapeutic agent, a non-chemotherapeutic antineoplastic agent and / or radiation are administered to the patient simultaneously, before or after the administration of the bispecific junction construct.

31. A pharmaceutical composition comprising the bispecific bonding construct of claim 1 or 2.

32. The use of the bispecific joint construction of claim 1 or 2, in the manufacture of a medicament for the prevention, treatment or improvement of a disease.