Variant CD3-binding domains and their use in combination therapies for treatment of disease

Abstract

To provide a vCD3 binding domain DA×CD3 binding molecule.SOLUTION: A vCD3- Binding Domain comprises a CDRH1 Domain, a CDRH2 Domain, a CDRH3 Domain, a CDRL1 Domain, a CDRL2 Domain, and a CDRL3 Domain, at least one of them differs in amino acid sequence from the amino acid sequence of the corresponding CDR of a rCD3- Binding Domain, and the DA×CD3 Binding Molecule comprising the vCD3-Binding Domain exhibits an altered affinity for CD3, relative to a DA×CD3 Binding Molecule comprising the rCD3-Binding Domain. The invention particularly concerns to such DA×CD3 Binding Molecules comprising a vCD3-Binding Domain which exhibit reduced affinity for CD3 and are capable of mediating redirected killing of target cells expressing a DA and exhibit lower levels of cytokine release relative to a DA×CD3 Binding Molecule comprising a rCD3-Binding Domain. The invention particularly concerns the use of DA×CD3 Binding Molecules comprising a vCD3 -Binding Domain in the treatment of cancer and pathogen-associated diseases.SELECTED DRAWING: Figure 17A

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Patent Application No. 62 / 631,043 (filed February 15, 2018; pending) and U.S. Patent Application No. 62 / 738,632 (filed September 28, 2018; pending), each of which is incorporated herein by reference in its entirety.

[0002] [Reference to sequence listing] This application contains one or more sequence listings in accordance with Title 37, Code of Federal Regulations, Sections 1.821 et seq., which are disclosed in a computer-readable medium (Filename: 1301_0150PCT_ST25.txt, created January 30, 2019, size: 295,037 bytes), which file is incorporated by reference in its entirety into this application.

[0003] The present invention is directed to multispecific binding molecules (e.g., bispecific antibodies, diabodies, bispecific scFvs, trivalent molecules, TandAb®, BiTE®, etc.) (e.g., "DAxCD3 binding molecules") that comprise a CD3-binding domain capable of binding to an epitope on CD3 and a disease antigen-binding domain capable of binding to an epitope on a disease antigen ("DA"). The present invention particularly relates to DAxCD3 binding molecules as described above that comprise a variant CD3-binding domain (a "vCD3 binding domain"), wherein the vCD3-binding domain comprises a CDR H 1 domain , CDR H 2 domains, CDR H 3 domains, CDR L 1 domain, CDR L 2 domains and CDRs L It contains three domains, at least one of which is a canonical CD3 binding domain ( The DA×CD3 binding molecules described above comprising a vCD3-binding domain as described above have an amino acid sequence that differs from the amino acid sequence of the corresponding CDR of a DA×CD3 binding domain ("rCD3-binding domain"), and exhibit altered affinity for CD3 compared to DA×CD3 binding molecules comprising an rCD3-binding domain as described above. The present invention particularly relates to DA×CD3 binding molecules as described above comprising a vCD3-binding domain that have reduced affinity for CD3, are capable of mediating targeted killing of DA-expressing target cells, and exhibit reduced levels of cytokine release compared to DA×CD3 binding molecules comprising an rCD3-binding domain. The present invention particularly relates to the use of DA×CD3 binding molecules comprising a vCD3-binding domain in the treatment of cancer and pathogen-related diseases. The present invention also relates to pharmaceutical compositions comprising one or more of the above molecules. [Background technology]

[0004] I. Mammalian immune system The mammalian immune system functions as a defense against a variety of conditions, including wounds, infections, and neoplasms. The efficiency with which humans and other mammals develop immune responses to pathogens, foreign substances, and cancer antigens is based on two characteristics: the exquisite specificity of the immune response for antigen recognition, and immunological memory, which allows for a more rapid and vigorous response to reactivation by the same antigen (Non-Patent Documents 1 to 3).

[0005] In healthy individuals, the immune system is quiescent and inhibited by a variety of inhibitory receptors and ligands. Upon recognition of cancer antigens, microbial pathogens, or allergens, an array of activating receptors and receptor ligands is triggered to induce immune system activation. Such activation leads to the activation of macrophages, natural killer (NK) cells, and antigen-specific cytotoxic T cells, promoting the release of various cytokines, all of which act to counteract perceived threats to the subject's health (Non-Patent Documents 4-6). The immune system maintains its normal function when counteracting inhibitory immune signals are greater than activating immune signals. It can return to a quiescent state.

[0006] Thus, cancer disease states (and indeed infectious disease states) can be thought of as reflecting a failure of a subject's immune system to be sufficiently activated. Such failure may reflect insufficient presentation of activating immune signals, or may reflect an insufficient ability in the subject to mitigate inhibitory immune signals. In some instances, researchers have found that cancer cells can evade detection by co-opting the immune system (Non-Patent Document 3).

[0007] The mammalian immune system is mediated by two distinct but interrelated systems: the humoral immune system and the cellular immune system. Generally, the humoral system is mediated by soluble molecules (antibodies or immunoglobulins) produced by B cells. These molecules have the ability to bind to and neutralize antigens recognized as foreign to the body. The cellular immune system involves the mobilization of specialized cells called "T cells," which fulfill a variety of therapeutic roles. T cells are lymphocytes that mature in the thymus and circulate among tissues, the lymphatic system, and the circulatory system. In response to the presence and recognition of foreign structures (antigens), T cells become "activated" and initiate an immune response. In many instances, these foreign antigens are expressed on host cells as a result of neoplasia or infection. While T cells themselves do not secrete antibodies, they are usually required for antibody secretion by a second class of lymphocyte, B cells, which are derived from the bone marrow. Importantly, T cells exhibit exquisite immunological specificity, allowing them to distinguish antigens from one another.

[0008] T cell activation requires two interactions (Non-Patent Documents 5 and 6). In the first interaction, a cell must present an appropriate target antigen that binds to its class I or class II major histocompatibility complex (MHC) so that it can be bound by the T cell receptor (TCR) of a naive T lymphocyte. While almost any cell type can act as an antigen-presenting cell, some cells, such as macrophages, B cells, and dendritic cells, are specialized for presenting foreign antigens and are considered "professional" antigen-presenting cells. Immunological detection of antigen bound to the MHC I molecule of an antigen-presenting cell leads to the production of cytotoxic T cells. Immunological detection of antigen bound to the MHC II molecule of an antigen-presenting cell leads to the production of cytotoxic T cells. In the second interaction, the ligand of the antigen-presenting cell must bind to a coreceptor on the T cell (Non-Patent Documents 4 and 7). T cells that receive both stimulatory signals can respond to cytokines (e.g., interleukin-2 and interleukin-12).

[0009] In the absence of both costimulatory signals during TCR engagement, T cells enter a functionally unresponsive state, which is called clonal anergy (Non-Patent Document 8). In pathological conditions, T cells play an important role in various organ-specific autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis (Non-Patent Document 4).

[0010] Such immune "checkpoint" pathways are important in maintaining self-tolerance (i.e., preventing a subject from mounting an immune system attack against their own cells (an "autoimmune response")) and in limiting collateral tissue damage during antibacterial or anti-allergic immune responses. If contacting T cells produces only one of the two necessary signals, the T cells will not be activated and an appropriate immune response will not be mounted. Thus, the "two-signal" mechanism of T cell activation provides a way for the immune system to avoid unwanted responses, such as responses to self-antigens that would result in the immune system attacking the subject's own cells (an "autoimmune" response).

[0011] II. Cell surface molecules of the cellular immune system A. CD3, CD4, and CD8 Cells of the immune system are characterized by the expression of specialized glycoprotein cell surface molecules. Interactions between these molecules and molecules on other cells trigger, maintain, or dampen immune responses. In particular, all T cells are characterized by the expression of CD3, a T cell coreceptor consisting of four distinct chains (Non-Patent Documents 9, 10, 2).

[0012] In mammals, the complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with the TCR to generate an activation signal in T lymphocytes (Non-Patent Document 11). In the absence of CD3, the TCR does not assemble properly and is degraded (Non-Patent Document 12). CD3 is bound to the membrane of all mature T cells and does not bind to virtually any other cell type (see Non-Patent Documents 13-15).

[0013] The invariant CD3ε signaling component of the TCR complex on T cells has been used as a target to force the formation of an immunological synapse between T cells and cancer cells. Co-engagement of CD3 and tumor antigens activates T cells and triggers the lysis of tumor antigen-expressing cancer cells (Non-Patent Document 16). This approach allows bispecific antibodies to globally interact with the T cell compartment with high specificity for cancer cells, and this approach is broadly applicable to a wide range of cell surface tumor antigens and has also been implemented to target pathogen-infected cells (see, for example, Non-Patent Document 17; Patent Documents 1 and 2).

[0014] The first subset of T cells, known as "helper T cells," are characterized by the expression of CD4 (i.e., they are "CD4 + " and CD3 + CD4 + T cells are the majority CD4 is an essential organizer of mammalian immune and autoimmune responses (Non-patent Document 4). + T thin Cell activation occurs when antigen-major histocompatibility class II (MHC II) molecule complexes arranged on the surface of antigen-presenting cells (e.g., B cells, macrophages, or dendritic cells) interact with naive CD4 + A complex of two molecules arranged on the surface of T cells: the T cell receptor ( It is known that activation of T helper cells is mediated by costimulatory interactions between TCR and CD3 cell surface receptor ligands. Activated T helper cells can expand into Th1 cells, which can mediate inflammatory responses against target cells.

[0015] A second subset of T cells, known as "cytotoxic T cells," are characterized by the expression of CD8 (i.e., they are "CD8 + " and CD3 + CD8 is a T cell co-receptor consisting of two distinct chains expressed on cytotoxic T cells (Non-Patent Document 18). CD8 + T cell activation occurs when a target cell reacts with an antigen, a major histocompatibility class I antigen, on its surface. Major histocompatibility complex (MHC I) and CD8 + CD8 and T cell surface It has been shown that the interaction is mediated by costimulatory interactions between the major histocompatibility class II (MHC) complex and the cellular receptor complex (Non-Patent Document 19). Unlike MHC II molecules, MHC I molecules are very broadly expressed. Therefore, cytotoxic T cells can bind to a wide range of cell types. Activated cytotoxic T cells mediate cell killing by releasing the cytotoxins perforin, granzymes, and granulysin. Through the action of perforin, granzymes enter the cytoplasm of target cells, where the serine protease function of granzymes triggers the caspase cascade, a series of cysteines that ultimately leads to apoptosis (programmed cell death) of the target cells.

[0016] BT cell receptor ("TCR") T cell receptors ("TCR") bind to CD4 + or CD8 + It is a natural development of T cells that enables these cells to recognize antigenic peptides bound and presented by class I or class II MHC proteins of antigen-presenting cells. Recognition of pMHC (peptide-MHC) by the TCR leads to cytokine production and lysis of the antigen-presenting cell. It initiates the propagation of cellular immune responses (see, for example, Non-Patent Documents 22 to 24). CD3 is a receptor that binds to TCR (Non-Patent Documents 12, 2, 26, 27, 11, 28).

[0017] The complex of TCR and CD3 with the CD3ζ chain zeta chain (also known as the T cell receptor T3 zeta chain or CD247) comprises the TCR complex (Non-Patent Documents 29, 9). This complex is particularly important because it contains a large number (10) of immunoreceptor tyrosine-based activation motifs (ITAMs). [Prior art documents] [Patent documents]

[0018]

Patent Document 1

Patent document 2

Non-licensed literature

[0019] [Non-licensed document 1] Portoles, P. et al. (2009) “The TCR / CD3 Complex: Opening the Gate to Successful Vaccination,” Current Pharmaceutical Design 15:3290-3300

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

[0020] Multispecific molecules comprising a CD3-binding domain and a binding domain specific for a disease antigen ("DA") expressed on target cells can mediate targeted T cell killing of such target cells. However, the affinity of such molecules for CD3 can make them so potent that they exhibit undesirable cytokine release from stimulated T cells. Thus, despite previous progress in identifying molecules involved in mammalian immune responses, there remains a need for improved therapies for the treatment of cancer and infectious diseases. The present invention provides a panel of variant CD3-binding domains with a range of binding kinetics that can be used to modulate the cell-killing and / or cytokine-releasing activity of such multispecific molecules to enhance the therapeutic window. The present invention is directed to this, and other, goals. [Means for solving the problem]

[0021] The present invention provides multispecific binding molecules (e.g., CD3-binding domains capable of binding to an epitope on CD3 and disease antigen-binding domains capable of binding to an epitope on a disease antigen (“DA”). The present invention is particularly directed to DAxCD3 binding molecules as described above that comprise a variant CD3 binding domain (a "vCD3 binding domain"), wherein the vCD3 binding domain is a CDR H 1 domain , CDR H 2 domains, CDR H 3 domains, CDR L 1 domain, CDR L 2 domains and CDRs L It contains three domains, at least one of which is a canonical CD3 binding domain ( The DA×CD3 binding molecules described above comprising a vCD3-binding domain as described above have an amino acid sequence that differs from the amino acid sequence of the corresponding CDR of a DA×CD3 binding domain ("rCD3-binding domain"), and exhibit altered affinity for CD3 compared to DA×CD3 binding molecules comprising an rCD3-binding domain as described above. The present invention particularly relates to DA×CD3 binding molecules as described above comprising a vCD3-binding domain that have reduced affinity for CD3, are capable of mediating targeted killing of DA-expressing target cells, and exhibit reduced levels of cytokine release compared to DA×CD3 binding molecules comprising an rCD3-binding domain. The present invention particularly relates to the use of DA×CD3 binding molecules comprising a vCD3-binding domain in the treatment of cancer and pathogen-related diseases. The present invention also relates to pharmaceutical compositions comprising one or more of the above molecules.

[0022] In particular, the present invention provides a DAxCD3 binding molecule comprising a CD3 binding domain capable of binding to an epitope on CD3 and a disease antigen binding domain capable of binding to an epitope on a disease antigen, wherein the CD3 binding domain is: (I) (A) a CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, and SEQ ID NO: 97; H 1 domain; (B) CDR comprising the amino acid sequence of SEQ ID NO: 58 H 2 domains; (C) CDR comprising the amino acid sequence of SEQ ID NO: 59H 3 domains; (D) CDR comprising the amino acid sequence of SEQ ID NO: 60 L 1 domain; (E) CDR comprising the amino acid sequence of SEQ ID NO: 61 L 2 domains; and (F) CDR comprising the amino acid sequence of SEQ ID NO: 62 L 3 domains; or (II) (A) a CDR comprising the amino acid sequence of SEQ ID NO: 57 H 1 domain; (B) CDR comprising the amino acid sequence of SEQ ID NO: 58 H 2 domains; (C) a CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, and SEQ ID NO: 107. H 3 domains; (D) CDR comprising the amino acid sequence of SEQ ID NO: 60 L 1 domain; (E) CDR comprising the amino acid sequence of SEQ ID NO: 61 L 2 domains; and (F) CDR comprising the amino acid sequence of SEQ ID NO: 62 L 3 domains; or (III) (A) CDR comprising the amino acid sequence of SEQ ID NO: 57 H 1 domain; (B) CDR comprising the amino acid sequence of SEQ ID NO: 58 H 2 domains; (C) CDR comprising the amino acid sequence of SEQ ID NO: 59 H 3 domains; (D) CDR comprising the amino acid sequence of SEQ ID NO: 60 L 1 domain; (E) CDR comprising the amino acid sequence of SEQ ID NO: 61 L 2 domains; and (F) a CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 109 and SEQ ID NO: 111 L3 Domains: or (IV) (A) CDR comprising the amino acid sequence of SEQ ID NO: 57 H 1 domain; (B) CDR comprising the amino acid sequence of SEQ ID NO: 58 H 2 domains; (C) CDR comprising the amino acid sequence of SEQ ID NO: 59 H 3 domains; (D) CDR comprising the amino acid sequence of SEQ ID NO: 60 L 1 domain; (E) a CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 113 and SEQ ID NO: 115; L 2 domains; and (F) CDR comprising the amino acid sequence of SEQ ID NO: 62 L 3 Domains Includes.

[0023] The present invention further provides a method for the production of a CD3 binding domain comprising: (I) (A) a VL domain comprising the amino acid sequence of SEQ ID NO: 56; (B) a VH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:98, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, and SEQ ID NO:106; or (II)(A) a VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114; (B) A VH domain comprising the amino acid sequence of SEQ ID NO: 55. The present invention relates to an embodiment of the DAxCD3 binding molecule described above, comprising:

[0024] The present invention further relates to the embodiment of the DAxCD3 binding molecule described above, wherein the DAxCD3 binding molecule is a bispecific antibody, a bispecific diabody, a bispecific scFv, a bispecific TandAb, or a trivalent binding molecule.

[0025] The present invention further relates to embodiments of the above-described DAxCD3 binding molecules, wherein the DAxCD3 binding molecules are capable of binding to two or more disease antigens and / or different cell surface molecules of effector cells, and in particular, the different cell surface molecules of effector cells are CD2, CD8, CD16, TCR, NKp46, or NKG2D.

[0026] The present invention further relates to embodiments of the above-described DAxCD3 binding molecules, wherein the disease antigen is a cancer antigen or a pathogen-associated antigen.

[0027] The present invention further relates to a method for treating cancer, comprising administering to a patient a cancer antigen selected from the group consisting of 19.9, 4.2, ADAM-9, AH6, ALCAM, B1, B7-H3, BAGE, β-catenin, and blood type ALe. b / Le y , Burkitt lymphoma antigen‐38.13, C14, CA125, carboxypeptidase M, CD5, CD19, CD20, CD22, CD23, CD25, CD27, CD28, CD33, CD36, CD40 / CD154, CD45, CD56, CD46, CD52, CD56, CD79a / CD79b, CD103, CD123, CD317, CDK4, CEA, CEACAM5 / CEACAM6, CO17‐1A, CO‐43, CO‐514, CTA‐1, CTLA-4, cytokeratin 8, D1.1, D156-22, DR5, E1 series, EGFR, ephrin receptor, EphA2, Erb, GAGE, GD2 / GD3 / GM2 ganglioside, GICA19-9, gp100, Gp37, gp75, gpA33, HER2 / neu, HMFG, human papillomavirus-E6 / human papillomavirus-E7, HMW-MAA, I antigen, IL13Rα2, integrin β6, JAM-3, KID3, KID31, KS 1 / 4 pan-carcinoma antigen, L6, L20, LEA, LUCA‐2, M1:22:25:8, M18, M39, MAGE, MART, mesothelin, MUC‐1, MUM‐1, Myl, N‐acetylglucosaminyltransferase, neoglycoprotein, NS‐10, OFA‐1, OFA‐2, oncostatin M, p15, p97, PEM, PEMA, PIPA, PSA, PSMA, prostatic acid phosphate, R 24, ROR1, sphingolipids, SSEA‐1, SSEA‐3, SSEA‐4, sTn, T cell receptor‐derived peptide, T5A7, TAG‐72, TL5, TNF‐receptor, TNF‐γ receptor, TRA‐1‐85, transferrin receptor, 5T4, TSTA, VEGF, VEGF receptor, VEP8, VEP9, VIM‐D5, and Y hapten, Le y Selected from the group consisting of The present invention relates to an embodiment of the above-mentioned DAxCD3 binding molecule, wherein

[0028] The present invention further relates to a method for treating cancer, comprising administering to a patient a therapeutically effective amount of a cancer antigen, the method ... In one embodiment of the above-mentioned DAxCD3 binding molecule, the DAxCD3 binding molecule is CD123, CD19, or ROR1.

[0029] The invention further relates to embodiments of the above-described DAxCD3 binding molecules, wherein the pathogen-associated antigen is selected from the group consisting of: herpes simplex virus infected cell protein (ICP) 47, herpes simplex virus gD, Epstein-Barr virus LMP-1, Epstein-Barr virus LMP-2A, Epstein-Barr virus LMP-2B, human immunodeficiency virus gp160, human immunodeficiency virus gp120, human immunodeficiency virus gp41, human papillomavirus E6, human papillomavirus E7, human T-cell leukemia virus gp64, human T-cell leukemia virus gp46, and human T-cell leukemia virus gp21.

[0030] The present invention further provides a DAxCD3 binding molecule comprising a first polypeptide chain and a second polypeptide chain covalently linked to each other: (A) The first polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) Domain 1, which: (1) The VL domain (VL) of a monoclonal antibody capable of binding to the above epitope of a disease antigen DA ), including the subdomain (1A); and (2) the VH domain (VH) of a monoclonal antibody capable of binding to the above epitope of CD3; CD3 ), including subdomains (1B) wherein said Subdomain 1A and said Subdomain 1B are separated from each other by a peptide linker; and (ii) Domain 2, a heterodimer-promoting domain Includes; (B) The second polypeptide chain comprises, in the N-terminal to C-terminal direction: (i) Domain 1, which: (1) The VL domain (VL) of a monoclonal antibody capable of binding to the above epitope of CD3 CD3 ), including the subdomain (1A); and (2) The VH domain (VH) of a monoclonal antibody capable of binding to the above epitope of a disease antigen DA ), including subdomains (1B) wherein said Subdomain 1A and said Subdomain 1B are separated from each other by a peptide linker; and (ii) Domain 2, which is a Heterodimer-Promoting Domain, wherein the Heterodimer-Promoting Domains of the first polypeptide chain and the second polypeptide chain are different. Including, (a) the VL Domain of the first polypeptide chain and the VH Domain of the second polypeptide chain associate to form the disease antigen-binding domain, and the VH Domain of the first polypeptide chain and the VL Domain of the second polypeptide chain associate to form the CD3-binding domain; or (b) The embodiment of the DA×CD3 binding molecule described above, wherein the VL Domain of the first polypeptide chain and the VH Domain of the second polypeptide chain associate to form the CD3-binding domain, and the VH Domain of the first polypeptide chain and the VL Domain of the second polypeptide chain associate to form the disease antigen-binding domain.

[0031] The present invention further comprises: (a) the Heterodimer-Promoting Domain of the first polypeptide chain is an E-coil domain and the Heterodimer-Promoting Domain of the second polypeptide chain is a K-coil domain; or (b) the Heterodimer-Promoting Domain of the first polypeptide chain is a K-coil domain and the Heterodimer-Promoting Domain of the second polypeptide chain is an E-coil domain; This relates to the above-described embodiment of the DAxCD3 binding molecule.

[0032] The present invention further relates to embodiments of the above-described DAxCD3 binding molecules, wherein the first polypeptide chain or the second polypeptide chain further comprises Domain 3, which comprises the CH2 and CH3 domains of an immunoglobulin Fc domain.

[0033] The present invention further relates to embodiments of the DAxCD3 binding molecules described above, wherein the DAxCD3 binding molecule further comprises a third polypeptide chain comprising the CH2 and CH3 domains of an immunoglobulin Fc domain.

[0034] The present invention further relates to embodiments of the DAxCD3 binding molecules described above, wherein said DAxCD3 binding molecule further comprises a CD8 binding domain.

[0035] The present invention further relates to a method for producing the DAxCD3 binding molecule comprising: (I)(A) a first polypeptide comprising SEQ ID NO: 179; (B) a second polypeptide comprising SEQ ID NO: 175; and (C) a third polypeptide comprising SEQ ID NO: 176; or (II)(A) a first polypeptide comprising SEQ ID NO: 184; (B) a second polypeptide comprising SEQ ID NO: 181; and (C) a third polypeptide comprising SEQ ID NO: 176; or (III) (A) a first polypeptide comprising SEQ ID NO: 196; (B) a second polypeptide comprising SEQ ID NO: 186; and (C) a third polypeptide comprising SEQ ID NO: 176; or (IV)(A) a first polypeptide comprising SEQ ID NO: 197; (B) a second polypeptide comprising SEQ ID NO: 192; and (C) a third polypeptide comprising SEQ ID NO: 176; or (V)(A) a first polypeptide comprising SEQ ID NO: 193; (B) a second polypeptide comprising SEQ ID NO: 194; and (C) a third polypeptide comprising SEQ ID NO: 176; or (VI)(A) a first polypeptide comprising SEQ ID NO: 179; (B) a second polypeptide comprising SEQ ID NO: 175; (C) a third polypeptide comprising SEQ ID NO: 187; and (D) a fourth polypeptide comprising SEQ ID NO: 188; or (VII) (A) a first polypeptide comprising SEQ ID NO: 184; (B) a second polypeptide comprising SEQ ID NO: 181; (C) a third polypeptide comprising SEQ ID NO: 187; and (D) a fourth polypeptide comprising SEQ ID NO: 188; or (VIII) (A) a first polypeptide comprising SEQ ID NO: 196; (B) a second polypeptide comprising SEQ ID NO: 186; (C) a third polypeptide comprising SEQ ID NO: 187; and (D) a fourth polypeptide comprising SEQ ID NO: 188; or (IX) (A) a first polypeptide comprising SEQ ID NO: 193; (B) a second polypeptide comprising SEQ ID NO: 194; (C) a third polypeptide comprising SEQ ID NO: 187; and (D) a fourth polypeptide comprising SEQ ID NO: 188 The present invention relates to an embodiment of the DAxCD3 binding molecule described above, comprising:

[0036] The present invention further relates to pharmaceutical compositions comprising any of the above-described DAxCD3 binding molecules and a pharmaceutically acceptable carrier.

[0037] The present invention further relates to a method for the treatment of a disease, comprising administering to a subject in need thereof a therapeutically effective amount of any of the above-described DAxCD3 binding molecules or the above-described pharmaceutical compositions.

[0038] The present invention further relates to embodiments of the above-described methods wherein the disease is cancer, including embodiments wherein the cancer is selected from the group consisting of adrenal gland cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer, blood cancer, multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal cell carcinoma, testicular cancer, and uterine cancer.

[0039] The present invention further relates to embodiments of the aforementioned methods wherein the disease is a pathogen-associated disease, including embodiments wherein the pathogen-associated antigen is selected from the group consisting of pathogen-associated antigens: herpes simplex virus infected cell protein (ICP) 47, herpes simplex virus gD, Epstein-Barr virus LMP-1, Epstein-Barr virus LMP-2A, Epstein-Barr virus LMP-2B, human immunodeficiency virus gp160, human immunodeficiency virus gp120, human immunodeficiency virus gp41, human papillomavirus E6, human papillomavirus E7, human T-cell leukemia virus gp64, human T-cell leukemia virus gp46, and human T-cell leukemia virus gp21.

[0040] The present invention further relates to the use of any of the above-mentioned DAxCD3 binding molecules or the above-mentioned pharmaceutical compositions in the treatment of disease.

[0041] The present invention further relates to embodiments of the above-described uses wherein the disease is cancer, including embodiments wherein the cancer is selected from the group consisting of adrenal gland cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer, blood cancer, multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal cell carcinoma, testicular cancer, and uterine cancer.

[0042] The present invention further relates to embodiments of the above-described uses wherein the disease is a pathogen-associated disease, including embodiments wherein the pathogen-associated antigen is selected from the group consisting of pathogen-associated antigens: herpes simplex virus infected cell protein (ICP) 47, herpes simplex virus gD, Epstein-Barr virus LMP-1, Epstein-Barr virus LMP-2A, Epstein-Barr virus LMP-2B, human immunodeficiency virus gp160, human immunodeficiency virus gp120, human immunodeficiency virus gp41, human papillomavirus E6, human papillomavirus E7, human T-cell leukemia virus gp64, human T-cell leukemia virus gp46, and human T-cell leukemia virus gp21. [Brief explanation of the drawings]

[0043] [Figure 1A-B] 1A-B are schematic diagrams of representative covalent diabodies having two epitope-binding domains composed of two polypeptide chains, each containing an E-coil or K-coil Heterodimer-Promoting Domain (optional Heterodimer-Promoting Domains are provided below). Cysteine ​​residues may be present in the linker (FIG. 1A) and / or in the Heterodimer-Promoting Domains (FIG. 1B). VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. [Figure 2] 2 is a schematic diagram of a representative covalently linked diabody molecule having two epitope-binding domains consisting of two polypeptide chains, each having a CH2 and CH3 domain, such that the linked chains form all or part of an Fc domain. VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern. [Figure 3A-3C]Figures 3A-3C are schematic diagrams of representative covalently linked tetravalent diabodies having four epitope-binding domains composed of two pairs of polypeptide chains (i.e., four total polypeptide chains). One polypeptide in each pair has a CH2 and CH3 domain, such that the linked chains form all or part of an Fc domain. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. The two pairs of polypeptide chains can be identical. In embodiments where the two pairs of polypeptide chains are identical and the VL and VH domains recognize different epitopes (as shown in Figures 3A-3B), the resulting molecule has four epitope-binding domains and is bispecific and bivalent for each epitope it binds. In embodiments where the VL and VH domains recognize the same epitope (e.g., the same VL domain CDRs and the same VH domain CDRs are used on both chains), the resulting molecule has four epitope-binding domains and is monospecific and tetravalent for a single epitope. Alternatively, the two pairs of polypeptides can be different. In embodiments in which the VL and VH domains of each pair of polypeptides recognize different epitopes (as indicated by the different shading and patterns in Figure 3C), the resulting molecule has four epitope-binding domains, is tetraspecific, and is monovalent for each epitope it binds. Figure 3A shows an Fc domain-containing diabody containing peptide Heterodimer-Promoting Domains containing cysteine ​​residues. Figure 3B shows an Fc domain-containing diabody containing E-coil and K-coil Heterodimer-Promoting Domains containing cysteine ​​residues and a linker (with an optional cysteine ​​residue). Figure 3C shows an Fc domain-containing diabody containing antibody CH1 and CL domains to promote heterodimerization. [Figure 4A-4B]4A-4B are schematic diagrams of representative covalently linked diabody molecules having two epitope-binding domains composed of three polypeptide chains. Two of the polypeptide chains have CH2 and CH3 Domains, such that the linked chains form all or part of an Fc domain. The polypeptide chain comprising the VL and VH Domains further comprises a Heterodimer-Promoting Domain. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. [Figure 5] Figure 5 is a schematic diagram of a representative covalently linked diabody molecule having four epitope-binding domains composed of five polypeptide chains. Two of the polypeptide chains have CH2 and CH3 Domains, such that the linked chains form an Fc domain containing all or part of an Fc domain. The polypeptide chain containing the VL and VH Domains further contains a Heterodimer-Promoting Domain. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. [Figures 6A-6F] Figures 6A-6F are schematic diagrams of representative Fc domain-containing trivalent binding molecules with three epitope-binding domains. Figures 6A and 6B each show a schematic diagram of a trivalent binding molecule containing two diabody-type binding domains and a Fab-type binding domain, with different domain orientations, in which the diabody-type binding domain is N-terminal or C-terminal to the Fc domain. The molecules in Figures 6A and 6B contain four chains. Figures 6C and 6D each show a schematic diagram of a trivalent binding molecule containing two diabody-type binding domains at the N-terminus of the Fc domain and a Fab-type binding domain or scFv-type binding domain in which the light and heavy chains are linked via a polypeptide spacer. The trivalent binding molecules in Figures 6E and 6F each show a schematic diagram of a trivalent binding molecule containing two diabody-type binding domains at the C-terminus of the Fc domain and a Fab-type binding domain or scFv-type binding domain in which the light and heavy chains are linked via a polypeptide spacer. The trivalent binding molecules in Figures 6C-6F each contain three chains. VL and VH domains that recognize the same epitope are shown using the same shading or filling pattern. [Figures 7A-7D] Figures 7A-7D show the results of CTL and binding assays. Figure 7A shows representative target-transduced cell killing results (% cytotoxicity in CTL assays) mediated by DART-A type diabody constructs containing the VL and VH domains of CD3 mAb 1; CD3 mAb 1 M1; CD3 mAb 1 M2; CD3 mAb 1 M15; CD3 mAb 1 M17; CD3 mAb 1 M18; CD3 mAb 1 M19; and CD3 mAb 1 M20. Figures 7B-7C plot the correlation between affinity constants (Figure 7B: KD; Figure 7C: ka; and Figure 7D: kd) and CTL activity (EC50 for cell lysis at 18 hours) reported in Tables 11-12. [Figures 8A-8E] Figures 8A-8E show the results of a representative study (CTL assay) of targeted cell killing mediated by DART-A type diabody constructs containing the VL and VH domains of CD3 mAb 1; CD3 mAb 1 M2; CD3 mAb 1 M7; CD3 mAb 1 M13; and CD3 mAb 1 M15 using Pan-T effector cells and MV-4-11 leukemia target cells. Percent cytotoxicity is plotted in Figure 8A. Cytokine responses are plotted in Figures 8B-8E (Figure 8B: IFN-γ; Figure 8C: TNF-α; Figure 8D: IL-6; Figure 8D: IL-2). NegCtrl: negative control. [Figure 9A-9B] Figures 9A-9B show the ability of DART-B diabodies to bind disease antigens. Figure 9A shows the ability of CD123-WT, CD123-M1, CD123-M2, and CD123-M18 DART-B diabodies to bind to CD123-expressing MOLM-13 cells. Figure 9B shows the ability of 5T4-WT, 5T4-M1, 5T4-M2, and 5T4-M18 DART-B diabodies to bind to 5T4-expressing A498 cells. Binding was detected using a biotinylated antibody specific for the E / K coil of the diabody and streptavidin-phycoerythrin (PE). [Figures 10A-10B]Figures 10A-10B show the ability of CD123-WT, CD123-M1, CD123-M2, and CD123-M18 DART-B type diabodies to bind to CD8+ T cells (Figure 10A) and CD4+ T cells (Figure 10B). [Figures 11A-11Q] Figures 11A-11Q show the results of a representative study (CTL assay) of target cell killing mediated by the CD123xCD3 DART type B diabody constructs (with Fc domain): CD123-WT (Figures 11B, 11F, 11J, and 11N), CD123-M2 (Figures 11C, 11G, 11K, and 11O), CD123-M18 (Figures 11D, 11H, 11L, and 11P), and HIV-WT (Figures 11E, 11I, 11M, and 11Q) using Pan-T effector cells and MOLM-13 acute monocytic leukemia (AML) target cells. Percent cytotoxicity is plotted in Figure 11A. Cytokine responses and percent cytotoxicity are plotted in Figures 11B-11Q (Figures 11B-11E: IFN-γ; Figures 11F-11I: TNF-α; Figures 11J-11M: IL-6; Figures 11N-11Q: IL-2). [Figures 12A-12E] Figures 12A-12E show the results of a representative study (CTL assay) of targeted cell killing mediated by the CD123xCD3 (with Fc domain) DART type B diabody construct using PBMC effector cells and MOLM-13 AML target cells. Percent cytotoxicity is plotted in Figure 12A (E:T=15:1, 24 hours). Cytokine responses are plotted in Figures 12B-12E (Figure 12B: IFN-γ; Figure 12C: TNF-α; Figure 12D: IL-6; Figure 12E: IL-2). [Figures 13A-13Q]Figures 13A-13Q show the results of a representative study (CTL assay) of target cell killing mediated by the 5T4xCD3 DART type B diabody constructs (containing the Fc domain): 5T4-WT (Figures 13B, 13F, 13J, and 13N), 5T4-M2 (Figures 13C, 13G, 13K, and 13O), 5T4-M18 (Figures 13D, 13H, 13L, and 13P), and HIV-WT (Figures 13E, 13I, 13M, and 13Q) using Pan-T effector cells and A498 renal cell carcinoma target cells (E:T = 5:1, 24 h). Cytotoxicity is plotted in Figure 13A. Cytokine responses and percent cytotoxicity are plotted in Figures 13B-13Q (Figures 13B-13E: IFN-γ; Figures 13F-13I: TNF-α; Figures 13J-13M: IL-6; Figures 13N-13Q: IL-2). [Figures 14A-14J] Figures 14A-14J show the results of a representative study (CTL assay) of targeted cell killing mediated by the CD19xCD3 DART type B diabody construct (with the Fc domain) using PBMCs (Figures 14A-14E) or Pan-T effector cells (Figures 14F-14J) (E:T = 30:1 for PBMCs, E:T = 10:1 for Pan-T cells, 24-48 hours). Percent cytotoxicity (48 hours) is plotted in Figure 14A (PBMCs) and Figure 14F (Pan-T cells). Cytokine responses at 48 hours using PBMCs are plotted in Figures 14B-14E (PBMCs) and Figures 14G-14J (Pan T cells) (Figures 14B and 14G: IFN-γ; Figures 14C and 14H: TNF-α; Figures 14D and 14I: IL-6; Figures 14E and 14J: IL-2). [Figures 15A-15E]Figures 15A-15E show the ability of representative CD123xCD3 DART-B diabodies to mediate T cell activation. T cell activation was measured by assessing the ability of the diabodies to affect CD25 and CD69 expression. Percent cytotoxicity is plotted in Figure 15A. CD4+ T cell activation, as determined by measuring CD25, is plotted in Figure 15B. CD4+ T cell activation, as determined by measuring CD69, is plotted in Figure 15C. CD8+ T cell activation, as determined by measuring CD25, is plotted in Figure 15D. CD8+ T cell activation, as determined by measuring CD69, is plotted in Figure 15E. [Figures 16A-16E] Figures 16A-16E show the ability of representative 5T4xCD3 DART-B diabodies to mediate T cell activation. T cell activation was measured by assessing the ability of the diabodies to affect CD25 and CD69 expression. Percent cytotoxicity is plotted in Figure 16A. CD4+ T cell activation, as determined by measuring CD25, is plotted in Figure 16B. CD4+ T cell activation, as determined by measuring CD69, is plotted in Figure 16C. CD8+ T cell activation, as determined by measuring CD25, is plotted in Figure 16D. CD8+ T cell activation, as determined by measuring CD69, is plotted in Figure 16E. [Figures 17A-17B] Figures 17A-17B show the results of an in vivo study on the ability of an exemplary CD123xCD3 DART type B diabody construct to mediate tumor regression in vivo. Mice receiving KG1A cells were fed CD123-WT (50 μg / kg) or CD123-M18 (5 μg / kg or 50 μg / kg), and tumor volume was assessed over 35 days. Figure 17A: CD4; Figure 17B: CD8. [Figures 18A-18D]Figures 18A-18D show the results of an in vivo study on the ability of the CD123xCD3 DART type B diabody construct to mediate tumor regression in vivo. Mice receiving KG1A cells were fed CD123-WT, CD123-M2, or CD123-M18 (0.5, 5, 50, or 500 μg / kg), and tumor volume was assessed over 35 days. Figure 18A: CD123-WT; Figure 18B: CD123-M2; Figure 18C: CD123-M18; Figure 18D: CD123-WT and CD123-M18, 50 μg / kg and 500 μg / kg treatment groups. [Figures 19A-19D] Figures 19A-19D show the results of an in vivo study on the ability of the CD123xCD3 DART type B diabody construct to mediate tumor regression in vivo. Mice receiving MV4-11 cells were fed CD123-WT, CD123-M2, or CD123-M18 (0.5, 5, 50, or 500 μg / kg), and tumor volume was assessed over 35 days. Figure 19A: CD123-WT; Figure 19B: CD123-M18; Figure 19C: CD123-M2; Figure 19D: CD123-WT, CD123-M2, and CD123-M18, 500 μg / kg treatment groups. [Figures 20A-20B] Figures 20A-20B show the results of an in vivo study on the ability of the 5T4xCD3 DART type B diabody construct to mediate tumor regression in vivo. Mice receiving SKOV3 cells were fed 5T4-WT (10, 50, 100, or 500 μg / kg), 5T4-M18 (10, 50, 100, or 500 μg / kg), or 5T4-M2 (500 μg / kg), and tumor volume was assessed over 45 days. Figure 20A: 5T4-WT; Figure 20B: 5T4-M18 and 5T4-M2. [Figures 21A-21D]Figures 21A-21D show the results of in vivo studies on the cytokine release profile induced by the CD123xCD3 DART-B type diabody. Serum cytokine levels (pg / ml) were assessed 6 hours after administration of CD123-WT, CD123-M2, or CD123-M18 (50 or 500 μg / kg) to mice receiving KG1A cells. Figure 21A: IFN-γ; Figure 21B: TNF-α; Figure 21C: IL-6; and Figure 21D: IL-2. [Figures 22A-22C] Figures 22A-22C show the ability of the CD123xCD3xCD8 trivalent molecules T-CD123-WT, T-CD123-M1, T-CD123-M2, and T-CD123-M18 to bind to cell surface antigens: Figure 22A: binding to CD123-expressing MOLM-13 cells; Figure 22B: binding to CD4+ T cells; Figure 22C: binding to CD8+ T cells. [Figures 23A-23G] Figures 23A-23G show the results of a representative study (CTL assay) of targeted cell killing mediated by the CD123xCD3xCD8 trivalent molecule using different T cell populations: T-CD123-WT, T-CD123-M1, T-CD123-M2, and T-CD123-M18. Percent cytotoxicity using CD3+ Pan-T cells (Figure 23A); CD4+ T cells (Figure 23B); and CD8+ T cells (Figure 23C) is plotted in Figures 23A-23C. Cytokine responses using CD3+ Pan-T cells are plotted in Figures 23D-23G. Figure 23D: IFN-γ; Figure 23E: TNF-α; Figure 23F: IL-6; and Figure 23G: IL-2. [Figures 24A-24J] Figures 24A-24J show serum cytokine levels, Ki67 expression, and clinical pathology marker levels observed in cynomolgus monkeys treated with CD123-M18 (10 mg / kg and 20 mg / kg) or CD123-WT (0.003 mg / kg). Figure 24A: IFN-γ; Figure 24B: TNF-α; Figure 24C: IL-6; Figure 24D: IL-2; Figure 24E: IL-15; Figure 24F: Ki67-positive CD4+ T cells; Figure 24G: Ki67-positive CD8+ T cells; Figure 24H: platelets; Figure 24I: C-reactive protein; Figure 24J: blood urea nitrogen. [Figures 25A-25G] Figures 25A-25G show the results of a representative study of AML blast depletion mediated by DART-A-WT, CD123-WT, CD123-M1, and CD123-M18 in peripheral blood samples from AML patients. Figure 25A: AML34+ blast counts as a percentage of control; Figure 25B: CD4+ cell proliferation; Figure 25C: CD8+ cell proliferation; Figures 25D-G: cytokine release (Figure 25D: IFN-γ; Figure 25E: TNF-α; Figure 25F: IL-6; and Figure 25G: IL-2). [Figures 26A-26E] Figures 26A-26E show the results of a representative study (CTL assay) of targeted cell killing mediated by the CD123xCD3 diabody constructs CD123-WT, CD123-M1, CD123-M13, CD123-M17, CD123-M18, and CD123-M19 using Pan-T effector cells and MOLM-13 AML target cells (E:T = 15:1, 48-96 hours). Cytotoxicity as a function of %LDH released is plotted in Figure 26A. Cytokine responses are plotted in Figures 26B-26E (Figure 26B: IFN-γ; Figure 26C: TNF-α; Figure 26D: IL-6; Figure 26E: IL-2). [Figures 27A-27D] Figures 27A-27D present cumulative results from 4-7 target-directed cell killing assays (CTL assays) and cytokine release studies mediated by the CD123xCD3 diabody constructs CD123-WT, CD123-M1, CD123-M13, CD123-M17, CD123-M18, CD123-M19, and DART-A-WT using Pan-T effector cells and MOLM-13 AML target cells (E:T = 15:1, 48-96 hours). EC50 values ​​(in pM) of CTL activity are plotted in Figure 27A. CTL activity as a multiple of the EC50 value of CD123-WT is plotted in Figure 27B. CTL activity Emax as a percentage of CD123-WT is plotted in Figure 27C. The calculated therapeutic index (TI=Emax(CTL):Emax(cytokine)), normalized to CD123-WT, is plotted in Figure 27D. [Figures 28A-28B]Figures 28A-28B show the results of an in vivo study on the ability of CD123xCD3 diabody constructs to mediate tumor regression in vivo. Mice receiving KG1A cells were fed CD123-WT (0.5 mg / kg), CD123-M18, or CD123-M13 (0.005, 0.05, 0.5, and 1 mg / kg), and tumor volume was assessed over 42 days. Figure 28A: CD123-WT and CD123-M18. Figure 28B: CD123-WT and CD123-M13. [Figures 29A-29B] Figures 29A-29B show the results of an in vivo study on the ability of CD123xCD3 diabody constructs to mediate tumor regression in vivo. Mice receiving KG1A cells were fed CD123-WT (0.05 mg / kg), CD123-M18, or CD123-M17 (0.005, 0.05, 0.5, and 1 mg / kg), and tumor volume was assessed over a 42-day period. Figure 29A: CD123-WT and CD123-M18. Figure 29B: CD123-WT and CD123-M17. [Figure 30A-30B] Figures 30A-30B show the results of an in vivo study on the interleukin-2 cytokine release profile induced by the CD123xCD3 DART-B diabody. Serum cytokine levels (pg / ml) were assessed 6 hours after administration of CD123-WT (0.5 mg / kg), CD123-M13, CD123-M17, or CD123-M18 (0.05, 0.5, and 1 mg / kg) to mice receiving KG1A cells. Figure 30A: CD123-WT, CD123-M13, and CD123-M18; and Figure 30B: CD123-WT, CD123-M17, and CD123-M18. [Figures 31A-31F]Figures 31A-31F show the results of a representative study of autologous B cell depletion with CD19-WT, CD19.1-M18, and HIV-M18 from human and cynomolgus monkey PBMCs. CD20+ B cell depletion is plotted in Figure 31A (human PBMCs) and Figure 31B (cyno PBMCs). Cytokine release from treated human PBMCs is plotted in Figures 31C-F (Figure 31C: IFN-γ; Figure 31D: TNF-α; Figure 31E: IL-6; and Figure 31F: IL-2). [Figures 32A-32D] Figures 32A-32D show the reduction in B cell levels observed in the peripheral blood of cynomolgus monkeys treated with CD19.1-M18 (1 mg / kg and 10 mg / kg) or CD123-WT (0.1 mg / kg). Pre-treatment B cell levels are shown in Figure 32A (B cell populations are indicated by ellipses). Levels on days 1, 8, and 15 are shown in Figures 32B-32D, respectively. [Figures 33A-33C] Figures 33A-33C show immunohistochemical staining of B cells in lymph nodes from cynomolgus monkeys before and 7 days after treatment with the positive control CD19-WT (Figure 33A: 0.1 mg / kg) or the CD3 variant CD19.1-M18 (Figure 33B: 10 mg / kg; and Figure 33C: 30 mg / kg). [Figure 34] FIG. 34 shows the reduction in B cell levels observed in the peripheral blood of cynomolgus monkeys treated with CD19.1-M13 (1 mg / kg), CD19.1-M17 (1 mg / kg), or CD19-WT (0.1 mg / kg). [Figures 35A-35E] Figures 35A-35E show serum cytokine levels observed in cynomolgus monkeys treated with CD19.1-M13 (1 mg / kg), CD19.1-M17 (1 mg / kg), or CD19-WT (0.1 mg / kg): Figure 35A: TNF-α, Figure 35B: IFN-γ, Figure 35C: IL-2, Figure 35D: IL-6; and Figure 35E: IL-15. [Figure 36A-36B]Figures 36A-36B show T cell proliferation observed in cynomolgus monkeys treated with CD19.1-M13 (1 mg / kg), CD19.1-M17 (1 mg / kg), or CD19-WT (0.1 mg / kg). Figure 36A: CD4+ T cells that are Ki67-positive; Figure 36B: Ki67-positive CD8+ T cells. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention is directed to multispecific binding molecules (e.g., bispecific antibodies, diabodies, bispecific scFvs, trivalent molecules, TandAb®, BiTE®, etc.) (e.g., "DAxCD3 binding molecules") that comprise a CD3-binding domain capable of binding to an epitope on CD3 and a disease antigen-binding domain capable of binding to an epitope on a disease antigen ("DA"). The present invention particularly relates to DAxCD3 binding molecules as described above that comprise a variant CD3-binding domain (a "vCD3 binding domain"), wherein the vCD3-binding domain comprises a CDR H 1 domain , CDR H 2 domains, CDR H 3 domains, CDR L 1 domain, CDR L 2 domains and CDRs L It contains three domains, at least one of which is a canonical CD3 binding domain ( The DA×CD3 binding molecules described above comprising a vCD3-binding domain as described above have an amino acid sequence that differs from the amino acid sequence of the corresponding CDR of a DA×CD3 binding domain ("rCD3-binding domain"), and exhibit altered affinity for CD3 compared to DA×CD3 binding molecules comprising an rCD3-binding domain as described above. The present invention particularly relates to DA×CD3 binding molecules as described above comprising a vCD3-binding domain that have reduced affinity for CD3, are capable of mediating targeted killing of DA-expressing target cells, and exhibit reduced levels of cytokine release compared to DA×CD3 binding molecules comprising an rCD3-binding domain. The present invention particularly relates to the use of DA×CD3 binding molecules comprising a vCD3-binding domain in the treatment of cancer and pathogen-related diseases. The present invention also relates to pharmaceutical compositions comprising one or more of the above molecules.

[0045] As noted above, therapeutic molecules of the present invention particularly include bispecific binding molecules comprising an epitope-binding domain capable of immunospecifically binding to an epitope on a cell surface molecule of an effector cell and an epitope-binding domain capable of immunospecifically binding to an epitope on a target cell expressing a disease antigen. As used herein, the term "disease antigen" (abbreviated "DA") refers to an antigen expressed on the surface of an abnormal or infected cell and characteristic of such an abnormality or infection, or an antigen expressed on the surface of a foreign cell and characteristic of such a foreign source. As used herein, a cell that expresses a disease antigen on its cell surface and thus can be bound by a therapeutic molecule of the present invention and thereby targeted for killing by the therapeutic molecule is a "target cell." Of particular relevance to the present invention are disease antigens that are "cancer antigens" or "pathogen-associated antigens."

[0046] I. Antibodies and Their Binding Domains The DAxCD3 binding molecules of the present invention may be antibodies or may be derivable from antibodies (e.g., by fragmenting, truncating, etc., an antibody polypeptide, or by using a polynucleotide (or sequence thereof) encoding the amino acid sequence of an antibody molecule or such a polynucleotide).

[0047] An antibody is an immunoglobulin molecule that can specifically bind to a particular domain or portion or conformation ("epitope") of a molecule, such as a carbohydrate, polynucleotide, lipid, or polypeptide. Epitope-containing molecules may have immunogenic activity; therefore, they elicit an antibody response in an animal. Such molecules are called "antigens." As used herein, the terms "antibody" and "antibodies" include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFvs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFvs), intrabodies, and epitope-binding domains of any of the above. In particular, the term "antibody" refers to immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an epitope-binding domain. Immunoglobulin molecules include any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, Antibodies can be of any class (IgA, IgB, IgC, IgD, IgE, IgF, IgH, IgIgA, IgF, IgH, IgF ... An antibody is capable of "immunospecifically binding" to a protein or non-protein molecule because of the presence of a particular domain, portion, or form (an "epitope") on such molecule.

[0048] The term "monoclonal antibody" refers to a homogeneous population of antibodies, which are composed of amino acids (naturally occurring or non-naturally occurring) involved in the selective binding of an antigen. Monoclonal antibodies are highly specific, being directed against a single epitope (or antigenic site). The term "monoclonal antibody" includes not only intact and full-length monoclonal antibodies, but also fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single chain (scFv), and mutants thereof. "Antibody" encompasses immunoglobulins, fusion proteins comprising antibody portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site with the required specificity and ability to bind to the antigen. No limitation is intended as to the source of the antigen or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins and fragments such as those set forth above in the definition of "antibody." Methods for making monoclonal antibodies are known in the art. One method that may be employed is that described by Kohler, G. et al. (1975) "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity," Nature 256:495-497. This method, or modifications thereof, is described. Typically, monoclonal antibodies are expressed in mice, rats, or rabbits. Such antibodies are produced by immunizing animals with an immunogenic amount of cells, cell extracts, or protein preparations containing the desired epitope. The immunogen can be, but is not limited to, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissues. Cells that may be used for immunization may be cultured for a period of time (e.g., at least 24 hours) before they are used as the immunogen. Cells may be used alone or in combination with a non-denaturing adjuvant, such as Ribi, as an immunogen (see Jennings, VM (1995) "Review of Selected Adjuvants Used in Antibody Production," ILARJ.37(3):119-125). Generally, when used as an immunogen, cells are cultured in a fully denatured state. The cells must be maintained intact, and preferably viable. Intact cells can better enable the immunized animal to detect the antigen than ruptured cells. The use of denaturing or strong adjuvants, such as Freund's adjuvant, can rupture the cells and is therefore not recommended. The immunogen may be administered multiple times at periodic intervals, such as once every two weeks or once a week, or may be administered to maintain viability in the animal (e.g., during tissue engineering). Alternatively, existing monoclonal antibodies and other equivalent antibodies with immunospecificity for the desired pathogenic epitope can be recombinantly sequenced and produced by any means known in the art. In one embodiment, such antibodies are sequenced, and the polynucleotide sequence is then cloned into a vector for expression or propagation. The sequence encoding the antibody of interest is maintained in the vector in host cells, which can then be expanded and frozen for future use. The polynucleotide sequences of such antibodies may be used for genetic engineering to improve the affinity or other characteristics of the antibodies by generating monospecific or multispecific (e.g., bispecific, trispecific and tetraspecific) molecules of the invention, as well as affinity-optimized, chimeric, humanized and / or caninized antibodies, as described in more detail below.

[0049] A binding molecule of the invention binds to an epitope via its binding domain in an "immunospecific" manner. As used herein, an antibody, diabody, or other epitope-binding molecule binds to a region (i.e., epitope) of another molecule more frequently, rapidly, for longer periods, and / or with higher affinity than to alternative epitopes. An antibody is said to "immunospecifically" bind to a molecule (i.e., an epitope) when it reacts with or associates with that molecule. For example, an antibody that immunospecifically binds to a viral epitope is one that binds with higher affinity, higher avidity, more rapidly, and / or for a longer period of time than the antibody binds to other viral or non-viral epitopes. Reading this definition, it is also understood that, for example, an antibody (or moiety or epitope) that immunospecifically binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "immunospecific binding" does not necessarily require (although it can include) exclusive binding. Generally, references to binding imply "immunospecific" binding, but this is not necessarily the case.

[0050] In recent decades, there has been a resurgence of interest in the therapeutic potential of antibodies, and antibodies have become one of the major classes of biotechnology-derived drugs (Chan, CE et al. (2009) “The Use of Antibodies in the Treatment of Infectious Diseases,” Singapore Med. J. 50(7):663-666). Over 200 antibody-based drugs have been approved for use or are currently in development. is under development.

[0051] Natural antibodies (such as IgG antibodies) consist of two "light chains" complexed with two "heavy chains." Each light chain contains a variable domain ("VL") and a constant domain ("CL"). Each heavy chain contains a variable domain ("VH"), three constant domains ("CH1," "CH2," and "CH3"), and a "hinge" region ("H") located between the CH1 and CH2 domains. In contrast, scFvs are single-chain molecules created by linking the light chain variable region and the heavy chain variable region via a short connecting peptide.

[0052] Thus, the basic structural unit of naturally occurring immunoglobulins (e.g., IgG) is a tetramer of two light chains and two heavy chains, usually expressed as a glycoprotein of approximately 150,000 Da. The amino-terminal ("N-terminal") portion of each chain contains approximately 100-110 variable domains primarily responsible for antigen recognition. The carboxy-terminal ("C-terminal") portion of each chain defines a constant region, with light chains having a single constant domain and heavy chains usually having three constant domains and a hinge domain. Thus, the structure of the light chain of an IgG molecule is n-VL-CL-c, and the structure of the IgG heavy chain is n-VH-CH1-H-CH2-CH3-c, where n and c represent the N- and C-termini of the polypeptide, respectively. The ability of an intact, unmodified antibody (e.g., an IgG antibody) to bind to an epitope on an antigen depends on the presence and sequence of the variable domains. Unless otherwise specified, the order of domains in protein molecules described herein is in the "N-terminal to C-terminal" direction.

[0053] A. Characteristics of Antibody Variable Domains The variable domain of an IgG molecule consists of three complementarity-determining regions ("CDRs"), which contain the amino acid residues of the antibody that contact the epitope, and four intervening non-CDR segments called framework regions ("FRs"), which separate the CDR segments and generally maintain the structure of the CDR residues and position the CDRs, enabling them to contact the epitope (although certain framework residues can also play a role in such contacts). Thus, the VL and VH domains have the structure n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c, where n and c represent the N- and C-termini of the domains, respectively. The amino acid sequences of the CDRs determine whether an antibody can bind to a particular epitope.

[0054] Amino acids from the mature heavy and light chain variable domains of immunoglobulins are expressed as amino acids in the chains. The amino acid sequence is specified by the amino acid position. Kabat et al. (Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, NH1, MD (1991) ("Kabat"), incorporated herein by reference, describes numerous amino acid sequences for antibodies, identifies amino acid consensus sequences for each subgroup, and assigns residue numbers to each amino acid. CDRs are identified as defined by Kabat (Chothia, C. & Lesk, AM (1987) "Canonical structures for the hypervariable regions of immunoglobulins," J. Mol. Biol. 196:901-917). H 1 is 5 residues (It will be understood that the Kabat numbering scheme begins at the beginning. The Kabat numbering scheme can be extended to antibodies not included in the compendium by aligning the antibody with one of the Kabat consensus sequences with reference to conserved amino acids. This method of assigning residue numbers has become standard in the art, and amino acids at identical positions in different antibodies, including chimeric or humanized variants, are readily identified. For example, the amino acid at position 50 of a human antibody light chain occupies the same position as the amino acid at position 50 of a murine antibody light chain.

[0055] Polypeptides that are (or can function as) the first, second, and third CDRs of the light chain of an antibody are referred to herein as CDRs 1, 2, and 3, respectively. L 1 domain, CDR L 2 domains and CDRs L Similarly, the first, second and third domains of an antibody heavy chain are called Polypeptides which are (or can function as) the first, second, and third CDRs of are referred to herein as CDRs 1, 2, and 3, respectively. H 1 domain, CDR H 2 domains and CDRs H 3 domains and Therefore, CDR L 1 domain, CDR L 2 domains, CDR L 3 domains, CD R H 1 domain, CDR H 2 domains and CDRs H The term 3-domain refers to a The present invention is directed to polypeptides that, when incorporated into a protein, enable the protein to bind to a specific epitope, regardless of whether the protein is an antibody having a light and heavy chain, or a diabody, or a single-chain binding molecule (e.g., scFv, BiTe, etc.), or another type of protein. Thus, as used herein, the term "epitope binding domain" refers to a domain comprising a fragment or portion of a binding molecule (or a polypeptide having the amino acid sequence of such a fragment or portion) that contributes to the ability of the binding molecule to immunospecifically bind to an epitope. An epitope binding domain may contain one, two, three, four, or five of the CDR domains of an antibody, or may contain all six of the CDR domains of an antibody, and may be capable of immunospecifically binding to such an epitope, but may exhibit immunospecificity, affinity, or selectivity for an epitope that is different from the epitope of such an antibody. An epitope-binding domain may contain only a portion of the CDRs, i.e., only the subset of CDR residues necessary for binding (these are called "specificity-determining residues" or "SDRs"; Kim, JH et al. (2012) "Humanization by CDR Grafting and Specificity-Determining Residues"). Residue Grafting,” Methods Mol. Biol. 907:237-245;Kim,KS et al. (2010) “Construction Of A HumanizedAntibodyTo Hepatitis B Surface Antigen By Specificity-DeterminingResidues(SDR)-Grafting And De-Immunization,” Biochem.Biophys.Res. Commun. 396(2):231-237; Kashmiri, SV et al. (2005) “SDR Grafting - A New Approach To Antibody Humanization,” Methods 36(1):25-34; Gonzales, NR et al. (2004) “SDR Grafting Of A Murine Antibody Using Multiple HumanGermlineTemplates To Minimize Its Immunogenicity,”Mol.Immunol. 41:863-872). Preferably, the epitope-binding domain will contain all six of the CDR domains of such an antibody. An epitope-binding domain of an antibody may be a single polypeptide chain (e.g., an scFv), or may be two or more polypeptide chains, each having an amino terminus and a carboxy terminus (e.g., a diabody, an Fab fragment, an Fab2 fragment, etc.). may include:

[0056] The present invention also relates to epitope-binding molecules comprising the VL and / or VH domains of humanized antibodies. The term "humanized antibody" refers to a chimeric molecule, generally prepared using recombinant techniques, that has an epitope-binding domain of an immunoglobulin from a non-human species and the remainder of the immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. Anti-human PD-1 antibodies of the invention include antibodies PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, PD-1 mAb 15, PD-1 mAb 16, PD-1 mAb 17, PD-1 mAb 18, PD-1 mAb 19, PD-1 mAb 20, PD-1 mAb 21, PD-1 mAb 22, PD-1 mAb 23, PD-1 mAb 24, PD-1 mAb 25, PD-1 mAb 26, PD-1 mAb 27, PD-1 mAb 28, PD-1 mAb 29, PD-1 mAb 30, PD-1 mAb 31, PD-1 mAb 32, PD-1 mAb 33, PD-1 mAb 34, PD-1 mAb 35, PD-1 mAb 36, PD-1 mAb 37, PD-1 mAb 38, PD-1 mAb 39, PD-1 mAb 40 These include humanized, chimeric, or caninized variants of PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15. The polynucleotide sequences of the variable domains of such antibodies can be used for genetic engineering to generate such derivatives and to improve the affinity or other characteristics of the antibodies. The general principle in humanizing an antibody involves retaining the nucleotide sequence of the epitope-binding domain of the antibody while replacing the non-human remainder of the antibody with human antibody sequences. There are four general steps to humanize a monoclonal antibody. The steps are as follows: (1) determining the nucleotide and predicted amino acid sequences of the light and heavy chain variable domains of the starting antibody; (2) designing the humanized or caninized antibody, i.e., determining the antibody framework regions to be used during the humanization or caninization process; (3) the actual humanization or caninization method / technique; and (4) transfection and expression of the humanized antibody. See, e.g., U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415.

[0057] Epitope-binding domains may comprise complete variable domains fused to constant domains, or only the complementarity-determining regions (CDRs) of such variable domains grafted onto appropriate framework regions. Epitope-binding domains may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant regions that are immunogenic in human individuals, but leaves the possibility of foreign variable domains (LoBuglio, AF et al. (1989) "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (USA) 86:4220-4224). Another approach is to provide human-derived constant regions. The focus of current research is not only on reshaping the variable domains but also on modifying them to resemble human forms as closely as possible. Both heavy and light chain variable domains are known to contain three complementarity-determining regions (CDRs) flanked by four framework regions (FRs), which vary in response to the antigen of interest and determine binding ability; the framework regions are presumed to be relatively conserved in a given species and to provide a scaffold for the CDRs. When preparing a non-human antibody against a specific antigen, the variable domain can be "reshaped" or "humanized" by grafting the CDRs from the non-human antibody onto the FRs present in the modified human antibody. The application of this approach to various antibodies has been reviewed in Sato, K. et al. (1993) "Reshaping a Human Antibody to Inhibit the Interleukin 6-Dependent Tumor Cell Growth," Cancer Res 53:851-856; Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting an Antilysozyme Activity," Science 239:1534-1536; Kettleborough, CA et al. (1991) "Humanization of a Mouse Monoclonal Antibody by CDR-Grafting: The Importance of Framework Residues on Loop Conformation," Protein Engineering 4:773-3783; Maeda, H. et al. (1991) “Construction Of Reshaped HumanAntibodies With HIV-NeutralizingActivity,” HumanAntibodies Hybridoma 2:124-134;Gorman, SD et al. (1991) “Reshaping A TherapeuticCD4 Antibody,” Proc. Natl. Acad. Sci. (USA)88:4181-4185; Tempest, PR et al. (1991) “Reshaping AHuman Monoclonal Antibody” To Inhibit Human Respiratory Syncytial Virus Infection in vivo,” Bio / Technology 9:266-271; Co, MS et al. (1991) “Humanized Antibodies For Antiviral Therap y,” Proc. Natl. Acad. Sci. (USA)88:2869-2873;Carter, P. et al. (1992) “Hum anization Of An anti-p185her2 Antibody For Human CancerTherapy,”Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co, MS et al. (1992) "Chimeric and Humanized Antibodies With Specificity for the CD33 Antigen," J. Immunol. 148:1149-1154 In some embodiments, humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from the mouse antibody). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that have been altered to differ in sequence relative to the original antibody.

[0058] A number of humanized antibody molecules containing epitope-binding domains derived from non-human immunoglobulins have been described, including chimeric antibodies with rodent or modified rodent variable domains and their associated complementarity-determining regions (CDRs) fused to human constant domains (see, e.g., Winter et al. (1991) "Man-made Antibodies," Nature 349:293-299; Lobuglio et al. (1989) "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989); Shaw et al. (1987) "Characterization Of A Mouse / Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor-Associated Antigen," J. Immunol. 138:4534-4538; and Brown et al. (1987) “Tumor-Specific Genetically Engineered Murine / Human Chimeric (See, “Monoclonal Antibody,” Cancer Res. 47:3577-3583). Other references are provided where appropriate. Other references describe rodent CDRs that are grafted onto human supporting framework regions (FRs) before fusion with human antibody constant domains (see, e.g., Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science 239:1534-1536; and Jones et al. (1986) "Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse," Nature 321:522-525). describes rodent CDRs supported by a nucleotide sequence. See, e.g., European Patent Publication No. 519,596. These "humanized" molecules are designed to minimize unwanted immunological responses to rodent anti-human antibody molecules that limit the duration and effectiveness of therapeutic applications of these moieties in human recipients. Other methods that may be utilized to humanize antibodies are described in Daugherty et al. (1991) "Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins," Nucl. Acids Res. 19:2471-2476, as well as U.S. Patent Nos. 6,180,377; 6,054,297; and U.S. Patent Nos. 6,180,377, 6,054,29 ... No. 5,997,867; and U.S. Pat. No. 5,866,692.

[0059] B. Characteristics of Antibody Constant Regions Throughout this specification, the numbering of residues in the constant region of IgG is based on Kabat et al., Sequences of Proteins of Immunological Interest, 5 th The numbering of the EU index is according to Ed. Public Health Service, NH1, MD (1991) ("Kabat", which is expressly incorporated herein by reference). The term "EU index as in Kabat" refers to the numbering of the constant domains of the human IgG1 EU antibody.

[0060] Polymorphisms have been observed at many different positions within antibody constant regions (e.g., Fc positions, including but not limited to, positions 270, 272, 312, 315, 356, and 358, numbered according to the EU index as set forth in Kabat), and therefore slight differences may exist between the sequences presented here and those of the prior art. Polymorphic forms of human immunoglobulins have been well characterized. Currently, 18 heavy chain allotypes ("Gm allotypes") are known: Glm(1,2,3,17) or Glm(a,x,f,z), G 2m(23) or G2m(n), G3m(5,6,10,11,13,14,15,16,21,24,26,27,28) or G3m(b1,c3,b3,b0,b3,b4,s,t,g1,c5,u,v,g5)(Lefranc, et al., The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78(1990);Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211). In particular, the present invention It is understood that the antibody can incorporate any allotype, isoallotype, or haplotype of any immunoglobulin gene and is not limited to the allotype, isoallotype, or haplotype of the sequences presented herein. Furthermore, depending on the expression system, the C-terminal amino acid residue of the CH3 domain (bold above) can be removed post-translationally. Thus, the C-terminal residue of the CH3 domain can be any amino acid residue in the binding molecules of the invention. Specifically encompassed by the invention are binding molecules lacking the C-terminal residue of the CH3 domain. Also specifically encompassed by the invention are constructs that include a C-terminal lysine residue in the CH3 domain.

[0061] 1. Heavy chain constant region: Fc domain The CH1 domains of the two heavy chains of an antibody are complexed with the "CL" constant region of the antibody's light chain, which is attached to the heavy chain CH2 domain via an intervening hinge domain.

[0062] An exemplary CH1 domain is a human IgG1 CH1 domain. The amino acid sequence of an exemplary human IgG1 CH1 domain is (SEQ ID NO:1): ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV is.

[0063] An exemplary CH1 domain is a human IgG2 CH1 domain. The amino acid sequence of an exemplary human IgG2 CH1 domain is (SEQ ID NO:2): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTV is.

[0064] An exemplary CH1 domain is a human IgG3 CH1 domain. The amino acid sequence of an exemplary human IgG3 CH1 domain is (SEQ ID NO:3): ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YTCNVNHKPS NTKVDKRV is.

[0065] An exemplary CH1 domain is a human IgG4 CH1 domain. The amino acid sequence of an exemplary human IgG4 CH1 domain is (SEQ ID NO:4): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV

[0066] One exemplary hinge domain is a human IgG1 hinge domain. The amino acid sequence of an exemplary human IgG1 hinge domain is (SEQ ID NO: 5): EPKSCDKTHTCPPCP.

[0067] Another exemplary hinge domain is a human IgG2 hinge domain. The amino acid sequence of an exemplary human IgG2 hinge domain is (SEQ ID NO: 6): ERKCCVECPPCP.

[0068] Another exemplary hinge domain is a human IgG3 hinge domain. The amino acid sequence of an exemplary human IgG3 hinge domain is (SEQ ID NO:7): ELKTPLGDTT HTCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCPEPK SCDTPPPCPR CP is.

[0069] Another exemplary hinge domain is a human IgG4 hinge domain. The amino acid sequence of an exemplary human IgG4 hinge domain is (SEQ ID NO: 8): ESKYGPPCPSCP. As described herein, the IgG4 hinge domain may include a stabilizing mutation, such as an S228P substitution. The amino acid sequence of an exemplary S228P-stabilized human IgG4 hinge domain is (SEQ ID NO: 9): ESKYGPPCPPCP.

[0070] The CH2 and CH3 domains of the two heavy chains of an antibody interact to form the "Fc domain" of an IgG antibody, which is recognized by cellular Fc receptors, including but not limited to Fcγ receptors (FcγRs). As used herein, the term "Fc domain" is used to define the C-terminal region of an IgG heavy chain. An Fc domain is said to be of a particular IgG isotype, class, or subclass if its amino acid sequence is most homologous to that IgG isotype than to other IgG isotypes. In addition to their known uses in diagnostics, antibodies have been shown to be useful as therapeutic agents.

[0071] The amino acid sequence of an exemplary human IgG1 CH2-CH3 domain is (SEQ ID NO: 10): 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X , which is numbered by the EU index in Kabat, X is a lysine (K) or is absent.

[0072] The amino acid sequence of an exemplary CH2-CH3 domain of human IgG2 is (SEQ ID NO: 11): 231 240 250 260 270 280 APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE 390 400 410 420 430 WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X which are numbered according to the EU index as set forth in Kabat, where X is lysine (K) or absent.

[0073] The amino acid sequence of an exemplary CH2-CH3 domain of human IgG3 is (SEQ ID NO: 12): : 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESSGQPENN YNTTPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE 440 447 ALHNRFTQKS LSLSPG X , which is numbered by the EU index in Kabat, X is a lysine (K) or is absent.

[0074] The amino acid sequence of an exemplary human IgG4 CH2-CH3 domain is (SEQ ID NO: 13): 231 240 250 260 270 280 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS 340 350 360 370 380 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSLG X , which is numbered by the EU index in Kabat, X is a lysine (K) or is absent.

[0075] 2. Light chain constant region As mentioned above, the light chain of an antibody contains one variable domain (“VL”) and one constant domain (“CL”).

[0076] A preferred CL domain is a human IgG CLκ domain. The amino acid sequence of an exemplary human CLκ domain is (SEQ ID NO: 14): RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC is.

[0077] Alternatively, an exemplary CL domain is a human IgG CLλ domain. The amino acid sequence of an exemplary human CLλ domain is (SEQ ID NO: 15): QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS is.

[0078] II. Multispecific molecules The ability of an antibody to bind to an epitope of an antigen depends on the presence and amino acid sequence of the VL and VH domains of the antibody. The interaction between the light and heavy chains of an antibody, particularly its VL domain, The interaction of the VH domain with the VH domain forms one of the two epitope-binding domains of a natural antibody such as IgG. While natural antibodies can only bind to one epitope species (i.e., they are monospecific), natural antibodies can bind to multiple copies of that species (i.e., they exhibit bivalency or multivalency).

[0079] Antibody functionality can be enhanced by generating multispecific antibody-based molecules that can simultaneously bind to two distinct and different antigens (or different epitopes of the same antigen) and / or by generating antibody-based molecules with even higher valency with respect to the same epitope and / or antigen (i.e., three or more epitope-binding domains).

[0080] A wide range of recombinant bispecific antibody formats have been developed to provide molecules with greater potency than natural antibodies (see, for example, WO 2008 / 003116, WO 2009 / 132876, WO 2008 / 003103, WO 2007 / 146968, WO 2009 / 018386, WO 2012 / 009544, WO 2013 / 070565), most of which use linker peptides to fuse additional epitope-binding domains (e.g. scFv, VL, VH, etc.) to or within the antibody core (IgA, IgD, IgE, IgG or IgM), or to fuse multiple epitope-binding domains (e.g. two Fab fragments or scFvs). An alternative format uses linker peptides to fuse epitope-binding domains (e.g., scFv, VL, VH, etc.) to dimerization domains such as CH2-CH3 domains or alternative polypeptides (WO 2005 / 070966, WO 2006 / 107786, WO 2006 / 107617, WO 2007 / 046893). WO 2013 / 174873, WO 2011 / 133886, and WO 2010 / 136172 teach trispecific antibodies in which the CL and CH1 domains are switched from their natural positions and the VL and VH domains are diversified to allow binding to more than one antigen (WO 2008 / 027236; WO 2010 / 108127). WO 2013 / 163427 and WO 2013 / 119903 disclose modifying the CH2 domain to contain a fusion protein adduct containing a binding domain. WO 2010 / 028797, WO 2010028796, and WO 2010 / 028795 disclose recombinant antibodies in which the Fc domain is replaced with additional VL and VH domains to form trivalent binding molecules. WO 2003 / 025018 and WO 2003012069 disclose recombinant diabodies in which each chain contains an scFv domain.WO 2013 / 006544 discloses multivalent fab molecules that are synthesized as single polypeptide chains and then subjected to proteolysis to obtain heterodimeric structures. and WO 1991 / 003493 disclose adding additional binding domains or functional groups to antibodies or antibody portions (e.g., adding a diabody to the light chain of an antibody, or adding additional VL and VH domains to the light and heavy chains of an antibody, or adding heterologous fusion proteins to each other or linking multiple Fab domains to each other).

[0081] The art has further noted the possibility of generating diabodies that differ from such natural antibodies in that they can bind two or more different epitope species (i.e., can exhibit dual or multispecificity in addition to or instead of bivalency or multivalency) (see, e.g., Holliger et al. (1993) "'Diabodies': Small Bivalent And Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci. (USA) 90:6444-6448; U.S. Patent Publication No. 2004 / 0058400 (Hollinger et al.); U.S. Patent Publication No. 2004 / 0220388; WO 02 / 02781 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both the Epidermal Growth Factor Receptor and the Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672; WO 02 / 02781 (Mertens et al.); Olafsen, T. et al. (2004) “CovalentDisulfide-Linked Anti-CEA Diabody Allows Site-SpecificConjugation AndRadiolabeling For Tumor Targeting Applications,” Protein Eng Des Sel. 17(1):21-27;Wu, A. etal. (2001) “Multimerization Of A Chimeric Anti-CD20 SingleChain Fv-Fv Fusion Protein Is Mediated Through VariableDomain Exchange, ” Protein Engineering 14(2):1025-1033;Asano et al. (2004) “A Diabody For CancerImmunotherapyAnd Its Functional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P-683, J. Biochem. 76(8):992;Takemura,S.et al. (2000) “Construction Of A Diabody(SmallRecombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588; Baeuerle, PAetal. (2009) “Bispecific T cell Engaging AntibodiesForCancer Therapy,” Cancer Res. 69(12):4941-4944).

[0082] In particular, stable covalent heterodimeric non-monospecific diabodies, called DART® diabodies, have been developed; see, e.g., Sloan, DD et al. (2015) “Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells,” PLoS Pathog. 11(11):e1005233. doi: 10.1371 / journal.ppat.1005233; Al Hussaini, M. etal.(2015) “Targeting CD123 In AML Using A T-CellDirectedDual-Affinity Re-Targeting (DART(R)) Platform,”Bloodpii: blood-2014-05-575704; Chichili, GR et al. (2015) “A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates,” Sci. Transl. Med. 7(289):289ra82; Moore, P.A. et al. (2011) “Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma,” Blood 117(17):4542-4551; Veri, M.C. et al. (2010) “Therapeutic Control Of B-Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold,” Arthritis Rheum. 62(7):1933-1943; Johnson, S. et al. (2010) “Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion,” J. Mol. Biol. 399(3):436-449); U.S. Patent No. 8,044,180; No. 8,133,982; U.S. Patent No. 8,187,593; U.S. Patent No. 8,193,318; U.S. Patent No. 8,530,627; U.S. Patent No. 8,669,349; U.S. Patent No. 8,778,339; U.S. Patent No. 8,784,808; U.S. Patent No. 8,795,667; U.S. Patent No. 8,802,091; U.S. Patent No. 8,802,093; U.S. Patent No. 8,946,387; U.S. Patent No. 8,968,730; and U.S. Patent No. 8,993,730; U.S. Patent Publication No. 2009 / 0060910; U.S. Patent Publication No. 2010 / 0174053; U.S. Patent Publication No. 2011 / 0081347; U.S. Patent Publication No. 2011 / 0097323; U.S. Patent No. 2011 / 0117089; U.S. Patent Publication No. 2012 / 0009186; U.S. Patent Publication No. 2012 / 0034221; U.S. Patent Publication No. 2012 / 0141476; U.S. Patent Publication No. 2012 / 0294796; U.S. Patent Publication No. 2013 / 0149236; U.S. Patent Publication No. 2013 / 0295121; U.S. Patent Publication No. 20 14 / 0017237; and U.S. Patent Publication No. 2014 / 0099318; European Patent No. 1868650; European Patent No. 2158221; European Patent No. 2247304; European Patent No. 2252631; European Patent No. 2282770; European Patent No. 2328934; European Patent No. 2376109; European Patent No. 2542256; European Patent Patent No. 2601216; European Patent No. 2714079; European Patent No. 2714733; European Patent No. 2786762; European Patent No. 2839842; European Patent No. 2840091; and International Publication Nos. 2006 / 113665; 2008 / 157379; 2010 / 027797; 2010 / 033279; and International Publication No. See WO2010 / 080538; WO2011 / 109400; WO2012 / 018687; WO2012 / 162067; WO2012 / 162068; WO2014 / 159940; WO2015 / 021089; WO2015 / 026892; and WO2015 / 026894. Such diabodies comprise two or more covalently complexed polypeptides and involve the incorporation of one or more cysteine ​​residues into each of the employed polypeptide species that are capable of forming disulfide bonds, thereby covalently linking one or more pairs of such polypeptide chains to one another. For example, the addition of a cysteine ​​residue to the C-terminus of such a structure has been shown to allow disulfide bonding between the participating polypeptide chains, which stabilizes the resulting diabody without interfering with the binding properties of the diabody.

[0083] The simplest DART® comprises two polypeptide chains, each containing three domains (FIGS. 1A-1B). The first polypeptide chain contains: (i) a Domain comprising the epitope-binding region of the light chain variable domain (VL1) of a first immunoglobulin; (ii) a second Domain comprising the epitope-binding region of the heavy chain variable domain (VH2) of a second immunoglobulin; and (iii) a third Domain ("Heterodimer-Promoting Domain"), which serves to promote heterodimerization with the second polypeptide chain and to promote covalent binding of the first polypeptide chain to the second polypeptide chain of the diabody. The second polypeptide chain contains: a complementary first domain (VL2 domain); a complementary second domain (VH1 domain); and a third domain ("Heterodimer-Promoting Domain") that complexes with the third domain of the first polypeptide chain to promote heterodimerization and covalent bonding with the first polypeptide chain. Such molecules are stable and potent, and have the ability to simultaneously bind two or more antigens. In one embodiment, the third domains of the first and second polypeptide chains each contain a cysteine ​​(represented in the drawings as a "circled C") residue, which forms a covalent disulfide bond. The third domain of one or both of the polypeptide chains may further comprise a CH2-CH3 domain sequence, which, upon complexation of one diabody polypeptide with another, forms an Fc domain. Such an Fc domain may serve to alter the biological half-life of the diabody, reduce its immunogenicity, and / or bind to Fc receptors on cells (such as B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells) to enhance or inhibit effector function.Numerous variants of such molecules have been described (e.g., U.S. Patent Publication Nos. 2015 / 0175697; 2014 / 0255407; 2014 / 0099318; 2013 / 0295121; 2010 / 0174053; 2009 / 0060910 ... 2007-0004909; European Patent Publication No. 2714079; European Patent Publication No. 2601216; European Patent Publication No. 2376109; European Patent Publication No. 2158221; European Patent Publication No. 1868650; and International Publication No. 2012 / 162068; International Publication No. 2012 / 018687; International Publication No. 2010 / 080538; International Publication No. 2006 / . 113665), and are provided herein.

[0084] Recently, trivalent constructs incorporating two diabody-type binding domains and one non-diabody-type domain and one Fc domain have been described (see, e.g., WO 2004 / 024994). (See WO 2015 / 15 / 184207 and WO 2015 / 184203.) Such trivalent binding molecules can be used to generate monospecific, bispecific, or trispecific molecules, which are provided in more detail below. The ability to bind three different epitopes provides enhanced potency.

[0085] For applications where a bispecific or tetravalent molecule is desired but an Fc is not required, alternative configurations are known in the art, including, but not limited to, bispecific T cell engager molecules, also referred to as "BiTEs" (see, e.g., WO 1993 / 11161; and WO 2004 / 106381), and tetravalent tandem antibodies, also referred to as "TandAbs®" (see, e.g., U.S. Patent Publication No. 2011-0206672; European Patent Publication No. 2371866; WO 1999 / 057150; WO 2003 / 025018; and WO 2013 / 013700). BiTEs are formed from a single polypeptide chain containing tandemly linked scFvs, and TandAbs are formed by the homodimerization of two identical chains containing VH1, VL2, VH2, and VL2 domains, respectively.

[0086] The ability to generate multispecific binding molecules (e.g., bispecific antibodies, bispecific diabodies, trivalent molecules, etc.) allows the diabodies to be used (in "trans") to co-ligate two cells by co-ligating receptors present on different cells (e.g., cross-linking a cytotoxic T cell with a target cell, such as a cancer cell or a pathogen-infected cell, that expresses a disease antigen) (Staerz et al. (1985) "Hybrid Antibodies Can Target Sites For Attack"). By T Cells,” Nature 314:628-631, and Holliger et al. (1996) “Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” Protein Eng. 9:299-305; Marvin et al. (2005) “Recombinant Approaches To IgG-Like Bispecific Antibodies,” Acta Pharmacol. Sin. 26:649-658; Sloan et al. (2015) “Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic TCells,” PLoS Pathog 11(11):e1005233.doi:10.1371 / journal.ppat.1005233). Multispecific molecules can be used (in cis) to co-ligate molecules such as receptors present on the surface of the same cell. Co-ligation of different cells and / or receptors is useful for modulating effector function and / or immune cell signaling. Multispecific molecules (e.g., bispecific diabodies) containing epitope-binding domains can be directed to surface determinants of any immune cell, such as CD2, CD3, CD8, CD16, TCR, natural killer group 2 member D receptor (NKG2D), etc., expressed on T lymphocytes, natural killer (NK) cells, antigen-presenting cells, or other mononuclear cells. In particular, epitope-binding domains directed to cell surface receptors present on immune effector cells are useful for generating multispecific binding molecules capable of mediating targeted cell killing.

[0087] The present invention provides binding molecules capable of mediating targeted killing of target cells (e.g., cancer cells, pathogen-infected cells, etc.) expressing a disease antigen ("DA"). Such binding molecules are capable of binding to a "first epitope" and a "second epitope," one of which is an epitope of CD3 and the other of which is an epitope of a disease antigen. Whether a particular epitope is designated as a first epitope or a second epitope is irrelevant; such designation relates only to the presence and orientation of domains in the polypeptide chains of the binding molecules of the invention. Thus, bispecific molecules of the invention comprise a "VL" capable of binding to an epitope of CD3. CD3 " / "VH CD3 " domain, and "VL" domain that can bind to an epitope of a disease antigen DA " / "VH DA " domain. The present invention specifically encompasses bispecific diabodies, bispecific scFvs, BiTEs, antibodies, TandAbs, and trivalent binding molecules produced using any of the methods provided herein.

[0088] A. Fc domain-free bispecific diabodies In one embodiment, the DAxCD3 binding molecules of the invention are bispecific diabodies, which contain domains capable of binding to both a first and a second epitope, but do not contain an Fc domain and are therefore incapable of binding to an FcγR molecule via an Fc-FcγR interaction. The first polypeptide chain of such an embodiment of a bispecific diabody comprises, in the N-terminal to C-terminal direction: an N-terminus; a VL domain (i.e., VL) of a monoclonal antibody capable of binding to the first or second epitope; CD3 or VL DA a first intervening spacer peptide (Linker 1); (see above) The first polypeptide chain is VL CD3 (if it contains) can bind to the epitope of the disease antigen a VH domain of a monoclonal antibody, or (wherein the first polypeptide chain is a VL DAa VH domain of a monoclonal antibody capable of binding to an epitope of CD3 (if containing a VH domain); optionally a second intervening spacer peptide (Linker 2) containing a cysteine ​​residue; a Heterodimer-Promoting Domain; and a C-terminus (Figures 1A-1B).

[0089] The second polypeptide chain of this embodiment of the bispecific diabody comprises, in the N-terminal to C-terminal direction: an N-terminus; a VL domain of a monoclonal antibody capable of binding to a first or second epitope (i.e., a VL CD3 or VL DA and said VL domain is said first domain of said diabody; an intervening spacer peptide (Linker 1); a VH domain of a monoclonal antibody capable of binding to the first or second epitope (i.e., VH CD3 or VH DA and said VH domain is said first polypeptide of said diabody; a second intervening spacer peptide (Linker 2), optionally containing a cysteine ​​residue; a Heterodimer-Promoting Domain; and a C-terminus (Figures 1A-1B). The employed VL and VH Domains specific for a particular epitope are preferably obtained from or derived from the same monoclonal antibody. However, the domains may also be derived from different monoclonal antibodies, provided that the domains assemble to form a functional binding site capable of immunospecifically binding to the epitope. Such different antibodies are referred to herein as "corresponding" antibodies.

[0090] The VL Domain of the first polypeptide chain interacts with the VH Domain of the second polypeptide chain to form a first functional epitope-binding domain specific for one of the epitopes (e.g., the first epitope). Similarly, the VL Domain of the second polypeptide chain interacts with the VH Domain of the first polypeptide chain to form a second functional epitope-binding domain specific for the other epitope (i.e., the second epitope). Thus, the selection of the VL and VH Domains of the first and second polypeptide chains is important so that the two polypeptide chains of the diabody, together, contain VL and VH Domains that can bind both the first epitope and the second epitope (i.e., together, the VL CD3 / VH CD3 and VL DA / VH DA The coordinates are "coordinated" so that the

[0091] Most preferably, the length of the intervening spacer peptide (i.e., "Linker 1" separating the VL and VH Domains) is selected to substantially or completely prevent the VL and VH Domains of the polypeptide chains from binding to each other (e.g., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 intervening linker amino acid residues). Thus, the VL and VH Domains of a first polypeptide chain are substantially or completely unable to bind to each other. Similarly, the VL and VH Domains of a second polypeptide chain are substantially or completely unable to bind to each other. A preferred intervening spacer peptide (Linker 1) has the sequence (SEQ ID NO: 16): GGGSGGGG.

[0092] The length and composition of the second intervening spacer peptide (Linker 2) is selected based on the selection of one or more polypeptide domains that promote dimerization (i.e., "Heterodimer-Promoting Domains"), as described above. Typically, the second intervening spacer peptide (Linker 2) contains 3 to 20 amino acid residues. In particular, when one or more of the Heterodimer-Promoting Domains employed does not contain a cysteine ​​residue, a cysteine-containing second intervening spacer peptide (Linker 2) is utilized. The cysteine-containing second intervening spacer peptide (Linker 2) contains one, two, three, or four or more cysteines. A preferred cysteine-containing spacer peptide (Linker 2) has the sequence GGCGGG (SEQ ID NO: 17). Alternatively, Linker 2 does not contain a cysteine ​​(e.g., GGG, GGGS (SEQ ID NO: 18), LGGGSG (SEQ ID NO: 19), or the like). SEQ ID NO: 19), GGGSGGGSGGG (SEQ ID NO: 20), ASTKG (SEQ ID NO: 21), LEPKSS (SEQ ID NO: 22), APSSS (SEQ ID NO: 23), etc.), cysteine-containing heterodimer promoters as described below. Optionally, both a cysteine-containing Linker 2 and a cysteine-containing Heterodimer-Promoting Domain are used.

[0093] The Heterodimer-Promoting Domain may comprise GVEPKSC (SEQ ID NO: 24) or comprises or consists of VEPKSC (SEQ ID NO: 25) or AEPKS C (SEQ ID NO: 26) and, on the other polypeptide chain, GFNRGEC (SEQ ID NO: 27) or FNRGEC (SEQ ID NO: 28). Good (US Patent No. 2007 / 0004909).

[0094] In certain preferred embodiments, the Heterodimer-Promoting Domain is a tandem repeat coil domain of opposite charge, e.g., an "E-coil" Heterodimer-Promoting Domain (SEQ ID NO:29), in which glutamic acid residues form a negative charge at pH 7. E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K ), or a "K-coil" Heterodimer-Promoting Domain (SEQ ID NO: 30: K VAALK E- K VAAL K E- K VAAL K E- K VAAL K E). The presence of the domain promotes association between the first and second polypeptides, thus promoting heterodimer formation. Heterodimer-Promoting Domains may be utilized that contain the E-coil and K-coil sequences described above, modified to include one or more cysteine ​​residues. The presence of such cysteine ​​residues allows the coil present in one polypeptide chain to covalently bond with a complementary coil present in the other polypeptide chain, thereby covalently linking the polypeptide chains to each other and improving the stability of the diabody. Particularly preferred examples include the amino acid sequence E VAA CE K- E VAAL E K- E VAAL E K- E VAAL E Modified nucleotide sequence having K (SEQ ID NO: 31) The E coil and the amino acid sequence K VAA CK E- K VAAL K E- K VAAL K E- K VAAL K E (SEQ ID NO: 32) and a modified K coil that binds to the ribosomal domain.

[0095] As disclosed in WO 2012 / 018687, to improve the in vivo pharmacokinetic properties of diabodies, diabodies may be modified to contain a serum-binding protein polypeptide moiety at one or more of the diabody's termini. Most preferably, such serum-binding protein polypeptide moiety will be located at the C-terminus of the diabody's polypeptide chain. Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin has multiple small molecule binding sites that allow it to non-covalently bind to other proteins to extend its serum half-life. The albumin-binding domain 3 (ABD3) of protein G of Streptococcus strain G148 consists of 46 amino acid residues that form a stable triple-helical bundle and has broad albumin-binding specificity (Johansson, MU et al. (2002) "Structure, Specificity, And Mode Of Action"). “Interaction For Bacterial Albumin-Binding Modules,” J. Biol. Chem. 277(10):8114-8120). Therefore, serum binding to improve the in vivo pharmacokinetic properties of diabodies is important. A particularly preferred polypeptide portion of the protein is the albumin binding domain (ABD) from streptococcal protein G, and more preferably the albumin binding domain 3 (ABD3) of protein G of streptococcal strain G148 (SEQ ID NO: 33): LAAKVLANR ELDKYGVSDY YKNLINNAKT VEGVKALIDE ILAALP.

[0096] As disclosed in WO 2012 / 162068 (incorporated herein by reference), "deimmunized" variants of SEQ ID NO: 33 bind to MHC It has the ability to attenuate or eliminate class II binding. Based on the results of combined mutations, the following substitution combinations are considered to be preferred substitutions for forming such deimmunized ABDs: 66D / 70S+71A; 66S / 70S+71A; 66S / 70S+79A; 64A / 65A / 71A; 64A / 65A / 71A+66S; 64A / 65A / 71A+66D; 64A / 65A / 71A+66E; 64A / 65A / 79A+66S; 64A / 65A / 79A+66D; 64A / 65A / 79A+66E. A mutant ABD with the modifications L64A, I65A and D79A, or N66S, T70S and D79A. Amino acid sequence: LAEAKVLANR ELDKYGVSDY YKNLI D 66 NAK S 70 A 71 EGVKALIDEILAALP (SEQ ID NO: 34) or the amino acid sequence: LAEAKVLANR ELDKYGVSDY YKN A 64 A 65 NNAKTVEGVKALI A 79 E ILAALP (SEQ ID NO: 35) or the amino acid sequence: LAEAKVLANR ELDKYGVSDY YKNLI S 66 NAK S 70 VEGVKALI A 79 E ILAALP (SEQ ID NO: 36) Particularly preferred is a mutant deimmunized ABD having the sequence:

[0097] B. Fc domain-containing diabodies One embodiment of the present invention relates to multispecific diabodies (e.g., bispecific, trispecific, tetraspecific, etc.) that comprise an Fc domain and can simultaneously bind to an epitope on CD3 and an epitope on a disease antigen. The Fc domain of such molecules can be of any isotype (e.g., IgG1, IgG2, IgG3, or IgG4). The molecules may further comprise a CH1 domain and / or a hinge domain. If present, the CH1 domain and / or hinge domain can be of any isotype (e.g., IgG1, IgG2, IgG3, or IgG4), preferably of the same isotype as the desired Fc domain.

[0098] The addition of an IgG CH2-CH3 domain to one or both of the diabody polypeptide chains, such that the diabody chains complex to form an Fc domain, increases the biological half-life and / or alters the valency of the diabody. Such diabodies comprise two or more polypeptide chains with sequences that allow the polypeptide chains to be covalently linked to one another to form a covalently linked diabody capable of simultaneously binding a first epitope and a second epitope. Incorporation of an IgG CH2-CH3 domain into both of the diabody polypeptides allows for the formation of a two-chain bispecific Fc domain-containing diabody (Figure 2).

[0099] Alternatively, incorporation of an IgG CH2-CH3 domain into one of the diabody polypeptides allows for the formation of more complex four-chain bispecific Fc domain-containing diabodies (Figures 3A-3C). Figure 3C shows a representative four-chain diabody having a constant light (CL) domain and a constant heavy CH1 domain, although fragments of such domains and other polypeptides may alternatively be employed (see, e.g., Figures 3A and 3B, U.S. Patent Publication Nos. 2013-0295121; 2010-0174053 and 2009-0060910; EP 2714079; EP 2601216; EP 2376109; EP 2158221; and WO 2012 / 162068; WO 2012 / 018687; WO 2010 / 080538). Thus, for example, in place of the CH1 domain, a human The amino acid sequence GVEPKSC (SEQ ID NO: 24), VEPKSC (SEQ ID NO: 25), derived from the hinge domain of IgG Peptides having the C-terminal six amino acids of the human kappa light chain, GFNRGEC (SEQ ID NO: 27) or FNRGEC (SEQ ID NO: 28) may be employed, and in place of the CL domain, peptides having the C-terminal six amino acids of the human kappa light chain, GFNRGEC (SEQ ID NO: 29) or FNRGEC (SEQ ID NO: 30) may be employed. A representative peptide containing four-chain diabody is shown in Figure 3A. Alternatively, or in addition, tandem coil domains of opposite charge, such as an "E-coil" helical domain (SEQ ID NO: 29: E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K or SEQ ID NO: 30 : E VAA CE K- E VAAL E K- E VAAL E K- E VAAL E K); and the "K coil" domain (SEQ ID NO: 31: K VAALK E- K VAAL K E- K VAAL K E- K VAAL K E or SEQ ID NO: 32: K VAA CK E- K VAAL K E- K VAAL K E- K VAAL K E) A representative coil domain containing four-chain diabody is shown in Figure 3B.

[0100] The Fc domain-containing diabody molecules of the present invention may include an additional intervening spacer peptide (linker); such linkers are generally incorporated between the Heterodimer-Promoting Domain (e.g., E coil or K coil) and the CH2-CH3 Domain, and / or between the CH2-CH3 Domain and the Variable Domain (i.e., VH or VL). Typically, this additional linker contains 3-20 amino acid residues and may optionally contain all or a portion of an IgG hinge domain (preferably, the cysteine-containing portion of an IgG hinge domain having one, two, three, or four or more cysteine ​​residues). Linkers that can be employed in the bispecific Fc domain-containing diabody molecules of the present invention include: GGGS (SEQ ID NO: 18), LGGGSG (SEQ ID NO: 19), GGGSGGGSGGG (SEQ ID NO: 20), ASTKG (SEQ ID NO: 21), LEPKSS (SEQ ID NO: 22), APSSS (SEQ ID NO: 23), and APSSSPME (SEQ ID NO: 37). , VEPKSADKTHTCPPCP (SEQ ID NO: 38), LEPKSADKTHTCPPCP (SEQ ID NO: 39), DKTHTCPPCP (SEQ ID NO: 40), scFv linker: GGGGSGGGSGGGGS (SEQ ID NO: 41); "long (l Examples of linkers that can be used include: GGGGSGGGSGGG (SEQ ID NO: 42), GGC, and GGG. For ease of cloning, LEPKSS (SEQ ID NO: 22) may be used in place of GGG or GGC. Additionally, the amino acids GGG or LEPKSS (SEQ ID NO: 22) may be immediately followed by DKTHTCPPCP (SEQ ID NO: 40) to form alternative linkers: GGGDKTHTCPPCP (SEQ ID NO: 43); and LEPKSSDKTHTCPPCP (SEQ ID NO: 44). The bispecific Fc domain-containing The molecule may incorporate an IgG hinge domain in addition to or instead of a linker. An exemplary hinge domain is: EPKSCDKTHTCPPCP (SEQ ID NO: 5) from IgG1. ERKCCVECPPCP (SEQ ID NO: 6) from IgG2; ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 7) from IgG3; ESKYGPPCPSCP (SEQ ID NO: 8) from IgG4; and ESKYGPPCPPCP (SEQ ID NO: 9) from an IgG4 hinge variant containing a stabilizing S228P substitution to reduce strand exchange (numbered according to the EU index as set forth in Kabat).

[0101] As shown in Figures 3A-3C, Fc domain-containing diabodies of the invention may comprise four distinct chains. The first and third polypeptide chains of such diabodies contain four domains: (i) a VL1-containing Domain; (ii) a VH2-containing Domain; (iii) a Heterodimer-Promoting Domain; and (iv) a Domain containing a CH2-CH3 sequence. The second and fourth polypeptide chains contain: (i) a VL2-containing Domain; (ii) a VH1-containing Domain; and (iii) a Heterodimer-Promoting Domain, which promotes dimerization of the first / third polypeptide chain with the second / fourth polypeptide chain. The VL and / or VH Domains of the third and fourth polypeptide chains and the VL and / or VH Domains of the first and second polypeptide chains may be the same or different, allowing for tetravalent binding of monospecific, bispecific, or tetraspecific polypeptides. The notation "VL3" and "VH3" refer to the light chain variable domain and heavy chain variable domain, respectively, that bind the "third" epitope of such a diabody. Similarly, the notation "VL4" and "VH4" refer to the light chain variable domain and heavy chain variable domain, respectively, that bind the "fourth" epitope of such a diabody. Representative four-chain bispecific F The general structure of the polypeptide chains of c-domain-containing diabodies is presented in Table 1.

[0102] [Table 1]

[0103] In a specific embodiment, diabodies of the invention are bispecific, tetravalent (i.e., having four epitope-binding domains), Fc-containing diabodies composed of a total of four polypeptide chains (Figures 3A-3C). Bispecific, tetravalent, Fc-containing diabodies of the invention comprise two first epitope-binding domains and two second epitope-binding domains.

[0104] In further embodiments, Fc domain-containing diabodies of the present invention may comprise three polypeptide chains. The first polypeptide of such diabodies contains three domains: (i) a VL1-containing domain; (ii) a VH2-containing domain; and (iii) a domain containing a CH2-CH3 sequence. The second polypeptide of such diabodies contains: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) a domain that promotes heterodimerization and covalent bonding with the first polypeptide chain of the diabody. The third polypeptide of such diabodies comprises a CH2-CH3 sequence. Thus, the first and second polypeptide chains of such diabodies link together to form a VL1 / VH1 epitope-binding domain capable of binding to either the first or second epitope, and a VL2 / VH2 epitope-binding domain capable of binding to the other of the epitope chains. The first and second polypeptides are linked to each other by disulfide bonds involving cysteine ​​residues in their respective third domains. In particular, the first and third polypeptide chains complex with each other to form an Fc domain stabilized by a disulfide bond. Such bispecific diabodies have enhanced potency. Figures 4A and 4B show the structure of such diabodies. Such Fc domain-containing diabodies can have either of two orientations (Table 2).

[0105] [Table 2]

[0106] In a specific embodiment, diabodies of the invention are bispecific, bivalent (i.e., have two epitope-binding domains), Fc-containing diabodies composed of a total of three polypeptide chains (Figures 4A-4B). Bispecific, bivalent Fc-containing diabodies of the invention comprise one epitope-binding domain immunospecific for a first or second epitope, and a VL2 / VH2 epitope-binding domain capable of specific binding to the other of the above epitopes.

[0107] In further embodiments, the Fc domain-containing diabody may comprise a total of five polypeptide chains. In certain embodiments, two of the five polypeptide chains have identical amino acid sequences. The first polypeptide chain of such a diabody contains: (i) a VH1-containing domain; (ii) a CH1-containing domain; and (iii) a domain containing a CH2-CH3 sequence. The first polypeptide chain may be a heavy chain of an antibody containing a VH1 and a heavy chain constant region. The second and fifth polypeptide chains of such a diabody contain: (i) a VL1-containing domain; and (ii) a CL-containing domain. The second and / or fifth polypeptide chains of such a diabody may be a light chain of an antibody containing a VL1 that is complementary to the VH1 of the first / third polypeptide chain. The first, second, and / or fifth polypeptide chains may be isolated from naturally occurring antibodies or may be constructed recombinantly. The third polypeptide chain of such diabodies contains: (i) a VH1-containing Domain, (ii) a CH1-containing Domain, (iii) a Domain containing a CH2-CH3 sequence, (iv) a VL2-containing Domain, (v) a VH3-containing Domain, and (vi) a Heterodimer-Promoting Domain, which promotes dimerization of the third chain with the fourth chain. The fourth polypeptide of such diabodies contains: (i) a VL3-containing Domain, (ii) a VH2-containing Domain, and (iii) a Domain that promotes heterodimerization and covalent bonding with the third polypeptide chain of the diabody.

[0108] Thus, the first and second polypeptide chains and the third and fifth polypeptide chains of such a diabody are linked together to form two VL1 / VH1 epitope-binding domains capable of binding to a first epitope. The third and fourth polypeptide chains of such a diabody are linked together to form a VL2 / VH2 epitope-binding domain capable of binding to a second epitope and a VL3 / VH3 binding domain capable of binding to a third epitope. The first and third polypeptides are linked together by disulfide bonds involving cysteine ​​residues in their respective constant regions. In particular, the first and third polypeptide chains complex with each other to form an Fc domain. Such multispecific diabodies have enhanced potency. Figure 5 shows the structure of such a diabody. It will be understood that the VL1 / VH1, VL2 / VH2, and VL3 / VH3 domains can be identical or different, thereby enabling monospecific, bispecific, or trispecific binding.

[0109] The VL and VH domains of the polypeptide chains are selected to form a VL / VH binding site specific for a desired epitope. The VL / VH binding sites formed by the linkage of the polypeptide chains can be identical or different, thereby enabling tetravalent binding that is monospecific, bispecific, trispecific, or tetraspecific. In particular, the VL and VH domains can be selected such that a multivalent diabody contains two binding sites for a first epitope and two binding sites for a second epitope, or three binding sites for a first epitope and one binding site for a second epitope, or (as shown in Figure 5) two binding sites for a first epitope, one binding site for a second epitope, and one binding site for a third epitope. The general structures of the polypeptide chains of representative five-chain Fc domain-containing diabodies of the invention are shown in Table 3.

[0110] [Table 3]

[0111] In a specific embodiment, a diabody of the present invention is a bispecific, tetravalent (i.e., having four epitope-binding domains), Fc-containing diabody composed of five total polypeptide chains, having two epitope-binding domains immunospecific for a first epitope and two epitope-binding domains specific for a second epitope. In another embodiment, a bispecific, tetravalent, Fc-containing diabody of the present invention comprises three epitope-binding domains immunospecific for a first epitope and one epitope-binding domain specific for a second epitope. As noted above, the VL and VH domains may be selected to enable trispecific binding. Accordingly, the present invention also encompasses trispecific, tetravalent Fc-containing diabodies. The trispecific tetravalent Fc-containing diabodies of the invention comprise two epitope-binding domains immunospecific for a first epitope, one epitope-binding domain immunospecific for a second molecule, and one epitope-binding domain immunospecific for a third epitope.

[0112] In classical immune function, the interaction of antibody-antigen complexes with cells of the immune system leads to a wide range of responses, ranging from effector functions such as antibody-dependent cellular cytotoxicity, mast cell degranulation, and phagocytosis to immunoregulatory signals such as those controlling lymphocyte proliferation and antibody secretion. All of these interactions are initiated by the binding of the Fc domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses triggered by antibodies and immune complexes is achieved by the structural heterogeneity of three Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A), and FcγRIII (CD16) are activating (i.e., immune system-enhancing) receptors; FcγRIIB (CD32B) is an inhibitory (i.e., immune system-decreasing) receptor. Furthermore, interaction with the neonatal Fc receptor (FcRn) mediates the recycling of IgG molecules from endosomes to the cell surface and their release into the circulation. The amino acid sequences of exemplary wild-type IgG1 (SEQ ID NO: 10), IgG2 (SEQ ID NO: 11), IgG3 (SEQ ID NO: 12), and IgG4 (SEQ ID NO: 13) have been presented previously.

[0113] Modification of the Fc domain can lead to phenotypic changes, such as altered serum half-life, altered stability, altered susceptibility to cellular enzymes, or altered effector function. It may be desirable to modify Fc domain-containing binding molecules of the present invention with respect to effector function to enhance the effectiveness of such molecules, for example, in the treatment of cancer. In certain cases, for example, in the case of antibodies whose mechanism of action involves blocking or antagonizing rather than killing cells bearing the target antigen, reducing or eliminating Fc domain-mediated effector function is desirable. Increased effector function is generally desirable when targeting unwanted cells, such as tumors and foreign cells that express low levels of FcγR, e.g., tumor-specific B cells (e.g., non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma) that have low levels of FcγRIIB. Such molecules of the present invention with conferred or altered effector function activity are useful for the treatment and / or prevention of diseases, disorders, or infections in which enhanced efficacy of effector function activity is desirable.

[0114] Thus, in certain embodiments, the Fc domain of an Fc domain-containing molecule of the invention may be an engineered variable Fc region. The Fc domain of a bispecific Fc domain-containing molecule of the invention may be capable of binding to one or more Fc receptors (e.g., one or more FcγRs), but more preferably, the variant Fc domain has altered binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) (relative to the binding exhibited by the wild-type Fc domain), e.g., enhanced binding to an activating receptor, and / or reduced or no ability to bind to one or more inhibitory receptors. Thus, the Fc domain of the bispecific Fc domain-containing molecules of the invention may comprise some or all of the CH2 domain and / or some or all of the CH3 domain of a complete Fc domain, or may comprise a variant CH2 and / or variant CH3 sequence (e.g., which may comprise one or more insertions and / or one or more deletions relative to the CH2 or CH3 domain of the complete Fc domain). Such an Fc domain may comprise non-Fc polypeptide portions, or may comprise a portion of a complete Fc domain that does not occur in nature, or may comprise a non-naturally occurring orientation of the CH2 and / or CH3 domain (e.g., two CH2 domains or two CH3 domains, or a CH3 domain joined to a CH2 domain in the N-terminal to C-terminal direction, etc.).

[0115] Fc domain modifications that result in altered effector function are known in the art and include modifications that increase binding to activating receptors (e.g., FcγRIIA (CD16A)) and modifications that decrease binding to inhibitory receptors (e.g., FcγRIIB (CD32B)) (see, e.g., Stavenhagen, JB et al. (2007) “Fc Optimization of Therapeutic Antibodies Enhances Their Ability to Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors,” Cancer Res. 57(18):8882-8890). Table 4 shows the effects of activating receptors on the cytotoxicity of ATP. and / or a list of exemplary modifications that reduce binding to inhibitory receptors, including single, double, triple, quadruple and quintuple substitutions (numbering and substitutions (according to the EU index) are relative to the amino acid sequence of SEQ ID NO: 10 presented above).

[0116] [Table 4]

[0117] Exemplary variants of the human IgG1 Fc domain with reduced binding to CD32B and / or increased binding to CD16A contain F243L, R292P, Y300L, V305I or P396L substitutions, where the numbering is the EU index number in Kabat. These amino acid substitutions may be present in any combination within the human IgG1 Fc domain. In one embodiment, the mutant human IgG1 Fc domain contains F243L, R292P, and Y300L substitutions. In another embodiment, the mutant human IgG1 Fc domain contains F243L, R292P, Y300L, V305I, and P396L substitutions.

[0118] In certain embodiments, the Fc domain of the Fc domain-containing binding molecule of the present invention preferably exhibits reduced (or substantially no) binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) (relative to the binding exhibited by a wild-type IgG1 Fc domain (SEQ ID NO: 10)). In a specific embodiment, the Fc domain-containing binding molecule of the present invention comprises an IgG Fc domain with reduced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function. In a preferred embodiment, such a binding molecule In another embodiment, the CH2-CH3 domain of the present invention comprises any one, two, three, or four of the following substitutions: L234A, L235A, D265A, N297Q, and N297G, where the numbering is that of the EU index in Kabat. The H3 domain contains an N297Q substitution, an N297G substitution, an L234A and an L235A substitution, or a D265A substitution, because these mutations abolish FcR binding. Alternatively, the CH2-CH3 domain of a native Fc domain is utilized, which inherently exhibits low (or almost no) binding to FcγRIIIA (CD16a) and / or low effector function (relative to the binding and effector function exhibited by the wild-type IgG1 Fc domain (SEQ ID NO: 10)). In a specific embodiment, the Fc domain-containing binding molecule of the present invention comprises an IgG2 Fc domain (SEQ ID NO: 11), an IgG3 Fc domain (SEQ ID NO: 12), or an IgG4 Fc domain (SEQ ID NO: 13). When an IgG4 Fc domain is utilized, the present invention also encompasses the introduction of a stabilizing mutation, such as the S228P substitution in the hinge region described above (see, for example, SEQ ID NO: 9). The N297G, N297Q, L234A, L235A and D265A substitutions eliminate effector function and are therefore preferably not employed in situations where effector function is desired.

[0119] A preferred IgG1 sequence for the CH2 and CH3 domains of an Fc domain-containing molecule of the invention, with reduced or abolished effector function, comprises the L234A / L235A substitutions (SEQ ID NO: 45): APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X where X is a lysine (K) or is absent.

[0120] The serum half-life of a protein containing an Fc domain can be increased by increasing the binding affinity of the Fc domain for FcRn. As used herein, the term "half-life" refers to a pharmacokinetic property of a molecule that is a measure of the average survival time of the molecule after administration. Half-life can be expressed as the time required for 50 percent (50%) of a known amount of a molecule to be cleared from a subject's body (e.g., a human patient or other mammal) or a particular body cavity thereof, as measured in serum (i.e., circulating half-life) or other tissues. Generally, an increase in half-life leads to an increase in the mean residence time (MRT) in the circulation of the administered molecule.

[0121] In some embodiments, the Fc domain-containing binding molecules of the invention comprise a variant Fc domain that comprises at least one amino acid modification relative to a wild-type Fc region and therefore has an increased half-life (relative to a wild-type Fc domain). In some embodiments, the Fc domain-containing binding molecules of the invention comprise a variant IgG Fc domain that comprises a half-life-extending amino acid substitution at one or more positions selected from the group consisting of 238, 250, 252, 254, 256, 257, 256, 265, 272, 286, 288, 303, 305, 307, 308, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, 428, 433, 434, 435, and 436, where the numbering is that of the EU index in Kabat. Numerous mutations that can increase the half-life of Fc domain-containing molecules are known in the art, including, for example, M252Y, S254T, T256E, and combinations thereof. For example, see U.S. Patent No. 6,277,375; U.S. Patent No. 7,083,784; U.S. Patent No. 7,217,797; U.S. Patent No. 8,088,376; U.S. Patent Publication No. 2002 / 0147311; U.S. Patent Publication No. 2007 / 0148164; and WO 98 / 23289; WO 2009 / 058492; and WO 98 / 23289. See the mutations described in US Pat. No. 2010 / 033279, which are incorporated by reference in their entirety into this application.

[0122] In some embodiments, the Fc domain-containing binding molecules of the invention with enhanced half-life comprise a variant Fc domain comprising substitutions at two or more of Fc domain residues 250, 252, 254, 256, 257, 288, 307, 308, 309, 311, 378, 428, 433, 434, 435, and 436, in particular two or more substitutions selected from T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K, and Y436I. (A) M252Y, S254T and T256E; (B) M252Y and S254T; (C) M252Y and T256E; (D) T250Q and M428L; (E) T307Q and N434A; (F) A378V and N434A; (G) N434A and Y436I; (H) V308P and N434A; or (I) K288D and H435K The IgG Fc region may have a variant comprising the substitution:

[0123] In a preferred embodiment, the Fc domain-containing binding molecule of the invention has a variant IgG Fc domain comprising any one, two or three of the following substitutions: M252Y, S254T and T256E. (A) one or more mutations that alter effector function and / or FcγR binding; and (B) One or more mutations that increase serum half-life The present invention also encompasses binding molecules as described above having a variant Fc domain comprising:

[0124] The IgG1 sequence for the CH2 and CH3 domains of the Fc domain-containing molecules of the invention (Dall'Acqua, WF et al. (2006) "Properties of Human IgG1s Engineered for Enhanced Binding to the Neonatal Fc Receptor (FcRn)," J. Biol. Chem. 281(33):23514-23524) that provides increased half-life (and a 10-fold increase in binding to both cynomolgus monkey FcRn and human FcRn) contains the substitutions M252Y / S254T / T256E (SEQ ID NO: 46): APELLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X where X is a lysine (K) or is absent.

[0125] Another IgG1 sequence for the CH2 and CH3 domains of an Fc domain-containing molecule of the invention, which combines reduced or eliminated effector function provided by substitutions L234A / L235A with increased serum half-life provided by substitutions M252Y / S254T / T256E, is SEQ ID NO: 47: APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHEDPEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X Provided by, here X is a lysine (K) or is absent.

[0126] For certain antibodies, diabodies, and trivalent binding molecules in which it is desirable to have multiple Fc domain-containing polypeptide chains that differ in amino acid sequence (e.g., the Fc domain-containing polypeptide chains are not identical), it is desirable to reduce or prevent homodimerization between the CH2-CH3 domains of identical chains (e.g., two first polypeptide chains or two third polypeptide chains). The CH2 and / or CH3 domains of such polypeptide chains need not be identical in sequence and are advantageously modified to promote complex formation between the two polypeptide chains. For example, amino acid substitutions (preferably with amino acids containing bulky side groups that form "knobs," e.g., tryptophan) can be introduced into the CH2 or CH3 domain to prevent interaction with similarly mutated domains due to steric hindrance, allowing the altered domain to pair with a domain containing a complementary or adaptive mutation (e.g., a glycine substitution), i.e., a "hole," to promote heterodimerization. Such a series of mutations can be made to any pair of polypeptides comprising CH2-CH3 domains that form an Fc domain. Methods for engineering proteins to discourage homodimerization and promote heterodimerization are known in the art, particularly for the engineering of immunoglobulin-like molecules, and are encompassed herein (e.g., Ridgway et al. (1996) "'Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization," Protein Engr. 9:617-621; Atwell et al. (1997) “Stable Heterodimers From Remodeling TheDomainInterface Of A Homodimer Using A Phage Display Library,” J. Mol. Biol. 270: 26-35; and Xie et al. (2005) “A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis,” J. Immunol. Methods 296: 95-101 (see, respectively). The entire contents of which are incorporated herein by reference).

[0127] A preferred knob is generated by modifying an IgG Fc domain to contain the modification group T366W. A preferred hole is generated by modifying an IgG Fc domain to contain the modifications group T366S, L368A, and Y407V. To aid in purifying hole-bearing polypeptide chain homodimers from bispecific heterodimeric Fc domain-containing molecules, the protein A binding sites of the hole-bearing CH2 and CH3 domains of the polypeptide chains are preferably mutated by an amino acid substitution at position 435 (H435R). In this way, the hole-bearing polypeptide chain homodimers do not bind to protein A, while the bispecific heterodimers remain capable of binding to protein A via the protein A binding site on the knob-bearing polypeptide chain. In an alternative embodiment, the hole-bearing polypeptide chain may incorporate amino acid substitutions at positions 434 and 435 (N434A / N435K).

[0128] A preferred IgG1 amino acid sequence for the CH2 and CH3 domains of one Fc domain-containing polypeptide chain of an Fc domain-containing molecule of the invention has a "knob-bearing" sequence (SEQ ID NO: 48): APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W CLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X where X is a lysine (K) or is absent.

[0129] CH2 and CH3 domains of certain Fc domain-containing polypeptide chains of Fc domain-containing molecules of the invention having M252Y / S254T / T256E substitutions and "knob-bearing" sequences Another IgG1 amino acid sequence for APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W CLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X where X is a lysine (K) or is absent.

[0130] A preferred IgG1 amino acid sequence for the CH2 and CH3 domains of the other Fc domain-containing polypeptide chain of the Fc domain-containing molecule of the invention has the "hole-bearing" sequence (SEQ ID NO: 50): APE AAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C A VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHE ALHN R YTQKS LSLSPG X where X is a lysine (K) or is absent.

[0131] Another IgG1 amino acid sequence for the CH2 and CH3 domains of the other Fc domain-containing polypeptide chain of the Fc domain-containing molecule of the invention, having the M252Y / S254T / T256E substitutions and the "hole-bearing" sequence, is SEQ ID NO: 51: APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C A VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPG X where X is a lysine (K) or is absent.

[0132] As described below, the CH2-CH3 domains of SEQ ID NOs:48, 49, 50, and 51 contain a substitution at position 234 with alanine and a substitution at position 235 with alanine, thus forming Fc domains with reduced (or substantially no) binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) (compared to the binding exhibited by the wild-type Fc domain (SEQ ID NO:10)). The present invention also encompasses such CH2-CH3 domains that contain wild-type alanine residues, alternative and / or additional substitutions that modify the effector function and / or FγR binding activity of the Fc domain. The present invention also encompasses such CH2-CH3 domains that further comprise one or more half-life-extending amino acid substitutions. In particular, the present invention encompasses such hole-bearing and knob-bearing CH2-CH3 domains that further comprise M252Y / S254T / T256E.

[0133] The IgG4 amino acid sequence for the CH2 and CH3 domains of one of the Fc domain-containing polypeptides of the Fc domain-containing molecules of the present invention has Y252 / T254 / E256, thereby increasing serum half-life (compared to IgG1 CH2 and CH3 domains) (SEQ ID NO: 52): APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG X where Xis a lysine (K) or is absent.

[0134] Such a "knob-bearing" variant of the IgG4 CH2-CH3 amino acid sequence is SEQ ID NO: 53: APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSL W CLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG X wherein X is a lysine (K) or is absent.

[0135] Such a "hole-bearing" variant of the IgG4 CH2-CH3 amino acid sequence is SEQ ID NO: 54: APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSL S CA V K GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SRL TVDKSRWQEG NVFSCSVMHE ALHN R YTQKS LSLSLG X wherein X is a lysine (K) or is absent.

[0136] It is preferred that the first polypeptide chain has a "knob-bearing" CH2-CH3 sequence such as that of SEQ ID NO: 48 or SEQ ID NO: 49. However, it will be understood that a "hole-bearing" CH2-CH3 Domain (e.g., SEQ ID NO: 50 or SEQ ID NO: 51) can be employed in the first polypeptide chain, in which case a "knob-bearing" CH2-CH3 Domain (e.g., SEQ ID NO: 48 or SEQ ID NO: 49) is employed in the second polypeptide chain of an Fc domain-containing molecule of the invention having two polypeptide chains (or in the third polypeptide chain of an Fc domain-containing molecule having three, four, or five polypeptide chains).

[0137] In other embodiments, the invention encompasses Fc domain-containing binding molecules that comprise CH2 and / or CH3 domains that have been engineered to favor heterodimerization over homodimerization using mutations known in the art, such as those disclosed in WO 2007 / 110205; WO 2011 / 143545; WO 2012 / 058768; WO 2013 / 06867 (all of which are incorporated herein by reference in their entireties).

[0138] III. Trivalent Binding Molecules Containing an Fc Domain Further embodiments of the present invention relate to trispecific trivalent binding molecules comprising an Fc domain capable of simultaneously binding to a first epitope, a second epitope, and a third epitope (wherein at least one of the epitopes is not identical to another epitope). Such trispecific trivalent binding molecules comprise three epitope-binding domains, two of which are diabody-type binding domains providing binding site A and binding site B, and one of which is a Fab-type binding domain (or scFv-type binding domain) providing binding site C (see, e.g., Figures 6A-6F and WO 2015 / 184207 and WO 2015 / 184203). Such a trivalent binding molecule therefore comprises a "VL1" / "VH1" domain capable of binding to a first epitope, a "VL2" / "VH2" domain capable of binding to a second epitope, and a "VL3" and "VH3" domain capable of binding to a "third" epitope of the trivalent binding molecule. A "diabody-type binding domain" is a type of epitope-binding domain present in diabodies, as described above. A "Fab-type binding domain" and an "scFv-type binding domain" are epitope-binding domains formed by the interaction of a VL domain of an immunoglobulin light chain with a VH domain of a complementary immunoglobulin heavy chain, respectively. A Fab-type binding domain is an epitope-binding domain formed by the interaction of a VL domain of an immunoglobulin light chain with a VH domain of a complementary immunoglobulin heavy chain. Fab-type and scFv-type binding domains differ from diabody-type binding domains in that the two polypeptide chains forming a Fab-type binding domain contain only a single epitope-binding domain, whereas the two polypeptide chains forming a diabody-type binding domain contain at least two epitope-binding sites. Similarly, scFv-type binding domains also differ from diabody-type binding domains in that they contain only a single epitope-binding domain. Thus, as used herein, Fab-type and scFv-type binding domains are distinct from diabody-type binding domains.

[0139] Typically, trivalent binding molecules of the invention comprise four distinct polypeptide chains (see Figures 6A-6B), although the molecules can comprise fewer or more polypeptide chains, e.g., by fusing the polypeptide chains to one another (e.g., via peptide bonds), by "dividing" the polypeptides to form additional polypeptide chains, or by linking fewer or additional polypeptide chains via disulfide bonds. Figures 6C-6F illustrate this aspect of the invention by schematically showing a molecule having three polypeptide chains. As presented in Figures 6A-6F, trivalent binding molecules of the invention can have alternate orientations in which the diabody-type binding domain is N-terminal (Figures 6A, 6C, and 6D) or C-terminal (Figures 6B, 6E, and 6F) to the Fc domain. CH2 and CH3 domains useful for generating trivalent binding molecules are described above and include knob-bearing and hole-bearing domains.

[0140] In certain embodiments, the first polypeptide chain of such trivalent binding molecules of the invention contains: (i) a VL1-containing Domain; (ii) a VH2-containing Domain; (iii) a Heterodimer-Promoting Domain; and (iv) a Domain containing a CH2-CH3 sequence. The VL1 and VL2 Domains are located N-terminal or C-terminal to the CH2-CH3-containing Domain, as set forth in Table 4 (see also Figures 6A and 6B). The second polypeptide chain of such embodiments contains: (i) a VL2-containing Domain; (ii) a VH1-containing Domain; and (iii) a Heterodimer-Promoting Domain. The third polypeptide chain of such embodiments contains: (i) a VH3-containing Domain; (ii) a CH1-containing Domain; and (iii) a Domain containing a CH2-CH3 sequence. The third polypeptide chain can be the heavy chain of an antibody containing a VH3 and a heavy chain constant region, or a polypeptide containing such domains. The fourth polypeptide of such embodiments contains: (i) a VL3-containing Domain; and (ii) a CL-containing Domain. The fourth polypeptide chain can be the light chain of an antibody containing a VL3 complementary to the VH3 of the third polypeptide chain, or a polypeptide containing the above domain. The third or fourth polypeptide chains can be isolated from a naturally occurring antibody, or they can be constructed recombinantly, synthetically, or by other means.

[0141] The light chain variable domains of the first and second polypeptide chains are separated from the heavy chain variable domains of such polypeptide chains by an intervening spacer peptide, the intervening spacer linker being too short in length to allow the VL1 / VH2 (or VL2 / VH1) domains to be linked together to form an epitope-binding domain capable of binding a first or second epitope. A preferred intervening spacer peptide (Linker 1) for this purpose has the sequence (SEQ ID NO: 16): GGGSGGGG. The other domains of the trivalent binding molecule may be separated by one or more intervening spacer peptides (linkers), optionally containing cysteine ​​residues. In particular, as noted above, such linkers are typically incorporated between the Variable Domain (i.e., VH or VL) and the peptide Heterodimer-Promoting Domain (e.g., E-coil or K-coil), and between the peptide Heterodimer-Promoting Domain (e.g., E-coil or K-coil) and the CH2-CH3 Domain. Exemplary linkers useful for generating trivalent binding molecules are provided above and also in PCT Application Nos. PCT / US15 / 33081 and PCT / US15 / 33076. Thus, the first and second polypeptide chains of such trivalent binding molecules are linked together to form a first The third and fourth polypeptide chains of such a trivalent binding molecule link together to form a VL3 / VH3 binding site capable of binding to a third epitope.

[0142] As described above, trivalent binding molecules of the present invention may comprise three polypeptides. A trivalent binding molecule comprising three polypeptide chains can be obtained by linking the N-terminal domain of a fourth polypeptide to the VH3-containing domain of a third polypeptide (e.g., using an intervening spacer peptide (Linker 4)). Alternatively, a third polypeptide chain of a trivalent binding molecule of the present invention may be utilized that contains the following three domains: (i) a VL3-containing domain; (ii) a VH3-containing domain; and (iii) a domain containing a CH2-CH3 sequence, where the VL3 and VH3 are separated from each other by an intervening spacer peptide of sufficient length (at least 9 amino acid residues) to allow these domains to link to form an epitope-binding domain. One preferred intervening spacer peptide for this purpose has the sequence: GGGSGGGGSGGGGS (SEQ ID NO: 41).

[0143] It will be understood that the VL1 / VH1, VL2 / VH2, and VL3 / VH3 domains of such trivalent binding molecules can be different, thereby allowing for monospecific, bispecific, or trispecific binding. In particular, the VL and VH domains are selected such that the trivalent binding molecule comprises two binding sites for a first epitope and one binding site for a second epitope, or one binding site for the first epitope and two binding sites for the second epitope, or one binding site for the first epitope, one binding site for the second epitope, and one binding site for a third epitope.

[0144] The general structures of the polypeptide chains of representative trivalent binding molecules of the invention are provided in FIGS.

[0145] [Table 5]

[0146] As noted above, such trivalent binding molecules may comprise three, four, five, six or more polypeptide chains.

[0147] IV. EMBODIMENTS OF THE INVENTION As mentioned above, the present invention is directed to a DAxCD3 binding molecule comprising a vCD3 binding domain, wherein the vCD3 binding domain comprises a CDR H 1 domain, CDR H 2 domains, CDR H 3 Domain, CDR L 1 domain, CDR L 2 domains and CDRs L It includes three domains, At least one of the CDRs of the rCD3-binding domain has an amino acid sequence that differs from the amino acid sequence of the corresponding CDR of the rCD3-binding domain. The rCD3-binding domain to be used in such a comparison with a particular vCD3-binding domain is the CD3-binding domain of an isolated CD3-binding domain that exhibits the greatest CDR sequence identity with that particular vCD3-binding domain. Preferably, the rCD3-binding domain also exhibits at least 95% to 100% identity in the framework regions. One preferred rCD3-binding domain has the CDRs of CD3 mAb-1 and CD3 mAb-2. H 1 domain, CDR H 2 domains, CDR H 3 domains, CD R L 1 domain, CDR L 2 domains and CDRs L Includes 3 domains. DAxCD3 binding molecules of the invention comprising such a vCD3-binding domain have altered affinity for CD3 compared to DAxCD3 binding molecules comprising an rCD3-binding domain as described above. The present invention particularly relates to DAxCD3 binding molecules as described above comprising a vCD3-binding domain that have reduced affinity for CD3, are capable of mediating targeted killing of disease antigen-expressing target cells, and have reduced levels of cytokine release compared to DAxCD3 binding molecules comprising an rCD3-binding domain. The present invention particularly relates to the use of DAxCD3 binding molecules comprising a vCD3-binding domain in the treatment of cancer and pathogen-related diseases. The present invention also relates to pharmaceutical compositions comprising one or more of the above molecules.

[0148] Thus, the present invention provides a method for the preparation of a vCD3 binding domain comprising one or more of the VH and / or VL domains, or more preferably the CDRs of such domains. H 1. CDR H 2, and CDR H 3 , and CDR L 1. CDR L 2, and CDR L The DAxCD3 binding molecule contains three parts. In certain preferred embodiments of the invention, such DAxCD3 binding molecules will further contain binding domains sufficient to bind such molecules to epitopes of one, two, or more disease antigens. In other preferred embodiments of the invention, such DAxCD3 binding molecules will further contain binding domains sufficient to bind to one or more epitopes of other molecules expressed on the surface of effector cells, such as CD2, CD8, CD16, T cell receptor (TCR), NKp46, NKG2D, etc., expressed on T lymphocytes, natural killer (NK) cells, antigen-presenting cells, or other mononuclear cells.

[0149] The present invention is also directed to pharmaceutical compositions comprising one or more such DAxCD3 binding molecules.

[0150] By possessing binding domains sufficient for immunospecific binding to CD3 and a disease antigen, the molecules of the invention are capable of mediating the targeted killing of target cells (e.g., cancer cells or pathogen-infected cells) that display the disease antigen on their surface. The coexistence of these two binding affinities serves to localize CD3-expressing effector cells to the site of the target cell (i.e., "redirect" the effector cells), allowing them to mediate target cell killing. As noted above, such molecules may be bispecific or capable of binding to three or more epitopes (e.g., trispecific).

[0151] Efforts to employ CD3-binding molecules have been hampered by the high levels of immune activation caused by such therapies and the concomitant harmful production of high levels of cytokines in some patients. Thus, although anti-CD3 therapy has resulted in a substantial degree of immune activation in recipient patients that correlates with a significant increase in efficacy, the use of such molecules has been associated with significant toxicity (Frey, NV et al. (2016) "Cytokine Release Syndrome"). ndrome With Novel Therapeutics For Acute Lymphoblastic Leukemia,” Hematol. Am. Soc. Hematol. Educ Program. (1):567-572; Teachey, D.T.et al. (2013) “Cytokine Release Syndrome After Blinatumomab Treatment Related To Abnormal Macrophage Activation AndAmeliorated With Cytokine-Directed Therapy,”Blood121(26):5154-5157; Le Jeune, C. et al. (2016) “Potential For Bispecific T-Cell Engagers: Role Of Blinatumomab In Acute LymphoblasticLeukemia,” Drug Des. Devel. Ther.10:757-765; Newman,M.J. et al. (2016) “A Review Of Blinatumomab, A Novel Immunotherapy,” J. Oncol. Pharm.Pract. 22(4):639-645;Fitzgerald, J.C. et al. (2017) “CytokineReleaseSyndrome After Chimeric Antigen Receptor T-Cell Therapy for Acute LymphoblasticLeukemia,” Crit. Care Med.45(2):e124-e131; Teachey,D.T. et al. (2016) “Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cellTherapy for Acute Lymphoblastic Leukemia, ” Cancer Discov. 6(6):664-679; Goebeler, ME et al. (2016) “Blinatumomab: A CD19 / CD3 Bispecific TCellEngager (Bite) With Unique Anti-Tumor Efficacy,” Leuk.Lymphoma 57(5):1021-1032; Barrett, DM et al. (2014) “ToxicityManagement For Patients Receiving Novel T-Cell Engaging Therapies,” Curr. Opin. Pediatr. 26(1):43-49).

[0152] The present invention addresses these obstacles by demonstrating that by engineering a parent CD3-binding domain (i.e., rCD3-binding domain) that exhibits both enhanced cytotoxicity and enhanced cytokine release when incorporated into a DAxCD3 binding molecule, it is possible to produce variants with altered affinity for CD3 (i.e., vCD3-binding domains) that are capable of mediating targeted killing and exhibit reduced levels of cytokine release relative to DAxCD3 binding molecules comprising the rCD3-binding domain. In particular, DAxCD3 binding molecules comprising the vCD3-binding domain according to the present invention exhibit reduced levels of release of any one or more of: IFN-γ, TNF-α, IL-2, and / or IL-6.

[0153] The present invention stems, in part, from the recognition that cytotoxicity and cytokine release are separable properties of DAxCD3 binding molecules. The present invention encompasses variant CD3-binding domains (i.e., vCD3-binding domains) that retain high levels of cytotoxicity while reducing the levels of cytokine release, and the use of DAxCD3 binding molecules comprising such vCD3-binding domains in the treatment of disease. As used herein, the term "variant" with respect to such CD3-binding domains refers to a variant that retains a "corresponding" CDR of a "reference" CD3-binding domain (i.e., rCD3-binding domain). Hand / or CDRs L At least one CDR that is different from H and / or At least one CDR L The term "CD3 binding domain" is intended to refer to a CD3 binding domain having the following structure: As used herein, "corresponding" CDR H and / or CDRs L The term refers to a comparison between two CDR sequences where both such CDRs are CDR H One domain, All such CDRs are CDRs H 2 domains, and both of these CDRs are C DR H 3 domains, and all of these CDRs are CDR L Any such CDR is a CDR L two domains, or both such CDRs are CD R L The exemplary vCD3 binding domains described herein are shown in Figure 1. A preferred rCD3 binding domain for this purpose has the following CDRs: CDRs of CD3 mAb 1 H 1. CDR H 2. CDR H 3. CDR L 1. CDR L 2, and CDR L At least one of the three Preferably, such exemplary vCD3 binding domains have the following CDRs: CDRs of CD3 mAb 1: H 1. CDR H 2. CDR H 3. CDR L 1. CDR L 2, and CDR LThe vCD3-binding domain can be obtained by chemical modification of one or more CDRs of the rCD3-binding domain, but more preferably, the rCD3-binding domain will have at least five of the CDRs except for the modifications to encode the desired vCD3-binding domain. Cytotoxicity can be achieved by generating one or more polynucleotides encoding one or more CDRs of the CD3 binding domain, followed by expression of such polynucleotides in a suitable protein expression system (e.g., a cell, or an in vitro translation system). Cytotoxicity can be achieved by any suitable method (e.g., EC 50 Cytokine release may be measured by any suitable method (e.g., EC 50 , CTL assay to determine maximum) by assaying for any one or more of: IFN-γ, TNF-α, IL-6, or IL-2.

[0154] In particular, maximum cytotoxicity and absolute levels of cytokine release are not the only criteria used to evaluate whether a candidate CD3 binding domain is a suitable vCD3 binding domain encompassed by the present invention. 50 As provided herein, suitable vCD3 binding domains, when incorporated into DAxCD3 binding molecules, may exhibit high levels of cytotoxicity (i.e., low EC 50 It can mediate the concentration.

[0155] In certain embodiments, the invention provides vCD3 binding domains that, when incorporated into a DAxCD3 binding molecule, mediate targeted cell killing of cells to a maximum cytotoxicity (e.g., as measured in a CTL assay at 18-48 hours) that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the cytotoxicity mediated by a DAxCD3 binding molecule comprising the rCD3 binding domain. Additionally, or alternatively, DAxCD3 binding molecules comprising the vCD3 binding domain of the invention exhibit an EC of cytotoxicity (e.g., as measured in a CTL assay at 18-48 hours) that is less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, less than about 100%, less than about 200%, less than about 300%, less than about 400%, or less than about 500% higher than that exhibited by DAxCD3 binding molecules comprising the rCD3 binding domain. 50 Additionally, or alternatively, the cytotoxic EC of a DAxCD3 binding molecule comprising a vCD3 binding domain of the present invention (e.g., as measured in a CTL assay at 18-24 hours) is 50 The ratio of DA×CD3 binding molecules containing the rCD3-binding domain (EC 50 Mutant / EC 50 The standard is less than about 2, less than about 5, less than about 10, less than about 20, less than about 40, less than about 60, less than about 80, less than about 100, or less than about 200.

[0156] In certain embodiments, DAxCD3 binding molecules comprising a vCD3 binding domain of the invention exhibit a maximum release of one or more cytokines (e.g., as measured in a CTL assay at 18-24 hours) that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% lower than that exhibited by a DAxCD3 binding molecule comprising an rCD3 binding domain. Additionally, or alternatively, DAxCD3 binding molecules comprising a vCD3 binding domain of the invention exhibit an EC of release of one or more cytokines (e.g., as measured in a CTL assay at 18-48 hours) that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more higher than that exhibited by a DAxCD3 binding molecule comprising an rCD3 binding domain. 50 In certain embodiments, the cytokines released are selected from the group consisting of: IFN-γ, TNF-α, IL-2, and IL-6. Additionally, or alternatively, the EC20 values ​​of the release of one or more cytokines (e.g., as measured in a CTL assay at 18-24 hours) of a DAxCD3 binding molecule comprising a vCD3 binding domain of the invention are 50 The ratio of DA×CD3 binding molecules containing the rCD3-binding domain (EC 50 Mutant / EC 50 The standard) is greater than about 1, greater than about 2, greater than about 5, greater than about 10, greater than about 20, greater than about 40, greater than about 60, greater than about 80, greater than about 100, or greater than about 200.

[0157] Furthermore, DAxCD3 binding molecules comprising a vCD3-binding domain of the present invention retain at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the in vivo activity (e.g., anti-tumor, anti-pathogen activity) exhibited by a DAxCD3 binding molecule comprising an rCD3-binding domain. In light of the present disclosure, DAxCD3 binding molecules comprising a vCD3-binding domain may be administered at relatively high doses to achieve at least about 50% or more of the in vivo activity exhibited by a DAxCD3 binding molecule comprising an rCD3-binding domain, although it will be understood that such relatively high doses will exhibit reduced levels of cytokine release compared to DAxCD3 binding molecules comprising an rCD3-binding domain.

[0158] In one embodiment, such DAxCD3 binding molecules of the invention are monospecific, and thus have the ability to bind only to a single epitope on CD3 and only to a single epitope on a disease antigen.

[0159] Alternatively, such DAxCD3 binding molecules may be multispecific, i.e., capable of binding to one, two, three, four, or more epitopes, which may be assigned in any manner to bind to one, two, or more epitopes of CD3 and one, two, three, four, or more epitopes of one or more disease antigens.

[0160] In certain embodiments in which such a DAxCD3 binding molecule can immunospecifically bind to only a single disease antigen, the DAxCD3 binding molecule may be capable of immunospecifically binding to: only one CD3 epitope and one, two epitopes of a disease antigen as described above (the two epitopes may be the same or different); or it may be capable of immunospecifically binding to only one CD3 epitope and three epitopes of a disease antigen as described above (the three disease antigen epitopes may be the same, different, or two identical epitopes and one different epitope).

[0161] In other embodiments where such a DAxCD3 binding molecule is capable of immunospecifically binding to two different disease antigens (e.g., a first disease antigen and a second disease antigen), the molecule may be capable of immunospecifically binding to: only one CD3 epitope, and one or two epitopes of the first disease antigen (the two first disease antigen epitopes may be the same or different), and two or one epitope of the second disease antigen (the two second disease antigen epitopes may be the same or different).

[0162] In yet other embodiments, such a DAxCD3 binding molecule may be capable of immunospecifically binding to three different disease antigens (e.g., a first disease antigen, a second disease antigen, and a third disease antigen) and only one CD3 epitope.

[0163] In yet other embodiments, such DAxCD3 binding molecules may be capable of immunospecifically binding to one or two different disease antigens (e.g., a first disease antigen and a second disease antigen), to only one CD3 epitope, and to one or two different cell surface molecules (which may be the same cell surface molecule or different surface molecules) of effector cells (which may be the same type of effector cell or different types of effector cells).

[0164] Thus, for example, such a DAxCD3 binding molecule: (1) A single epitope of CD3 and a single epitope of a disease antigen arrayed on the surface of a target cell; (2) a single epitope on CD3 and two epitopes of the same disease antigen arrayed on the surface of the target cell; (3) a single epitope of CD3, one epitope of a first disease antigen arranged on the surface of a target cell, and one epitope of a second disease antigen arranged on the surface of a target cell; (4) a single epitope of CD3 and three epitopes of the same disease antigen arrayed on the surface of the target cell; (5) a single epitope of CD3, two epitopes of a first disease antigen arranged on the surface of a target cell, and one epitope of a second disease antigen arranged on the surface of a target cell; (6) a single epitope of CD3, one epitope of a first disease antigen arranged on the surface of a target cell, and one epitope of a second disease antigen arranged on the surface of a target cell; (7) A single epitope of CD3, a single epitope of a disease antigen arranged on the surface of a target cell, and a single epitope of a cell surface molecule other than CD3 arranged on the surface of an effector cell (which may be the same type of effector cell as the CD3-arranging effector cell or a different type of effector cell); (8) A single epitope of CD3, two epitopes of a disease antigen arranged on the surface of a target cell, and a single epitope of a cell surface molecule other than CD3 arranged on the surface of an effector cell (which may be the same type of effector cell as the CD3-arranging effector cell or a different type of effector cell); (9) A single epitope of CD3, one epitope of a first disease antigen arranged on the surface of a target cell, one epitope of a second disease antigen arranged on the surface of a target cell, and a single epitope of a cell surface molecule other than CD3 arranged on the surface of an effector cell (which may be the same type of effector cell as the CD3-arranging effector cell or a different type of effector cell); (10) A single epitope of CD3, one epitope of a disease antigen arranged on the surface of a target cell, and two epitopes of a cell surface molecule other than CD3 arranged on the surface of an effector cell (which may be the same type of effector cell as the CD3-arranging effector cell or a different type of effector cell); or (11) A single epitope of CD3, one epitope of a disease antigen arranged on the surface of a target cell, one epitope of a first cell surface molecule other than CD3 arranged on the surface of an effector cell (which may be the same type of effector cell as the CD3-arranging effector cell or a different type of effector cell), and one epitope of a second cell surface molecule other than CD3 arranged on the surface of an effector cell (which may be the same type of effector cell as the CD3-arranging effector cell or a different type of effector cell). may be bonded to

[0165] Thus, the present invention contemplates a DAxCD3 binding molecule comprising a first epitope-binding domain capable of immunospecifically binding to an epitope on CD3, a second epitope-binding domain capable of immunospecifically binding to an epitope on a disease antigen arranged on the surface of a target cell, as described above, and a third epitope-binding domain capable of immunospecifically binding to an epitope on a different cell surface molecule on an effector cell (which may be the same type of effector cell or a different type of effector cell). In a specific embodiment, the different cell surface molecule on the effector cell is CD8. Table 6 shows possible combinations of binding specificities for exemplary molecules of the invention.

[0166] [Table 6]

[0167] Further complex molecules can be formed to obtain DAxCD3 binding molecules capable of binding to CD3 and one or more disease antigens, as well as different cell surface molecules on effector cells, and having five or more epitope-binding domains. No constraints are imposed on the nature of the epitopes or additional epitopes to which the molecules of the invention may bind, except that such additional binding capabilities do not prevent molecules or binding domains capable of binding to epitopes on CD3 from binding to the epitopes on CD3, or molecules or binding domains capable of binding to epitopes on disease antigens from binding to the epitopes on disease antigens, such that one or more of the above molecules can mediate targeted killing of target cells.

[0168] V. Exemplary Binding Molecules The present invention is directed to DAxCD3 binding molecules (e.g., diabodies, bispecific antibodies, bispecific trivalent molecules, BiTe, TandAb, etc.) that can bind to CD3 and disease antigens, such as cancer antigens or pathogen-associated antigens. Such binding molecules can be readily produced from antibody CDRs and from antibody VL and VH domains. Listed below are exemplary antibodies that can be used to produce the binding molecules and combination therapies of the invention.

[0169] A. Anti-CD3 antibody CD3 mAb 1 The present invention uses variant CD3-binding domains (i.e., vCD3-binding domains) that comprise the variable light (VL) and variable heavy (VH) domains of an anti-human CD3 antibody, or CD3-binding portions thereof, and that mediate variant binding to CD3. As used herein, the term "variant binding" is intended to refer to binding that is comparable to that exhibited by the CD3-binding domain of a reference antibody having CDRs with the highest sequence identity to the CDRs of the variant CD3-binding domain. An exemplary CD3-binding reference antibody for the vCD3-binding domain of the present invention is CD3 mAb 1, the rCD3-binding domain of which can bind to human CD3 and CD3 of non-human primates (e.g., cynomolgus monkeys).

[0170] The amino acid sequence of the VH domain of CD3 mAb 1 (SEQ ID NO: 55) is shown below (C DR H Residues are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMN WVRQA PGKGLEWVG R IRSKYNNYAT YYADSVKX RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAY WGQGTL VTVSS where X is aspartic acid (D) or glycine (G)

[0171] The amino acid sequence of the VL domain of CD3 mAb 1 (SEQ ID NO: 56) is shown below (CDR L Residues are underlined): QAVVTQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G GTNKRAP WT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWV F GGGTKLTVLG

[0172] [Table 7]

[0173] The rCD3-binding domain of "CD3 mAb 1" comprises a CD3 mAb 1 VH domain having an aspartic acid (D) or glycine (G) at Kabat position 65, which corresponds to residue 68 of SEQ ID NO: 55 (i.e., X in SEQ ID NO: 55 is aspartic acid (D) or glycine (G)), and a VL domain of CD3 mAb 1 (SEQ ID NO: 56). Thus, for example, if such a CD3 mAb 1 VH domain has a glycine (G) as residue 68 thereof, its sequence is SEQ ID NO: 63, as shown below (CDR H Residues are underlined and Kabat position 65 is double underlined): JPEG2025186382000009.jpg23147

[0174] The CD3 binding molecules having the vCD3 binding domain of the present invention can be recognized using a CTL assay, in which: (1) a bispecific cancer antigen x CD3 diabody (e.g., a CD123 x CD3 diabody or a 5T4 x CD3 diabody) potentially containing a vCD3 binding domain; and (2) a bispecific cancer antigen x CD3 diabody with the corresponding rCD3 binding domain (e.g., the rCD3 binding domain of CD3 mAb 1); are separately incubated with effector Pan-T cells (or PBMCs) and target tumor cells (e.g., MOLM-13 or A498 cells) for 18, 24, or 42 hours, e.g., at an effector:target cell ratio of 5:1 (or 15:1 for PBMCs), and percentage cytotoxicity is assessed (e.g., by measuring lactate dehydrogenase (LDH) release using the CytoTox96® Non-Radioactive Cytotoxicity Assay Kit (Promega)). Induction cytotoxicity (i.e., cell killing) and / or EC 50 In one embodiment, the release of IFN-γ, TNF-α, IL-6, and IL-2 cytokines can be determined at the end of the CTL assay. Also at the end of the CTL assay, CD4 + and CD8 + T lymphocyte populations can also be assessed. Percentage cytotoxicity and / or EC2 for bispecific cancer antigen x CD3 diabodies potentially containing a vCD3 binding domain. 50 and the percentage cytotoxicity and / or EC for a cancer antigen × CD3 diabody having a rCD3 binding domain. 50 By comparing the vCD3 binding domains to the desired mutant CD3 binding and / or reduced levels of cytokine release, vCD3 binding domains are identified.

[0175] Alternatively, CD3 binding molecules having a vCD3 binding domain of the present invention can be recognized using a binding assay, in which: (1) a bispecific cancer antigen x CD3 diabody potentially containing a vCD3 binding domain; and (2) A bispecific cancer antigen x CD3 diabody having an rCD3-binding domain (e.g., the rCD3-binding domain of CD3 mAb 1). The diabody molecules are separately assessed for their ability to bind to the surface of tumor antigen-expressing cell lines (MOLM-13 or A498 cells) by FACS analysis. Briefly, cells are incubated in microtiter plates with diabody molecules (in FACS buffer containing 10% human AB serum). Cells are then washed and incubated with a labeled anti-human Fc secondary antibody mixed with streptavidin-phycoerythrin or a biotin-labeled mouse anti-EK-coil antibody that recognizes the E-coil / K-coil (EK) heterodimer-promoting domain of the diabody. Cells are then washed, resuspended in FACS buffer, and analyzed by flow cytometry for comparison.

[0176] Alternatively, CD3-binding molecules having the vCD3-binding domain of the present invention can be recognized using a mixed xenograft model, such as NOD / SCID mice. In such an assay, mice are transfected with activated human CD4 + or CD8 + Tumor cells (e.g., KG1A (AML) cells) mixed with T cells (E:T = 1:5) are injected into these animals. Bispecific cancer antigen x CD3 diabodies potentially containing a vCD3-binding domain or a cancer antigen x CD3 diabody containing a rCD3-binding domain are then injected into these animals, and the extent of tumor growth is monitored and compared.

[0177] Alternatively, any one, two, or more of the exemplary variants of CD3 mAb 1, referred to herein as "CD3 mAb 1 M3" through "CD3 mAb 1 M26," can be used to provide the vCD3-binding domain of a DA×CD3 binding molecule of the invention. The present invention generally contemplates anti-CD3 antibodies having the VL and VH domains of any of CD3 mAb 1 M3 through CD3 mAb 1 M26, where the VH domain has aspartic acid (D) at Kabat position 65 or glycine (G) at Kabat position 65. Exemplary variants of CD3 mAb 1, CD3 mAb 1 M3 through CD3 mAb 1 M26, have CDRs H 1 domain, CDR H 2 domains, CDR H 3 Domains N, CDR L 1 domain, CDR L 2 domains and CDRs L vCD3 containing 3 domains The DAxCD3 binding domains comprise at least one CDR of the rCD3-binding domain (CD3 mAb 1), at least one of which has an amino acid sequence that differs from the amino acid sequence of the corresponding CDR of the rCD3-binding domain (CD3 mAb 1), and which comprises the vCD3-binding domain. Compared to the DAxCD3-binding domain that comprises the rCD3-binding domain, the DAxCD3-binding molecule binds to CD3 with altered affinity, can mediate targeted killing, and exhibits reduced levels of cytokine release.

[0178] The amino acid sequence of a preferred variant anti-CD3 VH domain of the present invention is a variant of SEQ ID NO: 55, and is SEQ ID NO: 207 (CDR H Residues are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS X 1 X 2 X 3 MN WVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKX 4RF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HX 5 NX 6X 7 NSX 8 ST X 9 FAX 10 WGQGTL VTVSS where: X1 is T, D, or E; X2 is Y, D, or T; and X3 is A or is G; X4 is D or G; X5 is G, D, E, or K; and X6 is F or I. X7 is G or I; X8 is Y, A, G, or Q; and X9 is W, F, or Y. Yes;X 10 is Y or E.

[0179] The amino acid sequence of a preferred variant anti-CD3 VL domain of the present invention is a variant of SEQ ID NO: 56, and is SEQ ID NO: 208 (CDR L Residues are underlined): QAVVTQEPSL TVSPGGTVTL TC RSSTGAVT TSNYAN WVQQ KPGQAPRGLI G X 1 TNX 2 RAP WT PARFSGSLLG GKAALTITGA QAEDEADYYC AX 3 WYSNLWV F GGGTKLTVLG X1 is G or D; X2 is K or G; and X3 is L, E, or Q. do.

[0180] B. Mutant Anti-CD3 Antibodies 1.CD3 mAb 1 M1 CD3 mAb 1 M1 is a low-affinity variant of CD3 mAb 1 and is therefore also referred to as "CD3 mAb 1 Low." The amino acid sequence of the VH domain of CD3 mAb 1 M1 is shown in SEQ ID NO: 64 (CDR H The residues are underlined) As shown below, compared to SEQ ID NO:55, SEQ ID NO:64 contains an S100dT substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO:64 (also shown double underlined) may be an aspartic acid (D) or a glycine (G): JPEG2025186382000010.jpg23147 where X is aspartic acid (D) or glycine (G).

[0181] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M1 is SEQ ID NO:56.

[0182] [Table 8]

[0183] 2.CD3 mAb 1 M2 CD3 mAb 1 M2 has a faster off-rate than CD3 mAb 1 and is therefore also referred to as "CD3 mAb 1 Fast." The amino acid sequence of the VH domain of CD3 mAb 1 M2 is shown in SEQ ID NO: 66 (CDR H residues are underlined) and Compared to SEQ ID NO:55, SEQ ID NO:66 contains a G96K and S100dT substitution (sequence residue 110, shown double underlined) numbered according to Kabat; furthermore, position 65 in the Kabat numbering of SEQ ID NO:66 (also shown double underlined) may be an aspartic acid (D) or a glycine (G): JPEG2025186382000012.jpg23147 where X is aspartic acid (D) or glycine (G).

[0184] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M2 is SEQ ID NO:56.

[0185] [Table 9]

[0186] 3. CD3 mAb 1 M3 The amino acid sequence of the VH domain of CD3 mAb 1 M3 (SEQ ID NO: 68) is shown below (CDR H Residues are underlined). contains a G99I substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 68 (also shown double underlined) may be an aspartic acid (D) or a glycine (G): JPEG2025186382000014.jpg23147 where X is aspartic acid (D) or glycine (G).

[0187] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M3 is SEQ ID NO:56.

[0188] [Table 10]

[0189] 4.CD3 mAb 1 M4 The amino acid sequence of the VH domain of CD3 mAb 1 M4 (SEQ ID NO: 70) is shown below (CDR H Residues are underlined). contains a Y100bA substitution (shown double underlined and numbered according to Kabat); further, position 65 of SEQ ID NO: 70 according to Kabat numbering (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000016.jpg23147 where X is aspartic acid (D) or glycine (G).

[0190] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M4 is SEQ ID NO:56.

[0191] [Table 11]

[0192] 5.CD3 mAb 1 M5 The amino acid sequence of the VH domain of CD3 mAb 1 M5 (SEQ ID NO: 72) is shown below (CDR H Residues are underlined). contains a Y100bG substitution (shown double underlined and numbered according to Kabat); further, position 65 in the Kabat numbering of SEQ ID NO: 72 (also shown double underlined) may be an aspartic acid (D) or a glycine (G): JPEG2025186382000018.jpg23147 where X is aspartic acid (D) or glycine (G).

[0193] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M5 is SEQ ID NO:56.

[0194] [Table 12]

[0195] 6.CD3 mAb 1 M6 The amino acid sequence of the VH domain of CD3 mAb 1 M6 (SEQ ID NO: 74) is shown below (CDR H Residues are underlined). contains a Y100bQ substitution (shown double underlined and numbered according to Kabat); further, position 65 in the Kabat numbering of SEQ ID NO: 74 (also shown double underlined) may be an aspartic acid (D) or a glycine (G): JPEG2025186382000020.jpg23147 where X is aspartic acid (D) or glycine (G)

[0196] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M6 is SEQ ID NO:56.

[0197] [Table 13]

[0198] 7. CD3 mAb 1 M7 The amino acid sequence of the VH domain of CD3 mAb 1 M7 (SEQ ID NO: 76) is shown below (CDR H Residues are underlined). SEQ ID NO: 76 compared to SEQ ID NO: 55 contains a G96D substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 76 (also shown double underlined) may be an aspartic acid (D) or a glycine (G): JPEG2025186382000022.jpg23147 where X is aspartic acid (D) or glycine (G)

[0199] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M7 is SEQ ID NO:56.

[0200] [Table 14]

[0201] 8. CD3 mAb 1 M8 The amino acid sequence of the VH domain of CD3 mAb 1 M8 (SEQ ID NO: 78) is shown below (CDR H Residues are underlined). contains a G99E substitution (shown double underlined and numbered according to Kabat); further, position 65 in the Kabat numbering of SEQ ID NO: 78 (also shown double underlined) may be an aspartic acid (D) or a glycine (G): JPEG2025186382000024.jpg20148 where X is aspartic acid (D) or glycine (G)

[0202] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M8 is SEQ ID NO:56.

[0203] [Table 15]

[0204] 9. CD3 mAb 1 M9 The amino acid sequence of the VH domain of CD3 mAb 1 M9 (SEQ ID NO: 80) is shown below (CDR H Residues are underlined). contains a G99K substitution (shown double underlined and numbered according to Kabat); further, position 65 in the Kabat numbering of SEQ ID NO: 80 (also shown double underlined) may be an aspartic acid (D) or a glycine (G): JPEG2025186382000026.jpg20148 where X is aspartic acid (D) or glycine (G)

[0205] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M9 is SEQ ID NO:56.

[0206] [Table 16]

[0207] 10. CD3 mAb 1 M10 The amino acid sequence of the VH domain of CD3 mAb 1 M10 (SEQ ID NO: 82) is shown below (CDR H Residues are underlined). 2 contains an F98I substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 82 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000028.jpg22148 where X is aspartic acid (D) or glycine (G)

[0208] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M10 is SEQ ID NO:56.

[0209] [Table 17]

[0210] 11. CD3 mAb 1 M11 The amino acid sequence of the VH domain of CD3 mAb 1 M11 (SEQ ID NO: 84) is shown below (CDR H Residues are underlined). 4 contains a W100eF substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 84 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000030.jpg22148 where X is aspartic acid (D) or glycine (G)

[0211] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M11 is SEQ ID NO:56.

[0212] [Table 18]

[0213] 12. CD3 mAb 1 M12 The amino acid sequence of the VH domain of CD3 mAb 1 M12 (SEQ ID NO: 86) is shown below (CDR H Residues are underlined). 6 contains a W100eY substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 86 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000032.jpg22148 where X is aspartic acid (D) or glycine (G)

[0214] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M12 is SEQ ID NO:56.

[0215] [Table 19]

[0216] 13.CD3 mAb 1 M13 The amino acid sequence of the VH domain of CD3 mAb 1 M13 (SEQ ID NO: 88) is shown below (CDR H Residues are underlined). 8 contains a Y102E substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 88 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000034.jpg22148 where X is aspartic acid (D) or glycine (G)

[0217] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M13 is SEQ ID NO:56.

[0218] [Table 20]

[0219] 14. CD3 mAb 1 M14 The amino acid sequence of the VH domain of CD3 mAb 1 M14 (SEQ ID NO: 90) is shown below (CDR H Residues are underlined). 90 contains a T31D substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 90 (also shown double underlined) may be an aspartic acid (D) or a glycine (G): JPEG2025186382000036.jpg22148 where X is aspartic acid (D) or glycine (G)

[0220] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M14 is SEQ ID NO:56.

[0221] [Table 21]

[0222] 15.CD3 mAb 1 M15 The amino acid sequence of the VH domain of CD3 mAb 1 M15 (SEQ ID NO: 92) is shown below (CDR H Residues are underlined). 2 contains a T31E substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 92 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000038.jpg22148 where X is aspartic acid (D) or glycine (G)

[0223] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M15 is SEQ ID NO:56.

[0224] [Table 22]

[0225] 16.CD3 mAb 1 M16 The amino acid sequence of the VH domain of CD3 mAb 1 M16 (SEQ ID NO: 94) is shown below (CDR H Residues are underlined). 4 contains a Y32D substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 94 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000040.jpg21151 where X is aspartic acid (D) or glycine (G)

[0226] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M16 is SEQ ID NO:56.

[0227] [Table 23]

[0228] 17.CD3 mAb 1 M17 The amino acid sequence of the VH domain of CD3 mAb 1 M17 (SEQ ID NO: 96) is shown below (CDR H Residues are underlined). 6 contains a Y32T substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 96 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000042.jpg21151 where X is aspartic acid (D) or glycine (G)

[0229] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M17 is SEQ ID NO:56.

[0230] [Table 24]

[0231] 18.CD3 mAb 1 M18 The amino acid sequence of the VH domain of CD3 mAb 1 M18 (SEQ ID NO: 98) is shown below (CDR H Residues are underlined). 8 contains an A33G substitution (shown double underlined and numbered according to Kabat); furthermore, position 65 in the Kabat numbering of SEQ ID NO: 98 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000044.jpg21151 where X is aspartic acid (D) or glycine (G)

[0232] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M18 is SEQ ID NO:56.

[0233] [Table 25]

[0234] 19.CD3 mAb 1 M19 The amino acid sequence of the VH domain of CD3 mAb 1 M19 (SEQ ID NO: 100) is shown below (CDR H Residues are underlined). 100 contains G96K and F98I substitutions (shown double underlined and numbered according to Kabat); further, position 65 in the Kabat numbering of SEQ ID NO: 100 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000046.jpg21151 where X is aspartic acid (D) or glycine (G)

[0235] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M19 is SEQ ID NO:56.

[0236] [Table 26]

[0237] 20. CD3 mAb 1 M20 The amino acid sequence of the VH domain of CD3 mAb 1 M20 (SEQ ID NO: 102) is shown below (CDR H Residues are underlined). 102 contains G96K and Y100bG substitutions (shown double underlined and numbered according to Kabat); further, position 65 in the Kabat numbering of SEQ ID NO: 102 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000048.jpg21151 where X is aspartic acid (D) or glycine (G)

[0238] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M20 is SEQ ID NO:56.

[0239] [Table 27]

[0240] 21.CD3 mAb 1 M21 The amino acid sequence of the VH domain of CD3 mAb 1 M21 (SEQ ID NO: 104) is shown below (CDR H Residues are underlined). 104 contains G96K and W100eF substitutions (shown double underlined and numbered according to Kabat); further, position 65 in the Kabat numbering of SEQ ID NO: 104 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000050.jpg21151 where X is aspartic acid (D) or glycine (G)

[0241] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M21 is SEQ ID NO:56.

[0242] [Table 28]

[0243] 22.CD3 mAb 1 M22 The amino acid sequence of the VH domain of CD3 mAb 1 M22 (SEQ ID NO: 106) is shown below (CDR H Residues are underlined). 106 contains G96K and W100eY substitutions (shown double underlined and numbered according to Kabat); further, position 65 in the Kabat numbering of SEQ ID NO: 106 (also shown double underlined) may be aspartic acid (D) or glycine (G): JPEG2025186382000052.jpg21151 where X is aspartic acid (D) or glycine (G)

[0244] A preferred amino acid sequence of the VL domain of CD3 mAb 1 M22 is SEQ ID NO:56.

[0245] [Table 29]

[0246] 23.CD3 mAb 1 M23 A preferred amino acid sequence of the VH domain of CD3 mAb 1 M23 is SEQ ID NO:55 or SEQ ID NO:63.

[0247] The amino acid sequence of the VL domain of CD3 mAb 1 M23 (SEQ ID NO: 108) is shown below (CDR L Residues are underlined). 108 contains an L95E substitution (shown double underlined and numbered according to Kabat): JPEG2025186382000054.jpg21151

[0248] [Table 30]

[0249] 24.CD3 mAb 1 M24 A preferred amino acid sequence of the VH domain of CD3 mAb 1 M24 is SEQ ID NO:55 or SEQ ID NO:63.

[0250] The amino acid sequence of the VL domain of CD3 mAb 1 M24 (SEQ ID NO: 110) is shown below (CDR L Residues are underlined). 110 contains the L95Q substitution (shown double underlined and numbered according to Kabat): JPEG2025186382000056.jpg22148

[0251] [Table 31]

[0252] 25.CD3 mAb 1 M25 A preferred amino acid sequence of the VH domain of CD3 mAb 1 M25 is SEQ ID NO:55 or SEQ ID NO:63.

[0253] The amino acid sequence of the VL domain of CD3 mAb 1 M25 (SEQ ID NO: 112) is shown below (CDR L Residues are underlined). 112 contains a G50D substitution (shown double underlined and numbered according to Kabat): JPEG2025186382000058.jpg25148

[0254] [Table 32]

[0255] 26.CD3 mAb 1 M26 A preferred amino acid sequence of the VH domain of CD3 mAb 1 M26 is SEQ ID NO:55 or SEQ ID NO:63.

[0256] The amino acid sequence of the VL domain of CD3 mAb 1 M26 (SEQ ID NO: 114) is shown below (CDR L Residues are underlined). 114 contains a K53G substitution (shown double underlined and numbered according to Kabat): JPEG2025186382000060.jpg25148

[0257] [Table 33]

[0258] C. Exemplary antibodies that bind to cell surface molecules on effector cells As used herein, the term "effector cell" refers to a cell that directly or indirectly mediates the killing of target cells (e.g., foreign cells, infected cells, or cancer cells). Examples of effector cells include helper T cells, cytotoxic T cells, natural killer (NK) cells, plasma cells (antibody-secreting B cells), macrophages, and granulocytes. Preferred cell surface molecules of such cells include CD2, CD3, CD8, CD16, TCR, and NKG2D receptors. Thus, molecules capable of immunospecifically binding to epitopes of the above molecules or to other effector cell surface molecules may be used in accordance with the principles of the present invention. Exemplary antibodies whose VH and VL domains may be used to construct molecules capable of mediating targeted killing of target cells are provided below.

[0259] 1. Exemplary Anti-CD2 Antibodies In one embodiment, a molecule of the invention capable of mediating targeted killing of a target cell binds to an effector cell by immunospecifically binding to an epitope of CD2 present on the surface of said effector cell. Molecules that specifically bind to CD2 include the anti-CD2 antibody "CD2 mAb Lo-CD2a."

[0260] The amino acid sequence of the VH domain of CD2 mAb Lo-CD2a (ATCC Accession No. 11423; SEQ ID NO: 116) is shown below (CDR H Residues are underlined ): EVQLQQSGPE LQRPGASVKL SCKASGYIFT EYYMY WVKQR PKQGLELVG R IDPEDGSIDY VEKFKK KATL TADTSSNTAY MQLSSLTSED TATYFCAR Goalkeeper FNYRFAY WGQ GTLVTVSS

[0261] The amino acid sequence of the VL domain of CD2 mAb Lo-CD2a (ATCC Accession No. 11423; SEQ ID NO: 117) is shown below (CDR L Residues are underlined ): DVVLTQTPPT LLATIGQSVS ISC RSSQSLL HSSGNTYLN W LLQRTGQSPQ PLIY LVSKLE S GVPNRFSGS GSGTDFTLKI SGBEAEDLGV YYC MQFTHYP YT FGAGTKLE LK

[0262] 2. Exemplary Anti-CD8 Antibodies In one embodiment, a molecule of the invention capable of mediating targeted killing of a target cell binds to an effector cell by immunospecifically binding to an epitope of CD8 present on the surface of said effector cell. Molecules that specifically bind to CD8 include the anti-CD8 antibodies "OKT8" and "TRX2."

[0263] The amino acid sequence of the VH domain of OKT8 (SEQ ID NO: 118) is shown below (CDR H Residue Groups are underlined): QVQLLESGPE LLKPGASVKM SCKA SGYTFT DYNMH WVKQS HGKSLEWIG Y IYPYTGGTGY NQKFKN KATL TVDSSSSTAY MELRSLTSED SAVYYCARNF RYTYWYFDVW GQGTTVTVSS

[0264] The amino acid sequence of the VL domain of OKT8 (SEQ ID NO: 119) is shown below (CDR L Residue Groups are underlined): DIVMTQSPAS LAVSLGQRAT ISCRASESVD SYDNSLMH WY QQKPGQPPKV LIY LASNLES GVPARFSGSG SRTDFTLTID PVEADDAATY YC QQNNEDPY T FGGGTKLEI KR

[0265] The amino acid sequence of the VH domain of TRX2 (SEQ ID NO: 120) is shown below (CDR H Residue Groups are underlined): QVQLVESGGG VVQPGRSLRL SCAASGFTFS DFGMN WVRQA PGKGLEWVA L IYYDGSNKFY ADSVKG RFTI SRDNSKNTLY LQMNSLRAED TAVYYCAK PH YDGYYHFFDS WGQGTLVTVS S

[0266] The amino acid sequence of the VL domain of TRX2 (SEQ ID NO: 121) is shown below (CDR L Residue Groups are underlined): DIQMTQSPSS LSASVGDRVT ITC KGSQDIN NYLA WYQQKP GKAPKLLIY N TDILHT GVPS RFSGSGSGTD FTFTISSLQP EDIATYYC YQ YNNGYT FGQG TKVEIK

[0267] VI. Exemplary Cancer- and Pathogen-Associated Antigens A. Exemplary cancer antigens arrayed on the surface of cancer cells As used herein, the term "cancer antigen" refers to an antigen that is characteristically expressed on the surface of cancer cells and thus may be treated with antibody-based or immunomodulatory molecules. Examples of cancer antigens include, but are not limited to, 19.9, identified in colon cancer, gastric cancer mucins; 4.2; ADAM-9 (U.S. Patent Publication No. 2006 / 0172350; WO 06 / 084075); AH6, identified in gastric cancer; ALCAM (WO 03 / 093443); APO-1 (malignant human lymphocyte antigen) (Trauth, BC et al. (1989) "Monoclonal Antibody-Mediated Tumor Regression By Induction Of Apoptosis," Science 245:301-304); Bl (Egloff, AM et al. (2006), "Cyclin B1 And Other Cyclins As Tumor AntigensIn Immunosurveillance And Immunotherapy Of Cancer,”Cancer Res. 66(l):6-9);B7-H3(Collins, M. et al. (2005) “The B7 Family Of Immune-Regulatory Ligands,”Genome Biol. 6:223.1-223.7). Chapoval, A. et al. (2001) “B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production,” NatureImmunol. 2:269-274; Sun, M. et al. (2002) “Characterization of Mouse and Human B7-H3 Genes,” J. Immunol.168:6294-6297);BAGE(Bodey, B. (2002) “Cancer-Testis Antigens: Promising Targets For Antigen Directed Antineoplastic Immunotherapy,” Expert Opin Biol Ther.2(6):577-584); Beta-catenin (Prange W. et al. (2003) "Beta-Catenin Accumulation in the Progression of Human Hepatocarcinogenesis Correlates with Loss of E-Cadherin and Acetaminophen" "P53 accumulation, but not with expression of conventional WNT-1 target genes," J. Pathol. 201(2):250-259; Blood type ALe identified in colon adenocarcinoma b / ALe y ; Burkitt lymphoma antigen-38.13; C14; CA125 (ovarian cancer antigen) identified in colon adenocarcinoma (Bast, R.C. Jr. et al. (2005) "New Tumor Markers: CA125 And Beyond," Int J Gynecol Cancer 15(Suppl 3):274-281; ​​Yu et al. (1991) "Coexpression Of Different Antigenic Markers On Moieties That Bear CA 125 Determinants," Cancer Res. 51(2):468-475); carboxypeptidase M (U.S. Patent Publication No. 2006 / 0166 291);CD5(Calin, GA et al. (2006) “Genomics OfChronic Lymphocytic Leukemia MicroRNAs As New PlayersWith ClinicalSignificance,” Semin Oncol. 33(2): 167-173);CD19(Ghetie et al. (1994) “Anti-CD19 InhibitsTheGrowth Of Human B-Cell Tumor Lines In Vitro And Of Daudi Cells In SCID MiceByInducing Cell Cycle Arrest,” Blood 83:1329-1336; Troussard, X.etal. 1998 Hematol Cell Ther. 40(4): 139-48);CD20(Reff et al. (1994) “Depletion Of B Cells In Vivo By A Chimeric Mouse HumanMonoclonalAntibody To CD20,” Blood 83:435-445; Thomas,DA etal. 2006 Hematol Oncol Clin North Am. 20(5): 1125-36);CD22 (Kreitman, R.J. (2006) “ImmunotoxinsForTargeted Cancer Therapy,” AAPS J. 18;8(3):E532-51);CD23(Rosati, S. et al. (2005) “ChronicLymphocyticLeukaemia: A Review Of The Immuno-Architecture,” CurrTop Microbiol Immunol. 294:91-107 );CD25(Troussard, X.et al. (1998) “Hairy Cell Leukemia. What Is New FortyYears After The FirstDescription?” Hematol Cell Ther.40(4): 139-148);CD27(Bataille, R. (2006) “The Phenotype OfNormal, Reactive And Malignant Plasma Cells.Identification Of "Many And Multiple Myelomas" And Of NewTargets ForMyeloma Therapy,” Haematologica 91(9):1234-1240);CD28(Bataille, R. (2006) “The Phenotype OfNormal, Reactive And Malignant PlasmaCells. Identification Of "Many AndMultiple Myelomas" And Of New Targets For Myeloma Therapy,” Haematologica 91(9): 1234-1240);CD33(Sgourosetal. (1993) “Modeling And Dosimetry Of Monoclonal Antibody M195 (Anti-CD33) In Acute MyelogenousLeukemia, ” J. Nucl. Med. 34:422-430); CD36(Ge, Y. (2005) “CD36: A Multiligand Molecule,” Lab Hematol. ll(l):31-7); CD40 / CD154(Messmer D.et al. 2005 A nn N Y Acad Sci. 1062:51-60); CD45(Jurcic, J.G. (2005) “Immunotherapy For Acute Myeloid Leukemia,” Curr Oncol Rep. 7(5):339-346); CD56(Bataille, R. (2006) “The Phenotype Of Normal, Reactive And Malignant Plasma Cells. IdentificationOf "Many And Multiple Myelomas" And Of New Targets For Myeloma Therapy,” Haematologica 91(9): 1234-40); CD46(U.S. Patent No. 7,148,038; International Publication No. 03 / 032814); CD52(Eketorp, S.S. et al. (2014) “Alemtuzumab (Anti-CD52 Monoclonal Antibody) As Single-Agent Therapy In Patients With Relapsed / Refractory Chronic Lymphocytic Leukaemia (CLL)-A Single Region Experience On Consecutive Patients,” Ann Hematol. 93(10):1725-1733; Suresh, T. et al. (2014) “New Antibody Approaches To Lymphoma Therapy,” J. Hematol. Oncol. 7:58; Hoelzer, D. (2013) “Targeted Therapy With Monoclonal Antibodies In Acute Lymphoblastic Leukemia,”Curr. Opin. Oncol. 25(6):701-706);CD56(Bataille, R. (2006) “The Phenotype OfNormal, Reactive And Malignant Plasma Cells.Identification Of “Many And Multiple Myelomas” And Of NewTargets For Myeloma Therapy,”Haematologica91(9):1234-1240);CD79a / CD79b(Troussard,X. et al. (1998) “Hairy CellLeukemia. What Is NewForty Years After The First Description?” Hematol. Cell. Ther. 40(4): 139-148;Chu, P.G. etal.(2001) “CD79: A Review, ” Appl. Immunohistochem. Mol. Morphol. 9(2):97-106);CD103(Troussard, X.et al. (1998)“Hairy CellLeukemia. What Is New FortyYears After The First Description?” Hematol. Cell.Ther. 40(4):139-148);CD317(Kawai,S. et al. (2008) “Interferon-ΑEnhances CD317 Expression AndThe Antitumor Activity Of Ant i-CD317 Monoclonal Antibody In Renal Cell Carcinoma Xenograft Models,” Cancer Science 99(12):2461-2466; Wang, W. et al. (2009)HM1.24(CD317) Is A Novel Target “抗HM1.24抗体用于肺癌免疫治疗”,《癌症免疫学与免疫治疗》58(6):967 - 976;王,W.等人(2009年)“嵌合和人源化抗HM1.24抗体介导对肺癌细胞的抗体依赖性细胞毒性。肺癌”,63(1):23 - 31;赛义德,A.等人(2013年)“BST2( tetherin)启动子的异常调节增强了高级别乳腺癌细胞的细胞增殖和凋亡逃避”,《公共科学图书馆·综合》8(6)e67191,第1 - 10页);CDK4(李,Y.M.等人(2006年)“靶向癌症中的细胞周期蛋白和细胞周期蛋白依赖性激酶:来自小鼠的经验教训,对人类治疗应用的希望”,《细胞周期》5(18):2110 - 2114);CEA(癌胚抗原;富恩等人(1995年)“用抗独特型抗体疫苗治疗的患者对癌胚抗原的免疫反应”,《临床研究杂志》9(1):334 - 42);马泰林,C.(2006年)“循环蛋白质生物标志物与乳腺癌”,《妇产科与生殖医学》34(7 - 8):638 - 646;特列斯 - 阿维拉,F.I.等人(2005年)“癌胚 Antigen: Apropos Of An Old Friend,” Rev. Invest.Clin.57(6):814-819);CEACAM5 / CEACAM6(Zheng, C. et al. (2011) “A Novel Anti-CEACAM5 Monoclonal Antibody, CC4,Suppresses ColorectalTumor Growth and Enhances NK Cells-Mediated TumorImmunity,” PLoS One 6(6):e21146, pp. 1-11);CO17-1A(Ragnhammaret al. (1993) “Effect OfMonoclonal Antibody 17-1A AndGM-CSF In Patients With Advanced ColorectalCarcinoma - Long-Lasting, CompleteRemissions Can Be Induced,” Int. J. Cancer 53:751-758);CO‐43(Blood type Le b CO-514 (blood group Le) confirmed in adenocarcinoma a );CTA‐1:CTLA‐4(Peggs, KS et al. (2006) “Principles and Use of Anti-CTLA4 Antibody in Human Cancer Immunotherapy,” Curr. Opin. Immunol. 18(2):206-13; Cytokeratin 8 (International Publication No. WO 03 / 024 191);Dl.l;D156‐22;DR5(Abdulghani, J. et al.(2010) "TRAIL Receptor Signaling AndTherapeutics," Expert Opin. Ther.Targets 14(10): 1091-1108; Andera, L.(2009) "Signaling Activated By TheDeath Receptors Of The TNFRFamily," Biomed. Pap. Med. Fac. Univ. PalackyOlomouc Czech. Repub.153(3): 173-180; Carlo-Stella, C. et al. (2007) "Targeting TRAIL Agonistic Receptors for Cancer Therapy," Clin, Cancer 13(8):2313-2317; Chaudhari, BR et al. (2006) "Following the TRAIL to Apoptosis," Immunologic Res. 35(3):249-262; confirmed in pancreatic cancer. EGFR (epidermal growth factor receptor; Adenis, A. et al. (2003) "Inhibitors of Epidermal Growth Factor Receptor and Colorectal Cancer," Bull. Cancer. 90 Spec No:S228-S232); ephrin receptors (and especially EphA2 ( U.S. Patent No. 7,569,672; International Publication No. WO 06 / 084226; Erb (ErbB1; ErbB3; ErbB4; Zhou, H. et al. (2002) "Lung Tumorigenesis Associated with Erb-B-2 and Erb-B-3 Overexpression in Human Erb-B-3 Transgenic Mice" Enhanced By Methylnitrosourea,” Oncogene21(57):8732-8740;Rimon, E. et al. (2004) “Gonadotropin-InducedGene Regulation In Human Granulosa CellsObtained From IVF Patients: ModulationOf Genes Coding For Growth Factors AndTheir Receptors And Genes Involved InCancer And Other Diseases,” Int J Oncol. 24(5):1325-1338 );GAGE(GAGE‐1;GAGE‐2;Akcakanat, A. et al. (2006) “HeterogeneousExpression Of GAGE, NY-ESO-1, MAGE-A and SSX ProteinsIn Esophageal Cancer:Implications For Immunotherapy,”Int J Cancer. 118(1):123-128);GD2 / GD3 / GM2(Livingston,P.O. et al. (2005) “Selection Of GM2, Fucosyl GM1,Globo H And Polysialic Acid AsTargets On Small Cell Lung Cancers ForAntibody-Mediated Immunotherapy,” Cancer Immunol Immunother. 54(10):1018-1025);ガング リオシドGD2(G D2 ;Salehetal. (1993) “Generation Of A Human Anti-Idiotypic Antibody That Mimics The GD2 Antigen,” J.Immunol.,151,3390-3398); ガングリオシドGD3(G D3;Shitaraetal. (1993) “A Mouse / Human ChimericAnti-(GangliosideGD3) Antibody With Enhanced Antitumor Activities,”Cancer Immunol. Immunother. 36:373-380);ガングリオシドGM2(G M2 ;Livingston et al. (1994) “Improved SurvivalIn Stage III Melanoma Patients WithGM2 Antibodies: A Randomized Trial OfAdjuvant Vaccination With GM2 Ganglioside,” J. Clin.Oncol.12:1036-1044);ガングリオシドGM3(G M3 ;Hoon et al. (1993) “Molecular Cloning Of AHuman Monoclonal Antibody Reactive ToGanglioside GM3 Antigen On Human Cancers,” CancerRes.53:5244-5250);GICA19‐9(Herlyn etal. (1982)"Monoclonal Antibody Detection Of A CirculatingTumor-Associated Antigen.I. Presence Of Antigen In Sera Of Patients WithColorectal, Gastric, And Pancreatic Carcinoma, " J. Clin. Immunol. 2:135-140);gp100(Lotem, M. etal.(2006) “Presentation Of Tumor Antigens By Dendritic Cells Genetically Modified With Viral AndNonviral Vectors,” J, Immunother. 29(6): 616-27); VaccinesAgainstHuman T Cell Leukemia. II Generation And Characterization Of AMonoclonalIdiotype Cascade (Abl, Ab2, And Ab3), " J. Immunol. 141:1398-1403); " J. Exp. Med.171(4): 1375-1380); gpA33 Is The Human Homologue Of The Mouse B (Brown) Locus Gene Product," J. Exp. Med (Heath, J.K. et al. (1997) “The Human A33 Antigen Is A Transmembrane Glycoprotein And A Novel Member Of The Immunoglobulin Superfamily,” Proc. Natl. Acad. Sci. (U.S.A.) 94(2):469-474; Ritter, G. et al. (1997) “Characterization Of Posttranslational Modifications Of Human A33 Antigen, A Novel Palmitoylated Surface Glycoprotein Of Human Gastrointestinal Epithelium,” Biochem. Biophys. Res. Commun. 236(3):682–686; Wong, N.A. et al. (2006) “EpCAM and gpA33 Are Markers Of Barrett's Metaplasia,” J. Clin. Pathol. 59(3):260–263; Almqvist, Y. (2006) “In vitro and in vivo Characterization of 177Lu-huA33: A Radioimmunoconjugate Against Colorectal Cancer,” Nucl. Med. Biol. x3(8):991–998); HER2 antigen (HER2 / n eu, p185 HER2 ; Pal, S.K. et al. (2006) “Targeting HER2 Epitopes,” Semin Oncol. 33(4):386–391); HMFG (human milk fat globule antigen; International Publication No. 1995 / 0151 No. 71); human papillomavirus E6 / human papillomavirus E7 (DiMaio, D. et al. (2006) “Human Papillomaviruses and Cervical Cancer,” Adv. Virus Res. 66: 125-59); HMW-MAA (high molecular weight melanoma antigen); Natali et al. (1987) “Immunohistochemical Detection of Antigens in Human Primary and Metastatic Melanomas by the Monoclonal Antibody 140 / 240 and Its Possible Prognostic Significance,” Cancer 59:55-63; Mittelman et al. (1990) “Active Specific Immunotherapy in Patients with Melanoma. A Clinical Trial with Mouse Antiidiotypic Monoclonal Antibodies Elicited with Syngeneic Anti-High-Molecular-Weight-Melanoma-Associated Antigen Monoclonal Antibodies,” J. Clin. Invest. 86:2136-2144);I antigen (differentiation antigen; Feizi (1985) "Demonstration By Monoclonal Antibodies That Carbohydrate Structures Of Glycoproteins And Glycolipids Are Onco-Developmental Antigens," Nature 314:53-57);IL1 3Rα2 (International Publication No. 2008 / 146911; Brown, C.E. et al. (2013) “Glioma IL13Rα2 Is Associated With Mesenchymal Signature Gene Expression And Poor Patient Prognosis,” PLoS One. 18;8(10):e77769; Barderas, R. et al. (2012) “High Expression Of IL-13 Receptor Α2 In Colorectal Cancer Is Associated With Invasion, Liver Metastasis, And Poor Prognosis,” Cancer Res. 72(11):2780-2790; Kasaian, M.T. et al. (2011) “IL-13 Antibodies Influence IL-13 Clearance In Humans By Modulating Scavenger Activity Of IL-13Rα2,” J. Immunol. 187(1):561-569; Bozinov, O. et al. (2010) “Decreasing Expression Of The Interleukin-13 Receptor IL-13Ralpha2 In Treated Recurrent Malignant Gliomas,” Neurol. Med. Chir. (Tokyo) 50(8):617-621; Fujisawa, T. et al.(2009)“A novel role of interleukin-13 receptor a lpha2 in pancreatic cancer invasion and metastasis,” Cancer Res. 69(22):8678-8685); Integrin β6 (International Publication No. 03 / 087340); JAM-3 (International Publication No. 06 / 084078); KID3 (WO 05 / 028498); KID31 (WO 06 / 076584); KS1 / 4 pan-cancer antigen (Perez et al. (1989) "Isolation and Characterization of AcDNA Encoding the Ksl / 4 Epithelial Carcinoma Marker," J. Immunol. 142:3662-3667; Moller et al. (1991) "Bi-specific Monoclonal Antibody-Directed Lysis of Ovarian Carcinoma Cells by Activated Human T Lymphocytes," Cancer Immunol. Immunother. 33(4):210-216; Ragupathi, G. 2005 Cancer Treat Res. 123:157-80; L6 and L20 (human lung cancer antigens) (Hellstrom et al. (1986) "Monoclonal Mouse Antibodies Raised Against Human Lung Carcinoma, "Cancer Res. 46:3917-3923; LEA; LUCA-2 (U.S. Patent Publication No. 2006 / 0172349; WO 06 / 083852); M1:22:25:8; M18; M39; MAGE (MAGE-1; MAGE-3) (Bodey, B. (2002) "Cancer-Tests Antigens: Promising Targets For Antigen Directed Antineoplastic Immunotherapy,” Expert Opin. Biol. Ther. 2(6):577-584);MART(Kounalakis, N. et al. (2005) “Tumor Cell And Circulating Markers In Melanoma: Diagnosis,Prognosis,And Management,” Curr. Oncol. Rep.7(5):377-382);Mesothelin(Chang, K. et al. (1996) “Molecular CloningOf Mesothelin, A Differentiation Antigen PresentOn Mesothelium, Mesotheliomas,And Ovarian Cancers,”Proc. Natl. Acad. Sci. (USA)93:136-140);MUC‐ 1 (Mathelin, C. (2006) "Circulating Proteinic Biomarkers And Breast Cancer," Gynecol. Obstet. Fertil. 34(7-8):638-646); MUM-1 (Castelli, C. et al. (2000) "T-Cell Recognition Of Melanoma-Associated Antigens," J. Cell. Physiol. 182(3):323-331); N-acetylglucosaminyltransferase (Dennis, J. W. (1999) "Glycoprotein Glycosylation and Cancer Progression," Biochim Biophys Acta. 6;1473(l):21-34; Neoglycoproteins NS-10 and OFA-1 identified in adenocarcinoma OFA-2; oncostatin M (oncostatin receptor beta; U.S. Pat. No. 7,572,896; WO 06 / 084092); p15 (Gil, J. et al. (2006) "Regulation of the INK4b-ARF-INK4a Tumor Suppressor Locus: All for One or One for All," Nat. Rev. Mol. Cell Biol. 7(9):667-677); p97 (melanoma-associated antigen; Estin et al. al. (1989) “Transfected MouseMelanoma Lines ThatExpress Various Levels Of Human Melanoma-Associated Antigenp97,” J. Natl. Cancer Instit. 81(6):445-454) PEM (Polymorphic Epithelial Mucin; Hilkens et al. (1992) "Cell Membrane-Associated Mucins and Their Adhesion-Modulating Property," Trends in Biochem. Sci. 17:359-363) PEMA (polymorphic epithelial mucin antigen); PIPA (U.S. Patent No. 7,405,061; International Publication No. 04 / 043239; PSA (prostate-specific antigen; Henttu et al. (1989) “cDNA Coding For The Entire Human ProstateSpecificAntigen Shows High Homologies To The Human Tissue Kallikrein Genes,” Biochem. Biophys. Res. Comm.10(2):903-910; Israeli et al. (1993) “Molecular Cloning Of A Complementary DNA Encoding A Prostate-Specific Membrane Antigen,” Cancer Res. 53:227-230; Cracco, C. M. et al. (2005) “Immune Response In Prostate Cancer,” Minerva Urol Nefrol. 57(4):301-311; PSMA (prostate-specific membrane antigen; Ragupathi, G. (2005) “Antibody Inducing Polyvalent Cancer Vaccines,” Cancer Treat. Res. 123:157-180); prostatic acid phosphate phospholipase (Tailor et al. (1990) "Nucleotide Sequence of Human Prostatic Acid Phosphatase Determined From a Full-Length cDNA Clone," Nucl. Acids Res. 18(16):4928); R identified in melanoma 24 ;ROR1 (U.S. Patent No. 5,843,749); Sphingolipids; SSEA-1; SSEA-3; SSEA-4; sTn (Holmberg, LA (2001) “Theratope Vaccine (STn-KLH),” Expert OpinBiol Ther. 1(5):881-91); T-cell receptor-derived peptides from cutaneous T-cell lymphoma (see Edelson (1998) "Cutaneous T-Cell Lymphoma: A Model For Selective Immunotherapy," Cancer J Sci Am. 4:62-71); T5A7 and TAG-72 identified in bone marrow cells (see Yokota et al. (1992) "Rapid Immunotherapy," Cancer J Sci Am. 4:62-71); d Tumor Penetration Of A Single-Chain Fv And Comparison WithOther Immunoglobulin Forms, " Cancer Res. 52:3402-3408; TL5 (blood type A); TNF receptor (TNF -α receptor, TNF-β receptor; TNF-γ receptor) (van Horssen, R. et al. (2006) “TNF-Alpha In Cancer Treatment: Molecular Insights, Antitumor Effects, And Clinical Utility,” Oncologist 11(4):397-408; Gardnerova, M. et al. (2000) “The Use of TNF-Family Ligands and Receptors and Agents Which Modify Their Interaction As Therapeutic Agents,” Curr. Drug Targets 1(4):327-364); TRA-1-85 (blood type H); transferrin receptor (U.S. Pat. No. 7,572,895; WO 05 / 121179); 5T4 (TPBG, trophoblast glycoprotein; Boghaert, E.R. et al. (2008) “The Oncofetal Protein, 5T4, Is a Suitable Target For Antibody-Guided Anti-Cancer Chemotherapy With Calicheamicin,” Int. J. Oncol. 32(1):221-234; Eisen, T. et al. al. (2014) “Naptumomab Estafenatox:Targeted Immunotherapy with a Novel Immunotoxin,” Curr. Oncol. Rep. 16:370, pp. 1-6); T antigen of DNA tumor virus and virally induced tumor antigens, including the envelope antigens of RNA tumor viruses; carcinoembryonic antigens such as CEA, colon and bladder oncofetal antigen; and tumor-specific transplantation antigens identified in embryonic carcinoma cells, such as alpha-fetoprotein (TSTA) (Hellstrom et al. (1985) "Monoclonal Antibodies To Cell Surface Antigens Shared By Chemically Induced Mouse Bladder Carcinomas," Cancer. Res. 45:2210-2188); VEGF (Pietrantonio, F. et al. (2015) “Bevacizumab-Based Neoadjuvant ChemotherapyForColorectal Cancer Liver Metastases: Pitfalls And Helpful Tricks In A ReviewForClinicians,” Crit. Rev. Oncol. Hematol.95(3):272-281;Grabowski, JP (2015) “Current ManagementOf OvarianCancer,” Minerva Med. 106(3):151-156; Field, KM(2015) “Bevacizumab And Glioblastoma:ScientificReview, Newly Reported Updates, And Ongoing Controversies,” Cancer 121(7):997-1007; Suh, DH et al. (2015) “Major Clinical Research Advances In Gynecologic Cancer In 2014,” J. Gynecol. Oncol. 26(2):156-167; Liu, KJ et al. (2015) “Bevacizumab In Combination With Anticancer Drugs For Previously Treated Advanced Non-Small Cell Lung Cancer,” Tumour Biol. 36(3):1323-1327; Di Bartolomeo, M. et al. (2015) “Bevacizumab Treatment In The Elderly Patient WithMetastaticColorectal Cancer,” Clin. Interv. Aging10:127-133); VEGF receptor (O'Dwyer. PJ (2006) “The Present And Future Of Angiogenesis-Directed TreatmentsOfColorectal Cancer,” Oncologist. 11(9):992-998);VEP8 ;VEP9;VIM-D5; and Y heptane, Le identified in embryonal carcinoma cells y Further cancer antigens and molecules (e.g., antibodies) that bind to them are disclosed in Table 7. 5T4, B7-H3, CEACAM5 / CEACAM6, CD123, DR5, EGFR, ephrin receptors, gpA33, HER2 / neu, IL13Rα2, ROR1, and VEGF are particularly preferred "cancer antigens" of the present invention.

[0268] [Table 34] JPEG2025186382000063.jpg249155 JPEG2025186382000064.jpg250157 JPEG2025186382000065.jpg241156 JPEG2025186382000066.jpg232164

[0269] Exemplary antibodies whose VH and VL domains can be used to construct binding molecules of the invention that can bind to cancer antigens arranged on the surface of cancer cells and mediate targeted killing of said cancer cells are listed in the table above, and additional antibodies that can be used to construct molecules that can bind to cancer antigens arranged on the surface of cancer cells and mediate targeted killing of said cancer cells are provided below. do.

[0270] 1. Exemplary anti-B7-H3 antibodies B7-H3 is a cancer antigen overexpressed in a wide variety of solid tumors and a member of the B7 family of molecules involved in immune regulation (U.S. Patent No. 8,802,091; U.S. Patent No. 2014 / 0328750; U.S. Patent No. 2013 / 0149236; Loo, D. et al. (2012) "Development of AnFc-Enhanced Anti-B7-H3 Monoclonal Antibody With Potent (See "Antitumor Activity," Clin. Cancer Res. 18(14):3834-3845). Multiple independent studies have shown that malignant cancer cells (e.g., neuroblastoma and gastric, ovarian, pancreatic, and non-small cell lung cancer cells) exhibit significantly increased expression of B7-H3 protein, and that this increased expression is associated with increased disease severity (Zang, X. et al. (2007) "The B7 Family and Cancer Therapy: Costimulation and Coinhibition," Clin. Cancer Res. 13:5271-5279), suggesting that B7-H3 is involved in immune evasion pathways. These findings suggest that B7-H3 may be utilized by tumors as a marker for B7-H3 signaling (Hofmeyer, K. et al. (2008) “The Contrasting Role of B7-H3,” Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278).

[0271] B7‐H3 also binds to CD4 +and CD8 + B7-H3 has been shown to costimulate T cell proliferation. B7-H3 also stimulates IFN-γ production and CD8+ lytic activity (Chapoval, A. et al. (2001) "B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production," Nature Immunol. 2:269-274; Sharpe, AH et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2:116-126). However, this protein It may also act to inhibit T cell activation through NFAT (nuclear factor for activated T cells), NF-κB (nuclear factor kappa B), and AP-1 (activator protein 1) factors (Yi. KH et al. (2009) "Fine Tuning The Immune Response Through B7-H3 and B7-H4,” Immunol. Rev. 229:145-151), B7-H3 also inhibits Th1, Th2, or Th It is thought that B7-H3 inhibits B7-H3 expression in vivo (Prasad, DV et al. (2004) "Murine B7-H3 Is a Negative Regulator of T Cells," J. Immunol. 173:2500-2506; Fukushima, A. et al. (2007) "B7-H3 Regulates the Development of Experimental Allergic Conjunctivitis in Mice," Immunol. Lett. 113:52-57; Yi, KH et al. (2009) "Fine Tuning The Immune Response Through B7-H3 And B7-H4,”Immunol.Rev. 229:145-151).

[0272] Preferred B7-H3 binding molecules comprise the VL and / or VH domains of the humanized anti-human B7-H3 monoclonal antibodies "B7-H3 mAb-B," "B7-H3 mAb-C," "B7-H3 mAb-D," or any of the anti-B7-H3 antibodies provided herein; more preferably, the CDRs of the VL domain of such anti-B7-H3 monoclonal antibodies L and / or the CDRs of the VH domain. H It has one, two or all three of the following:

[0273] Upon humanization, antibody B7-H3 mAb-B yields two variant VH domains: B7-H3 mAb-B VH1 and B7-H3 mAb-B VH2, and two variant VL domains: B7-H3 mAb-B VL1 and B7-H3 mAb-B VL2, which can be used in any combination of VH / VL domains to obtain functional B7-H3 binding domains.

[0274] The amino acid sequence of the VH domain of B7-H3 mAb-B VH1 is SEQ ID NO: 122 (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMQ WVRQA PGQGLEWMG T IYPGDGDTRY TQKFKG RVTI TADKSTSTAY MELSSLRSED TAVYYCAR RG IPRLWYFDV W GQGTTVTVSS

[0275] The amino acid sequence of the VH domain of B7-H3 mAb-B VH2 is SEQ ID NO: 123 (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMQ WVRQA PGQGLEWMG T IYPGGGDTRY TQKFQG RVTI TADKSTSTAY MELSSLRSED TAVYYCAR RG IPRLWYFDV W GQGTTVTVSS

[0276] The amino acid sequence of the VL domain of B7-H3 mAb-B VL1 is SEQ ID NO: 124 (CDR L residues are underlined). DIQMTQSPSS LSASVGDRVT ITC RASQDIS NYLN WYQQKP GKAPKLLIY Y TSRLHS GVPS RFSGSGSGTD FTLTISSLQP EDIATYYC QQ GNTLPPT FGG GTKLEIK

[0277] The amino acid sequence of the VL domain of B7-H3 mAb-B VL2 is SEQ ID NO: 125 (CDR L residues are underlined). DIQMTQSPSS LSASVGDRVT ITC RASQSIS SYLN WYQQKP GKAPKLLIY Y TSRLQS GVPS RFSGSGSGTD FTLTISSLQP EDIATYYC QQ GNTLPPT FGG GTKLEIK

[0278] The amino acid sequence of the VH domain of humanized B7-H3 mAb-C is SEQ ID NO: 126 (CDR H Residues are underlined): EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMS WVRQA PGKGLEWVA T INSGGSNTYY PDSLKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HD GGAMDY WGQG TTVTVSS

[0279] The amino acid sequence of the VL domain of humanized B7-H3 mAb-C is SEQ ID NO: 127 (CDR L residues are underlined). DIQMTQSPSS LSASVGDRVT ITC RASESIY SYLA WYQQKP GKAPKLLVY N TKTLPE GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QH HYGTPPWT FG QGTRLEIK

[0280] The amino acid sequence of the VH domain of B7-H3 mAb-D (SEQ ID NO: 128) is shown below (CDR H residues are underlined). EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMH WVRQA PGKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HG YRYEGFDY WG QGTTVTVSS

[0281] The amino acid sequence of the VL domain of B7-H3 mAb-D (SEQ ID NO: 129) is shown below (CDR L residues are underlined). DIQMTQSPSF LSASVGDRVT ITC KASQNVD TNVA WYQQKP GKAPKALIY S ASYRYS GVPS RFSGSGSGTD FTLTISSLQP EDFAEYFC QQ YNNYPFT FGQ GTKLEIK

[0282] Particularly preferred are B7-H3 binding molecules with humanized VH and / or VL domains, including, but not limited to, "enoblitutuzumab" (also known as MGA271; CAS Registry Number: 1353485-38-7). Enoblituzumab is an Fc-optimized monoclonal antibody that binds to HER2 / neu and mediates enhanced ADCC activity. The amino acid sequences of the complete heavy and light chains of enoblituzumab are known in the art (see, e.g., WHO Drug Information, 2017, Recommended INN: List 77, 31(1):49). The amino acid sequence of the VH domain of enoblituzumab is SEQ ID NO: 130 (CDRs: 1353485-38-7). H Residues are below (shown with a line): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMH WVRQA PGKGLEWVA Y ISSDSSAIYY ADTVKG RFTI SRDNAKNSLY LQMNSLRDED TAVYYCGR GR ENIYYGSRLD Y WGQGTTVTV SS The amino acid sequence of the VL domain of enoblituzumab is SEQ ID NO: 131 (CDR L Residues are underlined): DIQLTQSPSF LSASVGDRVT ITC KASQNVD TNVA WYQQKP GKAPKALIY S ASYRYS GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ YNNYPFT FGQ GTKLEIK

[0283] In addition to the preferred anti-B7-H3 binding molecules identified above, the present invention also provides the following anti-B7-H3 binding molecules: LUCA1; BLA8; PA20; or SKN2 (see U.S. Pat. No. 7,527,969; U.S. Pat. No. 8,779,098 and WO 2004 / 001381); M30; cM30; M30-H1-L1; M30-H1-L2; M30-H1-L3; M30-H1-L4; M30-H1-L5; M30-H1-L6; M30-H1-L7; M30-H4-L1; M30-H4-L2; M30-H4-L3; and M Use of any of 30-H4-L4 (see U.S. Patent Application Publication No. 2013 / 0078234 and WO 2012 / 147713); and 8H9 (see U.S. Patent Nos. 7,666,424; 7,737,258; 7,740,845; 8,148,154; 8,414,892; 8,501,471; 9,062,110; U.S. Patent Application Publication No. 2010 / 0143245, and WO 2008 / 116219) is also contemplated.

[0284] 2. Exemplary Anti-CEACAM5 and Anti-CEACAM6 Antibodies Carcinoembryonic antigen-related cell adhesion molecules 5 (CEACAM5) and 6 (CEACAM6) are known to inhibit the growth of various cancers, including thyroid cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, lung cancer, head and neck cancer, bladder cancer, prostate cancer, uterine cancer, endometrial cancer, breast cancer, hematopoietic cancer, leukemia, and ovarian cancer (WO 2011 / 034660), and in particular, colorectal cancer, gastrointestinal cancer, pancreatic cancer, non-small cell lung cancer (NSCL), breast cancer, thyroid cancer, gastric cancer, ovarian cancer, and uterine carcinoma (Zheng, C. et al. (2011) “A Novel Anti-CEACAM5 Monoclonal Antibody, CC4, Suppresses Colorectal Tumor Growth and Enhances NK Cells-Mediated Tumor Immunity,” PLoS One 6(6):e21146, pp. 1-11). It has been shown to be associated with various cancers.

[0285] CEACAM5 has been found to be overexpressed in 90% of gastrointestinal, colorectal, and pancreatic cancers, 70% of non-small cell lung cancer cells, and 50% of breast cancers (Thompson, JA et al. (1991) "Carcinoembryonic Antigen Gene Family: Molecular Biology And Clinical Perspectives," J. Clin. Lab. Anal. 5:344-366). Overexpressed carcinoembryonic antigen-associated cells Cellular adhesion molecule 6 (CEACAM6) plays an important role in the invasion and metastasis of various human cancers, including medullary thyroid carcinoma, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, lung cancer, head and neck cancer, bladder cancer, prostate cancer, uterine cancer, endometrial cancer, breast cancer, hematopoietic cancer, leukemia, and ovarian cancer (WO 2011 / 034660; Deng, X. et al. (2014) "Expression Profiling of CEACAM6 Associated With The Tumorigenesis And Progression In Gastric Adenocarcinoma," Genet. Mol. Res. 13(3):7686-7697; Cameron, S. et al. (2012) "Focal Overexpression Of CEACAM6 Contributes To Enhanced Tumourigenesis In Head And Neck CancerViaSuppression Of Apoptosis,” Mol. Cancer 11:74, pp.1-11;Chapin, C. et al. (2012) “Distribution And Surfactant AssociationOfCarcinoembryonic Cell Adhesion Molecule 6 In Human Lung,” Amer. J. Physiol. Lung Cell. Mol. Physiol. 302(2):L216-L25;Riley,CJ et al. (2009) “Design And Activity Of AMurine AndHumanized Anti-CEACAM6 Single-Chain Variable Fragment In TheTreatment OfPancreatic Cancer,” Cancer Res.69(5):1933-1940;Lewis-Wambi, JS et al. (2008) “OverexpressionOfCEACAM6 Promotes Migration And Invasion Of Oestrogen-Deprived Breast Cancer Cells,” Eur. J. Cancer 44(12):1770-1779; Blumenthal, RD et al. (2007) “Expression Patterns of CEACAM5 and CEACAM6 in Primary and Metastatic Cancers,” BMC Cancer. 7:2, pp. 1-15. Binds immunospecifically to CEACAM5 and CEACAM6. Antibodies are commercially available (Santa Cruz Biotechnology, Inc., Novus Biologicals LLC; Abnova Corporation).

[0286] The amino acid sequence (SEQ ID NO: 132) of the VH domain of the humanized anti-CEACAM5 / anti-CEACAM6 antibody 16C3 (EP 2585476) is shown below (CDR H Residues are below (shown with a line): QVQLQQSGPE VVRPGVSVKI SCKGS GYTFT DYAMH WVKQS HAKSLEWIGL ISTYSGDTKY NQNFKG KATM TVDKSASTAY MELSSLRSED TAVYYCAR GD YSGSRYWFAY WGQGTLVTVS S

[0287] The amino acid sequence (SEQ ID NO: 133) of the VL domain of the humanized anti-CEACAM5 / anti-CEACAM6 antibody 16C3 (EP 2585476) is shown below (CDR L Residues are below (shown with a line): DIQMTQSPSS LSASVGDRVT ITC GASENIY GALN WYQRKP GKSPKLLIW G ASNLAD GMPS RFSGSGSGRQ YTLTISSLQP EDVATYY CQN VLSSPYT FGG GTKLEIK

[0288] The amino acid sequence (SEQ ID NO: 134) of the VH domain of the humanized anti-CEACAM5 / CEACAM6 antibody hMN15 (WO 2011 / 034660) is shown below (CDR H Residues are underlined): QVQLVESGGG VVQPGRSLRL SC SSSGFALT DYYMS WVRQA PGKGLEWLG F IANKANGHTT DYSPSVKG RF TISRDNSKNT LFLQMDSLRP EDTGVYFCAR DMGIRWNFDV WGQGTPVTVS S

[0289] The amino acid sequence (SEQ ID NO: 135) of the VL domain of the humanized anti-CEACAM5 / CEACAM6 antibody hMN15 (WO 2011 / 034660) is shown below (CDR L Residues are underlined): DIQLTQSPSS LSASVGDRVT MTC SASSRVS YIHWYQQKPG KAPKRWIY GT STLAS GVPAR FSGSGSGTDF TFTISSLQPE DIATYYC QQW SYNPPT FGQG TKVEIKR

[0290] The present invention specifically relates to CEACAM5 / CEACAM6 binding molecules (e.g., CEACAM5 / CEACAM6×CD3 bispecific binding molecules) capable of binding to CEACAM5 and / or CEACAM6, in particular the VL and / or VH domains, and / or CDRs of the VL domains, of the anti-CEACAM5 / CEACAM6 monoclonal antibodies 16C3 or hMN15. L and / or the CDRs of the VH domain. H The term "antibody" includes and encompasses such molecules that contain one, two or all three of:

[0291] 3. Exemplary Anti-EGRF Antibodies Epidermal growth factor receptor (EGFR) is a cancer antigen in certain metastatic colorectal cancers, metastatic non-small cell lung cancers, and head and neck cancers. Exemplary antibodies that bind to human EGFR are "cetuximab" and "panitumumab." Cetuximab is a recombinant human-mouse chimeric epidermal growth factor receptor (EGFR) IgG1 monoclonal antibody (Govindan R. (2004) "Cetuximab In Advanced Non-Small Cell Lung Cancer," Clin. Cancer Res. 10(12 Pt 2):4241s-4244s; Bou-Assaly, W. et al. (2010) “Cetuximab (Erbitux),” Am. J.Neuroradiol.31(4):626-627). Panitumumab (Vectibix) (R), Amgen, is a fully humanized epidermal growth factor receptor (EGFR) IgG2 monoclonal antibody. It is a noclonal antibody. Trials on EGFR Inhibitors SuchasCetuximab and Panitumumab as Monotherapy and in Combination for Treatment of Metastatic Colorectal Cancer,” Avicenna J. Med.Biotechnol.7(4):134-144).

[0292] The amino acid sequence of the VH domain of the chimeric anti-EGFR antibody cetuximab (SEQ ID NO: 136) is shown below (CDR H Residues are underlined): QVQLKQSGPG LVQPSQSLSI TCTVS GFSLT NYGVH WVRQS PGKGLEWLG V IWSGGNTDYN TPFTS RLSIN KDNSKSQVFF KMNSLQSNDT AIYYCAR ALT YYDYEFAY WG QGTLVTVSA

[0293] The amino acid sequence of the VL domain of the chimeric anti-EGFR antibody cetuximab (SEQ ID NO: 137) is shown below (CDR L Residues are underlined): DILLTQSPVI LSVSPGERVS FSC RASQSIG TNIH WYQQRT NGSPRLLIK Y ASESISGIPS RFSGSGSGTD FTLSINSVES EDIADYYC QQ NNNWPTT FGA GTKLELKR

[0294] The amino acid sequence of the VH domain of panitumumab (SEQ ID NO: 138) is shown below (CDR H Residues are underlined): QVQLQESGPG LVKPSETLSL TCTVS GGSVS SGDYY WTWIR QSPGKGLEWI G HIYYSGNTN YNPSLKS RLT ISIDTSKTQF SLKLSSVTAA DTAIYYCVR D RVTGAFDI WG QGTMVTVSS

[0295] The amino acid sequence of the VL domain of panitumumab (SEQ ID NO: 139) is shown below (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC QASQDIS NYLN WYQQKP GKAPKLLIY D ASNLET GVPS RFSGSGSGTD FTFTISSLQP EDIATYFC QH FDHLPLA FGG GTKVEIKR

[0296] The present application specifically relates to EGFR binding molecules (e.g., EGFRxCD3 bispecific binding molecules) capable of binding to EGFR, in particular the VL and / or VH domains, and / or CDRs of the VL domains, of the anti-EGFR monoclonal antibodies cetuximab or panitumumab. L Of and / or the CDRs of the VH domain. H One of the two younger The term "antibody" includes and encompasses such binding molecules, including all three.

[0297] 4. Exemplary Anti-EphA2 Antibodies The receptor tyrosine kinase, ephrin type A receptor 2 (EphA2), is normally expressed at cell-cell contact sites in adult epithelial tissues. Recent studies have shown that EphA2 is also overexpressed in various types of epithelial cancers, with the highest levels of EphA2 expression observed in metastatic lesions. High levels of EphA2 expression have been identified in a wide range of cancers and numerous cancer cell lines, including prostate cancer, breast cancer, non-small cell lung cancer, and melanoma (Xu, J. et al. (2014) "High EphA2 Protein Expression in Renal Cell Carcinoma Is Associated with Renal Cell Carcinoma"). With A Poor Disease Outcome,” Oncol. Lett. Aug2014;8(2): 687-692; Miao, B. et al. (2014) “EphA2 isaMediator of Vemurafenib Resistance and a Novel Therapeutic Target in Melanoma,” Cancer Discov. pii: CD-14-0295). Although not considered a cancer marker, EphA2 appears to be persistently overexpressed and functionally altered in many human cancers (Chen, P. et al. (2014) "EphA2 Enhances the Proliferation and Invasion Ability of LnCap Prostate Cancer Cells," Oncol. Lett. 8(1):41-46). Exemplary antibodies that bind to EphA2 are "EphA2 mAb 1," "EphA2 mAb 2," and "EphA2 mAb 3."

[0298] The amino acid sequence of the VH domain of EphA2 mAb 1 (SEQ ID NO: 140) is shown below (CDR H Residues are underlined): QVQLKESGPG LVAPSQSLSI TCTVSGFSLS RYSVH WVRQP PGKGLEWLG M IWGGGSTDYN SALKSRLSIS KDNSKSQVFL KMNSLQTDDT AMYYCAR KHG NYYTMDY WGQ GTSVTVSS

[0299] The amino acid sequence of the VL domain of EphA2 mAb 1 (SEQ ID NO: 141) is shown below (CDR L Residues are underlined): DIQMTQTTSS LSASLGDRIT ISC RASQDIS NYLN WYQQKP DGTVKLLIY Y TSRLHS GVPS RFSGSGSGTD YSLTISNLEQ EDIATYFC QQ GYTLYT FGGG TKLEIK

[0300] The amino acid sequence of the VH domain of EphA2 mAb 2 (SEQ ID NO: 142) is shown below (CDR H Residues are underlined): QIQLVQSGPE LKKPGETVKI SCKASGFTFT NYGMN WVKQA PGKGLKWMG W INTYIGEPTY ADDFKG RFVF SLETSASTAY LQINNLKNED MATYFCAR EL GPYYFDY WGQ GTTLTVSS

[0301] The amino acid sequence of the VL domain of EphA2 mAb 2 (SEQ ID NO: 143) is shown below (CDR L Residues are underlined): DVVMTQTPLS LPVSLGDQAS ISC RSSQSLV HSSGNTYLH W YLQKPGQSPK LLIY KVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFC SQSTHVP T FGSGTKLEI K

[0302] The amino acid sequence of the VH domain of EphA2 mAb 3 (SEQ ID NO: 144) is shown below (CDR H Residues are underlined): EVQLVESGGG SVKPGGSLKL SCAASGFTFT DHYMY WVRQT PEKRLEWVA T ISDGGSFTSY PDSVKG RFTI SRDIAKNNLY LQMSSLKSED TAMYYCTR DE SDRPFPY WGQ GTLVTVSS

[0303] The amino acid sequence of the VL domain of EphA2 mAb 3 (SEQ ID NO: 145) is shown below (CDR L Residues are underlined): DIVLTQSHRS MSTSVGDRVN ITC KASQDVT TAVA WYQQKP GQSPKLLIF W ASTRHA GVPD RFTGSGSGTD FTLTISSVQA GDLALYYC QQ HYSTPYT FGG GTKLEIK

[0304] The present application specifically relates to EphA2 binding molecules (e.g., EphA2xCD3 bispecific molecules) capable of binding to EphA2, particularly the VL and / or VH domains, and / or CDRs of the VL domains, of anti-EphA2 monoclonal antibodies EphA2 mAb 1, EphA2 mAb 2, and EphA2 mAb 3. L one, two or all three of the following: and / or CDRs of the VH domain H including one, two, or all three of the following: It includes and encompasses binding molecules.

[0305] 5. Exemplary Anti-gpA33 Antibodies The 43 kD transmembrane glycoprotein A33 (gpA33) is expressed in >95% of all colorectal cancers (Heath, JK et al. (1997) "The Human A33 Antigen Is a Transmembrane Glycoprotein and a Novel Member of the Immunoglobulin Superfamily," Proc. Natl. Acad. Sci. (USA) 94(2):469-474; Ritter, G. et al. (1997) “Characterization Of Posttranslational Modifications Of HumanA33Antigen, A Novel Palmitoylated Surface Glycoprotein Of HumanGastrointestinalEpithelium,” Biochem. Biophys. Res. Commun. 236(3):682-686; Wong, NA et al. (2006) “EpCAM and gpA33 Are Markers of Barrett's Metaplasia,” J. Clin. Pathol. 59(3):260-263). An exemplary antibody that binds is "gpA33 mAb 1."

[0306] The amino acid sequence of the VH domain of gpA33 mAb 1 (SEQ ID NO: 146) is shown below (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT GSWMN WVRQA PGQGLEWIG R IYPGDGETNY NGKFKD RVTI TADKSTSTAY MELSSLRSED TAVYYCAR IY GNNVYFDV WG QGTTVTVSS

[0307] The amino acid sequence of the VL domain of gpA33 mAb 1 (SEQ ID NO: 147) is shown below (CDR L Residues are underlined): DIQLTQSPSF LSASVGDRVT ITC SARSSIS FMY WYQQKPG KAPKLLIY DT SNLAS GVPSR FSGSGSGTEF TLTISSLEAE DAATYYC QQW SSYPLT FGQG TKLEIK

[0308] The present application specifically relates to gpA33 binding molecules (e.g., gpA33xCD3 bispecific binding molecules) capable of binding to gpA33, in particular the VL and / or VH domains, and / or CDRs of the VL domain, of the anti-gpA33 monoclonal antibody gpA33 mAb 1, or any of the anti-gpA33 monoclonal antibodies provided in WO 2015 / 026894. L and / or C of the VH domain. DR H and the like. The present invention further includes and encompasses the exemplary gpA33xCD3 bispecific binding molecules provided in WO 2015 / 026894.

[0309] 6. Exemplary Anti-HER2 / neu Antibodies HER2 / neu is a 185 kDa receptor originally identified as a transforming gene product from neuroblastomas in chemically treated rats. HER2 / neu has been extensively studied because of its function in multiple human carcinomas (including breast and gastric cancer) and in mammalian development (Hynes et al. (1994) Biochim. Biophys. Acta 1198:165-184; Dougallet al. (1994) Oncogene 9:2109-2123; Lee et al. (1995) Nature 378:394-398). Human HER2 / neu Exemplary antibodies that bind to HER2 / neu include "margetuximab," "trastuzumab," and "pertuzumab." Margetuximab (also known as MGAH22; CAS Registry Number: 1350624-75-7) is an Fc-optimized monoclonal antibody that binds to HER2 / neu and mediates enhanced ADCC activity. Trastuzumab (also known as rhuMAB4D5, commercially available as Herceptin®; CAS Registry Number: 180288-69-1; see U.S. Patent No. 5,821,337) is a humanized version of the antibody 4D5, with an IgG1 / κ constant region. Pertuzumab (also known as rhuMAB2C4, commercially available as Perjeta™; CAS Registry Number: 380610-27-5; see, for example, WO 2001 / 000245) is a humanized version of the antibody 2C4, with an IgG1 / κ constant region.

[0310] The present application relates to Her2 / Neu binding molecules (e.g., Her2 / Neu x CD3 bispecific binding molecules) capable of binding to Her2 / Neu, in particular the VL and / or VH domains, and / or CDRs of the VL domains, of the anti-Her2 / Neu monoclonal antibodies margetuximab, trastuzumab, or pertuzumab. L one, two or all three of the following and / or is the CDR of the VH domain H Such combinations include one, two, or all three of It includes and encompasses molecules.

[0311] The amino acid sequence of the VH domain of margetuximab is SEQ ID NO: 148 (CDR H Residues are underlined): QVQLQQSGPE LVKPGASLKL SCTASGFNIK DTYIH WVKQR PEQGLEWIG R IYPTNGYTRY DPKFQD KATI TADTSSNTAY LQVSRLTSED TAVYYCSR WG GDGFYAMDY W GQGASVTVSS

[0312] The amino acid sequence of the VL domain of margetuximab is SEQ ID NO: 149 (CDR L Residues are underlined): DIVMTQSHKF MSTSVGDRVS ITC KASQDVN TAVA WYQQKP GHSPKLLIY S ASFRYT GVPD RFTGSRSGTD FTFTISSVQA EDLAVYYC QQ HYTTPPT FGG GTKVEIK

[0313] The complete heavy and light chain amino acid sequences of margetuximab are known in the art (see, e.g., WHO Drug Information, 2014, Recommended INN: List 71, 28(1):93-94). reference).

[0314] The amino acid sequence of the VH domain of trastuzumab is SEQ ID NO: 150 (CDR H Residue Groups are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIH WVRQA PGKGLEWVA R IYPTNGYTRY ADSVKG RFTI SADTSKNTAY LQMNSLRAED TAVYYCSR WG GDGFYAMDY W GQGTLVTVSS

[0315] The amino acid sequence of the VL domain of trastuzumab is SEQ ID NO: 151 (CDR L Residue Groups are underlined): DIQMTQSPSS LSASVGDRVT ITC RASQDVN TAVA WYQQKP GKAPKLLIY S ASFLY SGVPS RFSGSRSGTD FTLTISSLQP EDFATYYC QQ HYTTPPT FGQ GTKVEIK

[0316] The amino acid sequence of the VH domain of Pertuzumab is SEQ ID NO: 152 (CDR H residue are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMD WVRQA PGKGLEWVA D VNPNSGGSIY NQRFKG RFTL SVDRSKNTLY LQMNSLRAED TAVYYCAR NL GPSFYFDY WG QGTLVTVSS

[0317] The amino acid sequence of the VL domain of Pertuzumab is SEQ ID NO: 153 (CDR L residue are underlined): DIQMTQSPSS LSASVGDRVT ITC KASQDVS IGVA WYQQKP GKAPKLLIY S ASYRYT GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ YYIYPYT FGQ GTKVEIK

[0318] In addition to the preferred anti-HER2 / neu binding molecules identified above, the present invention also provides the following anti-Her-2 / neu binding molecules: 1.44.1; 1.140; 1.43; 1.14.1; 1.100.1; 1.96; 1.18.1; 1.20; 1.39; 1.24; and 1.71.3 (U.S. Pat. No. 8,350,011; U.S. Pat. No. 8,858,942; and International Publication No. WO 2004 / 023994). 2008 / 019290); F5 and C1 (U.S. Patent Nos. 7,892,554; 8,173,424; 8,974,792; and WO 99 / 55367); and the anti-Her-2 binding moieties of U.S. Patent Application Publication Nos. 2013017114 and WO 2011 / 147986 and WO 2012 / 143524. The VL and / or VH domains and / or CDRs of the VL domains of any of the children L and / or the CDRs of the VH domain. H The present invention further contemplates HER2 / Neu binding molecules that comprise one, two, or all three of the following: The present invention further includes and encompasses the exemplary HER2 / NeuxCD3 bispecific binding molecules provided in WO 2012 / 143524.

[0319] 7. Exemplary Anti-VEGF Antibodies VEGF-A is a chemical signal that stimulates angiogenesis in a variety of diseases, particularly in certain metastatic cancers such as metastatic colorectal cancer, as well as certain lung, kidney, and ovarian cancers and glioblastoma multiforme of the brain. An exemplary antibody that binds to human VEGF-A is "bevacizumab" (Avastin®). Bevacizumab is a recombinant humanized IgG1 monoclonal antibody (Midgley, R. et al. (2005) "Bevacizumab - Current Status And Future Directions," Ann. Oncol. 16(7):999-1004; Hall, RD et al. (2015) "Angiogenesis Inhibition As A Therapeutic Strategy In Non-Small Cell Lung Cancer (NSCLC)," Transl. Lung Cancer Res. 4(5):515-523; Narita, Y. (2015) "Bevacizumab For Glioblastoma, ” Ther. Clin. Risk Manag. 11:1759-1765).

[0320] The amino acid sequence of the VH domain of bevacizumab (SEQ ID NO: 154) is shown below (CDR H Residues are underlined): EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMN WVRQA PGKGLEWVG W INTYTGEPTY AADFKR RFTF SLDTSKSTAY LQMNSLRAED TAVYYCA KYP HYYGSSHWYF DV WGQGTLVT VSS

[0321] The amino acid sequence of the VL domain of bevacizumab (SEQ ID NO: 155) is shown below (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC SASQDIS NYLN WYQQKP GKAPKVLIYF TSSLHS GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ YSTVPWT FGQ GTKVEIKR

[0322] The present application specifically relates to VEGF binding molecules (e.g., VEGFxCD3 bispecific binding molecules) capable of binding to VEGF, in particular the VL and / or VH domains, and / or CDRs of the VL domain, of the anti-VEGF monoclonal antibody bevacizumab. L One or two of or all three and / or the CDRs of the VH domain H One, two or all three of The term "conjugate" includes, includes, and encompasses such binding molecules.

[0323] 8. Exemplary Anti-5T4 Antibodies The oncofetal protein 5T4 is a tumor-associated protein expressed on the cell membrane of many carcinomas, including kidney, colon, prostate, and lung cancer, and in acute lymphoblastic leukemia (Boghaert, ER et al. (2008) "The Oncofetal Protein, 5T4, Is A Suitable Target For Antibody-Guided Anti-Cancer ChemotherapyWith Calicheamicin,” Int. J. Oncol. 32(1):221-234; Eisen, T. et al. (2014) “Naptumomab Estafenatox: TargetedImmunotherapy with a NovelImmunotoxin,” Curr. Oncol.Rep. 16:370, pp. 1-6). Exemplary antibodies that bind to human 5T4 include "5T4 mAb 1" and "5T4 mAb 2."

[0324] The amino acid sequence of the VH Domain of 5T4 mAb 1 (SEQ ID NO: 156) is shown below (CDR residues are underlined): QVQLVQSGAE VKKPGASVKV SCKAS GYTFT SFWMH WVRQA PGQGLEWMG R IDPNRGGTEY NEKAKS RVTM TADKSTSTAY MELSSLRSED TAVYYCAG GN PYYPMDY WGQ GTTVTVSS

[0325] The amino acid sequence of the VL Domain of 5T4 mAb 1 (SEQ ID NO:157) is shown below (CDR residues are underlined): DIQMTQSPSS LSASVGDRVT ITC RASQGIS NYLA WFQQKP GKAPKSLIY R ANRLQS GVPS RFSGSGSGTD FTLTISSLQP EDVATYYC LQ YDDFPWT FGQ GTKLEIK

[0326] The amino acid sequence of the VH Domain of 5T4 mAb 2 (SEQ ID NO: 158) is shown below (CDR residues are underlined): QVQLQQPGAE LVKPGASVKM SCKAS GYTFT SYWIT WVKQR PGQGLEWIG D IYPGSGRANY NEKFKS KATL TVDTSSSTAY MQLSSLTSED SAVYNCAR YG PLFTTVVDPN SYAMDY WGQG TSVTVSS

[0327] The amino acid sequence of the VL domain of 5T4 mAb 2 (SEQ ID NO: 159) is shown below (CDR residues are underlined): DVLMTQTPLS LPVSLGDQAS ISC RSSQSIV YSNGNTYLE W YLQKPGQSPK LLIY KVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYC FQGSHVP FT FGSGTKLE IK

[0328] The present application specifically relates to an anti-5T4 monoclonal antibody, 5T4 mAb, capable of binding to 5T4. the VL and / or VH domains, and / or CDRs of the VL domains of 5T4 mAb 1 or 5T4 mAb 2, or of any of the anti-5T4 antibodies provided in WO 2013 / 041687 or WO 2015 / 184203; L One of the Two or all three and / or CDRs of the VH domain H One, two or three of The present invention further includes and encompasses exemplary 5T4xCD3 bispecific binding molecules provided in WO 2015 / 184203.

[0329] Further, the present application specifically relates to 5T4xCD3xCD8 trispecific binding molecules capable of binding to 5T4, CD3, and CD8, and in particular to the VL and / or VH domains, and / or CDRs of the VL domains, of the anti-5T4 monoclonal antibodies 5T4 mAb 1 or 5T4 mAb 2, or of any of the anti-5T4 monoclonal antibodies provided herein. L and / or the CDRs of the VH domain. Hand / or the VL and / or VH domains, and / or the CDRs of the VL domains, of any of the anti-CD8 monoclonal antibodies provided in WO 2015 / 184203. L One, two or all three of and / or CDRs of the VH domain H Including one, two or all three of the above This includes and encompasses trispecific binding molecules.

[0330] 9. Exemplary Anti-IL-13Rα2 Antibodies Interleukin-13 receptor α2 (IL-13Rα2) is overexpressed in a variety of cancers, including glioblastoma, colorectal cancer, cervical cancer, pancreatic cancer, multiple melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer, and lung cancer (WO 2008 / 146911; Brown, C.E. et al. (2013) "Glioma IL13Rα2 Is Associated With Mesenchymal Signature Gene Expression And Poor Patient Prognosis," PLoS One.18;8(10):e77769; Barderas, R. et al. (2012) "High Expression of IL-13Receptor α2 In Colorectal Cancer Is Associated With Invasion, Liver Metastasis, and Poor Prognosis," Cancer Res. 72(11):2780-2790; Kasaian, M.T. et al. (2011) “IL-13Antibodies Influence IL-13 Clearance In Humans By ModulatingScavenger ActivityOf IL-13Rα2,” J. Immunol.187(1):561-569; Bozinov, O. et al. (2010) “DecreasingExpressionOf The Interleuki Interleukin-13 Receptor IL-13Ralpha2 in Treated Recurrent Malignant Gliomas,” Neurol. Med. Chir. (Tokyo) 50(8):617-621; Fujisawa, T. et al.(2009)“A Novel Role of Interleukin-13 Receptor Alpha2 in Pancreatic Cancer Invasion and Metastasis,” Cancer Res.69(22):8678-8685). Antibodies that immunospecifically bind to IL-13Rα2 are commercially available. and have been previously described in the art (Abnova Corporation, Biorbyt, LifeSpan BioSciences, United States Biologicals; see also WO 2008 / 146911). Exemplary antibodies that bind to human IL-13Rα2 include "hu08." (See, for example, WO 2014 / 072888).

[0331] The amino acid sequence of the VH domain of hu08 (SEQ ID NO: 160) is shown below (CDR residues are underlined): EVQLVESGGG LVQPGGSLRL SCAAS GFTFS RNGMS WVRQA PGKGLEWVA T VSSGGSYIYY ADSVKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR QG TTALATRFFD V WGQGTLVTV SS

[0332] The amino acid sequence of the VL domain of hu08 (SEQ ID NO: 161) is shown below (CDR residues are underlined): DIQMTQSPSS LSASVGDRVT ITC KASQDVG TAVA WYQQKP GKAPKLLIY S ASYRSTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QH HYSAPWT FGG GTKVEIK

[0333] The present application specifically relates to IL13Rα2 binding molecules (e.g., IL13Rα2×CD3 bispecific binding molecules) capable of binding to IL13Rα2, in particular, the VL and / or VH domains, and / or CDRs of the VL domain, of the anti-IL13Rα2 monoclonal antibody hu08. L and / or the CDRs of the VH domain. H One of the It includes and encompasses CD16xIL13Rα2 binding molecules that contain two or all three.

[0334] 10. Exemplary Anti-CD123 Antibodies CD123 (interleukin-3 receptor alpha, IL-3Ra) is a 40 kDa molecule and is part of the interleukin-3 receptor complex (Stomski, FC et al. (1996) "Human Interleukin-3 (IL-3) Induces Disulfide-Linked IL-3 Receptor Alpha- and Beta-Chain Heterodimerization, Which Is Required For Receptor Activation But Not High-Affinity Binding," Mol. Cell. Biol. 16(6):3035-3046). CD123 promotes the early differentiation of pluripotent stem cells into erythroid, myeloid, and lymphoid progenitor cells. CD123 has been reported to be overexpressed on malignant cells in a wide range of hematological malignancies, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute B-lymphoblastic leukemia (B-ALL), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), acute B-lymphoblastic leukemia (B-ALL), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), and myelodysplastic syndromes (MDS) (Munoz, L. et al. (2001) "Interleukin-3 Receptor Alpha Chain (CD123) Is Widely Expressed in Hematologic Malignancies,” Haematologica 86(12):1261-1269). Overexpression of CD123 is associated with poor prognosis in AML (Tettamanti, MS et al. (2013) “Targeting of Acute Myeloid Leukemia by Cytokine-Induced Killer Cells Redirected with a Novel CD123-Specific Chimeric Antigen Receptor,” Br. J. Haematol. 161:389-401).

[0335] An exemplary antibody that binds to human CD123 and can be employed in the present invention is "CD123 mAb 1" (see, e.g., WO 2015 / 026892).

[0336] The amino acid sequence of the VH domain of CD123 mAb 1 (SEQ ID NO: 162) is shown below (CDR H Residues are underlined): EVQLVQSGAE LKKPGASVKV SCKASGYTFT DYYMK WVRQA PGQGLEWIG D IIPSNGATFY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCAR SH LLRASWFAY W GQGTLVTVSS

[0337] The amino acid sequence of the VL domain of CD123 mAb 1 (SEQ ID NO: 163) is shown below (CDR L Residues are underlined): DFVMTQSPDS LAVSLGERVT MSC KSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIY WASTR ES GVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYC QNDYSY PYT FGQGTKL EIK

[0338] The present application specifically relates to CD123 binding molecules (e.g., CD123xCD3 bispecific binding molecules) capable of binding to CD123, in particular the VL and / or VH domains, and / or CDRs of the VL domains, of the anti-CD123 monoclonal antibody CD123 mAb 1, or any of the anti-CD123 antibodies disclosed in U.S. Patent No. 2017 / 081424 and WO 2016 / 036937. L one, two or all three of the following: and / or CDRs of the VH domain H including one, two, or all three of the following: The present invention further includes and encompasses exemplary CD123xCD3 bispecific binding molecules, including flotetuzumab (known as MGD007; CAS Registry Number 1664355-28-5), JNJ-63709178 (Johnson & Johnson, see also WO 2016 / 036937), and XmAb14045 (Xencor, see also U.S. Patent No. 2017 / 081424).

[0339] 11. Exemplary Anti-CD19 Antibodies CD19 (B-lymphocyte surface antigen B4, GenBank accession number: M28170) is a component of the B-cell receptor (BCR) complex and a positive regulator of B-cell signaling, modulating the threshold for B-cell activation and humoral immunity. CD19 is one of the most ubiquitously expressed antigens in the B-cell lineage and is expressed in >95% of B-cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL). Notably, CD19 expression persists in B-cell lymphomas that have become resistant to anti-CD20 therapy (Davis et al. (1999) "Therapy of B-cell Lymphoma With Anti-CD20 Antibodies Can Result in the Loss of CD20 Antigen Expression." Clin Cancer Res, 5:611-615, 1999). CD19 has also been suggested as a target for treating autoimmune diseases (Tedder (2009) "CD19: A Promising Cell Target For Rheumatoid Arthritis," Nat. Rev. Rheumatol. 5:572-577).

[0340] An exemplary humanized antibody that binds to human CD19 and may be employed in the present invention is the anti-CD19 antibody disclosed in WO 2016 / 048938 (referred to herein as "CD19 (referred to as "mAb 1").

[0341] The amino acid sequence of the VH domain of CD19 mAb 1 (SEQ ID NO: 164) is shown below (CDR H Residues are underlined): QVTLRESGPA LVKPTQTLTL TCTFSGFSLS TSGMGVG WIR QPPGKALEWL A HIWWDDDKR YNPALKS RLT ISKDTSKNQV FLTMTNMDPV DTATYYCAR M ELWSYYFDYW GQGTTVTVSS

[0342] The amino acid sequence of the VL domain of CD19 mAb 1 (SEQ ID NO: 165) is shown below (CDR L Residues are underlined): ENVLTQSPAT LSVTPGEKAT ITC RASQSVS YMH WYQQKPG QAPRLLIY DA SNRAS GVPSR FSGSGSGTDH TLTISSLEAE DAATYYC FQG SVYPF TFGQG TKLEIK

[0343] The amino acid sequence of an alternative VL domain of CD19 mAb 1 (SEQ ID NO: 195) is shown below (CDR L Residues are underlined): ENVLTQSPAT LSVTPGEKVT ITC SASSSVS YMH WYQQKPG QAPRLLIY DT SKLAS GVPSR FSGSGSGTDH FLTISSLEAE DAATYYC FQG SVYPFT FGQG TKLEIK

[0344] The present invention specifically relates to CD19 binding molecules (e.g., CD19xCD3 bispecific binding molecules) capable of binding to CD19, in particular the VL and / or VH domains, and / or CDRs of the VL region, of the anti-CD19 monoclonal antibody CD19 mAb 1, or any of the anti-CD19 antibodies disclosed in U.S. Pat. No. 7,112,324. L One of the two one or all three and / or the CDRs of the VH domain H One, two or three of The present invention specifically includes and encompasses such binding molecules, including blinatumomab (BLINCYTO®; amino acid sequence found in WHO Drug Information, 2009, Recommended INN: List 62, 23(3):240-241), and zuvortuximab. Exemplary CD19x antibodies that can be employed in the present invention include CD19x (known as MGD011, amino acid sequence found in WHO Drug Information, 2016, Proposed INN:List 116, 30(4):627-629). This includes and encompasses CD3 bispecific binding molecules.

[0345] B. Exemplary Pathogen-Associated Antigens As used herein, the term "Pathogen Antigen" refers to an antigen that is characteristically expressed on the surface of a pathogen-infected cell and therefore can be treated with antibody-based or immunomodulatory molecules. Examples of pathogen antigens include, but are not limited to: herpes simplex virus (e.g., infected cell protein (ICP) 47, gD, etc.); varicella-zoster virus; Kaposi's sarcoma-associated herpesvirus; Epstein-Barr virus (e.g., LMP-1, LMP-2A, LMP-2B, etc.); cytomegalovirus (e.g., UL11, etc.); human immunodeficiency virus (e.g., env proteins gp160, gp120, gp41, etc.); human papillomavirus (e.g., E6, E7, etc.); human T-cell leukemia virus (e.g., env proteins gp64, gp46, gp21, etc.); hepatitis A virus; hepatitis B virus; hepatitis C virus; vesicular stomatitis virus (VSV); bacillus; Citrobacter; cholera; diphtheria; Enterobacter; Neisseria gonorrhoeae; Helicobacter pylori; Klebsiella; Legionella; meningococcus; mycobacteria. ;Pseudomonas;Streptococcus pneumoniae;Rickettsia;Salmonella;Serratia;Staphylococcus;Streptococcus;Tetanus;Aspergillus (Fumigatus, Aspergillus niger, etc.);Blastomyces dermatitidis;Candida (Albicans, Krusei, Glabrata, Tropicalis, etc.);Cryptococcus neoformans;Mucorae (Mucora mucor, Rhizopus, Rhizopus);Sporothrix schenckii;Paracoccus Antibodies include antigens expressed on the surface of cells infected with Dioides brasiliensis; Coccidioides immucis; Histoplasma capsulatum; Leptospirosis; Borrelia burgdorferi; helminthic parasites (hookworms, cestodes, trematodes, flatworms (e.g., Schistosoma)); Giardia lamblia; Trichinella spp.; Dientamoeba; Trypanosoma brucei; Trypanosoma cruzi; and Leishmania donovani. Such antibodies are commercially available from a number of sources or can be obtained by immunizing mice or other animals (including for the production of monoclonal antibodies) with the antigens.

[0346] Exemplary antibodies are provided below in which the VH and VL domains may be used to construct molecules capable of binding to pathogen antigens arrayed on the surface of pathogen-infected cells. Additional antibodies are known in the art.

[0347] 1. Exemplary Anti-HIV-env Antibodies The env protein of HIV is an exemplary pathogen-associated antigen, and an antibody that binds to the env protein of HIV is an exemplary antibody that can bind to a pathogen-associated antigen.

[0348] The first step in HIV-1 infection occurs through the binding of cell surface CD4 to a heterodimer of the trimeric HIV-1 envelope glycoprotein (env), transmembrane glycoprotein (gp41), and surface glycoprotein (gp120). The gp120 and gp41 glycoproteins are initially synthesized as a single gp160 polypeptide, which is subsequently cleaved to generate a noncovalent gp120 / gp41 complex. The env ectodomain is a heterodimer of approximately 140 kDa, consisting of the entire gp120 component and approximately 20 kDa of gp41 (Harris, A. et al. (2011) "Trimeric HIV-1 Glycoprotein Gp140 Immunogens And Native HIV-1 Envelope Glycoproteins Display The Same Closed And Open Quaternary Molecular Architectures," Proc. Natl. Acad. Sci. (USA) 108(28):11440-11445). Antibodies that immunospecifically bind to the env protein are commercially available and have been previously described in the art (e.g., GenBank Accession No. AFQ31503; Buchacher, A. et al. (1994) "Generation Of Human Monoclonal Antibodies Against HIV-1 Proteins; Electrofusion AndEpstein-Barr Virus Transformation For Peripheral Blood Lymphocyte Immortalization,” AIDS Res. Hum. Retroviruses 10(4):359-369; Shen, R. (2010) “GP41-Specific Antibody Blocks Cell-Free HIV Type 1 Transcytosis Through Human Rectal Mucosa And Model Colonic Epithelium,” J. Immunol. 184(7):3648-3655; WO 2012 / 162068; and WO 2016 / 054101 (See U.S. Patent Publication No. WO 2014 / 159940). Exemplary antibodies that bind to HIV env include "7B2" (GenBank Accession No. AFQ31503) and "A32" (WO 2014 / 159940). Multiple VH domains of antibody A32 with minor changes in framework regions 1 and / or 4 have been reported in the art (see, e.g., Protein Database Accession No. PDB:4YBL_H, U.S. Patent Publication No. 2015 / 0239961, and WO 2006 / 044410). Any of these mutant antibody A32 VH domains may be used in accordance with the present invention.

[0349] The amino acid sequence of the VH domain of 7B2 (SEQ ID NO: 166) is shown below (CDR residues are underlined): QVQLVQSGGG VFKPGGSLRL SCEASGFTFT EYYMT WVRQA PGKGLEWLAY ISKNGEYSKY SPSSNG RFTI SRDNAKNSVF LQLDRLSADD TAVYYCAR AD GLTYFSELLQ YIFDL WGQGA RVTVSS

[0350] The amino acid sequence of the VL domain of 7B2 (SEQ ID NO: 167) is shown below (CDR residues are underlined): DIVMTQSPDS LAVSPGERAT IHCK SSQTLL YSSNNRHSIA WYQQRPGQPP KLLLY WASMR LS GVPDRFSG SGSGTDFTLT INNLQAEDVA IYYC HQYSSH PPT FGHGTRV EIK

[0351] The amino acid sequence of an exemplary VH domain of A32 (SEQ ID NO: 168) is shown below (CDR residues are underlined): QVQLQESGPG LVKPSQTLSL SCTVSGGSSS SGAHYWS WIR QYPGKGLEWI G YIHYSGNTY YNPSLKS RIT ISQHTSENQF SLKLNSVTVA DTAVYYCAR G TRLRTLRNAF DI WGQGTXVT VSS Here, X is L or M.

[0352] The amino acid sequence of such an exemplary VH domain of A32, where X is L (SEQ ID NO:2) 09) is shown below (CDR residues are underlined): QVQLQESGPG LVKPSQTLSL SCTVSGGSSS SGAHYWS WIR QYPGKGLEWI G YIHYSGNTY YNPSLKS RIT ISQHTSENQF SLKLNSVTVA DTAVYYCAR G TRLRTLRNAF DI WGQGTLVT VSS

[0353] The amino acid sequence of the VL domain of A32 (SEQ ID NO: 169) is shown below (CDR residues are underlined): QSALTQPPSA SGSPGQSVTI SC TGTSSDVG GYNYVS WYQH HPGKAPKLII S EVNNRPSGV PDRFSGSKSG NTASLTVSGL QAEDEAEYYC SSYTDIHNFV FGGGTKLTVL

[0354] The present application specifically relates to HIV-binding molecules (e.g., HIVxCD3 bispecific binding molecules) capable of binding to HIV, and in particular to the VL and / or VH domains, and / or CDRs of the VL domains, of the anti-HIV monoclonal antibodies 7B2, A32, and any of the anti-HIV antibodies disclosed in WO 2016 / 054101, WO 2017 / 011413, and WO 2017 / 011414. L One, two or All three and / or the CDRs of the VH domain H Contains one, two or all three of the following: The present invention specifically includes and encompasses the exemplary HIVxCD3 bispecific binding molecules provided in WO 2014 / 159940, WO 2015 / 184203, WO 2017 / 011413, and WO 2017 / 011414.

[0355] The present invention further specifically relates to HIVxCD3xCD8 trispecific binding molecules capable of binding to HIV, CD3 and CD8, and in particular to the VL and / or VH domains, and / or CDRs of the VL domains, of the anti-HIV monoclonal antibodies 7B2 or A32 or any of the anti-HIV antibodies disclosed in WO 2015 / 184203, WO 2016 / 054101, WO 2017 / 011413, and WO 2017 / 011414. L and / or C of the VH domain. DR H and / or WO 2015 / 1 The VL and / or VH domains, and / or CDRs of the VL domains, of any of the anti-CD8 monoclonal antibodies provided in US Pat. No. 84203. LOne of the two one or all three and / or the CDRs of the VH domain H One, two or three of The invention includes and encompasses all of the above trispecific binding molecules.

[0356] 2. Exemplary Anti-RSV Glycoprotein F Antibodies A further exemplary pathogen-associated antigen is RSV glycoprotein F. An exemplary anti-RSV glycoprotein F antibody is palivizumab (see, e.g., Protein Data Bank (PDB) ID No. 2HWZ). Another anti-RSV glycoprotein F antibody is motavizumab (see, e.g., PDB ID No. 3IXT), and the CDR of palivizumab. L 1 to shi A variant of palivizumab engineered to remove stearate residues is included. The amino acid sequence of the VH domain of the variant of palivizumab (SEQ ID NO: 170) is shown below (CDR residues are underlined): QVTLRESGPA LVKPTQTLTL TCTFSGFSLS TSGMSVG WIR QPPGKALEWL A DIWWDDKKD YNPSLKS RLT ISKDTSKNQV VLKVTNMDPA DTATYYCAR S MITNWYFDV W GAGTTVTVSS

[0357] The amino acid sequence of the VL domain of the variant of Palivizumab (SEQ ID NO: 171) is shown below (CDR residues are underlined): DIQMTQSPST LSASVGDRVT ITC RASQSVG YMH WYQQKPG KAPKLLIY DT SKLAS GVPSR FSGSGSGTEF TLTISSLQPD DFATYYC FQG SGYPFT FGGG TKLEIK

[0358] VII. Exemplary Binding Molecules of the Invention As described below, the invention is exemplified using several DAxCD3 binding moieties with different structures, including molecules capable of mediating targeted killing of tumor cells (e.g., "DART-A" type diabodies or "DART-B" type diabodies or trivalent molecules described below).

[0359] A. DART-A type diabody DART-type A diabodies are bispecific diabodies that do not contain an Fc domain and can bind to CD3 and a disease antigen (e.g., a cancer antigen). Provided herein is an exemplary DART-type A diabody consisting of two polypeptide chains with one binding site for CD3 and one binding site for the cancer antigen CD123 (see, e.g., Figure 1).

[0360] An exemplary DART-A type diabody (referred to as "DART-A-WT") is SEQ ID NO: 172: JPEG2025186382000067.jpg35150 The polypeptide has a first polypeptide chain having an amino acid sequence of

[0361] Residues 1-113 of the first polypeptide chain of such exemplary DART-A type diabodies correspond to the VL domain of CD123 mAb 1 (SEQ ID NO: 162). Residues 114-121 (double underlined) of the first polypeptide chain of such exemplary DART-A type diabodies correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain of such exemplary DART-A type diabodies correspond to the VH domain of CD3 mAb 1 (SEQ ID NO: 55), where Kabat position 65 (double underlined) is aspartic acid (D). Residues 247-252 (underlined) of the first polypeptide chain of such exemplary DART-A type diabodies correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 253-280 of the first polypeptide chain of such exemplary DART-A type diabodies correspond to the heterodimer-promoting "K-coil" ( K VAALK E- K VAAL K E- K VAAL K E- K VAAL K E; SEQ ID NO: 30).

[0362] The second polypeptide chain of such an exemplary DART-A type diabody DART-A-WT is set forth in SEQ ID NO: 173: JPEG2025186382000068.jpg35149 It has the amino acid sequence:

[0363] Residues 1-110 of the second polypeptide chain of such exemplary DART-A type diabodies, DART-A-WT, correspond to the VL domain of CD3 mAb 1 (SEQ ID NO: 56). Residues 111-118 (double underlined) of the second polypeptide chain of such exemplary DART-A type diabodies correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 119-238 of the second polypeptide chain of such exemplary DART-A type diabodies correspond to the VH domain of CD123 mAb 1 (SEQ ID NO: 163). Residues 239-244 (underlined) of the second polypeptide chain of such exemplary DART-A type diabodies correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 245-272 of the second polypeptide chain of such exemplary DART-A type diabodies comprise a heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29).

[0364] As will be appreciated in light of the present disclosure, additional DART-A type diabodies can be similarly constructed (by substituting the VL and VH domains of such antibodies for the VL and VH domains of the exemplary DART-A type diabodies) that have binding sites for other disease antigens and / or have the CD3 binding domain of a variant anti-CD3 antibody (i.e., a vCD3 binding domain). Similarly, other DART-A type molecules incorporating alternative linkers and / or alternative Heterodimer-Promoting Domains, as provided herein, can be similarly constructed. For example, an exemplary panel of CD123xCD3 DART-A type diabodies was generated that has the same structure as the DART-A-WT diabody provided above, but includes the VL and VH domains of one of the CD3 mAb 1 variants (M1-M26) provided above.

[0365] The exemplary CD123xCD3 DART-A type diabodies in the above panel each have the sequence SEQ ID NO: 189: JPEG2025186382000069.jpg41152 wherein: X1 is T, D, or E; X2 is Y, D, or T; X3 is A or G; X4 is D or G; and X5 is G , D, E, or K; X6 is F or I; X7 is G or I; X8 is Y, A, G or Q; X9 is S or T; X 10 is W, F, or Y; X 11 is Y or It's E.

[0366] Residues 1-113 of the first polypeptide chain of this panel of exemplary DART-A type diabodies correspond to the VL Domain of CD123 mAb 1 (SEQ ID NO: 162). Residues 114-121 (double underlined) of the first polypeptide chain of this panel of exemplary DART-A type diabodies correspond to Linker 1 (GGGSGGGG; SEQ ID NO: 16; double underlined). Residues 122-246 of the first polypeptide chain of this panel of exemplary DART-A type diabodies correspond to the VH Domain of CD3 mAb 1 M1 through CD3 mAb 1 M22 (SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, or 106). Residues 247-252 (underlined) in the above panel of exemplary DART-A type diabodies correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 253-280 of the first polypeptide chain in the above panel of Di are heterodimer-promoting "K coil" ( K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E; SEQ ID NO: 30).

[0367] The second polypeptide chain of such an exemplary DART-A type diabody is set forth in SEQ ID NO: 190: JPEG2025186382000070.jpg42145 wherein: X1 is G or D; X2 is K or G; and X3 is L. , E or Q.

[0368] Residues 1-110 of the second polypeptide chain of this panel of exemplary DART-A type diabodies correspond to the VL domain of CD3 mAb 1 M23 through CD3 mAb 1 M26 (SEQ ID NOs: 108, 110, 112, and 114). Residues 111-118 (double underlined) of the second polypeptide chain of this panel of exemplary DART-A type diabodies correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16; double underlined). Residues 119-238 of the second polypeptide chain of this panel of exemplary DART-A type diabodies correspond to the VH domain of CD123 mAb 1 (SEQ ID NO: 163). Residues 239-244 (underlined) of the second polypeptide chain of this panel of exemplary DART-A type diabodies correspond to linker 2 (GGCGGG; SEQ ID NO: 17; underlined). Residues 245-272 of the second polypeptide chain of the above panel of exemplary DART-A type diabodies are heterodimer-promoting "E coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29).

[0369] The amino acid sequences and designations of a panel of exemplary DART-A type diabodies comprising the VL and VH domains of CD3 mAb 1 variants are shown in Table 8 below.

[0370] [Table 35] JPEG2025186382000072.jpg247155 JPEG2025186382000073.jpg69162

[0371] B.DART-B type diabody DART-B type diabodies are bispecific diabodies that contain an Fc domain and can bind to CD3 and a disease antigen (e.g., a cancer or infectious disease antigen). Provided herein is an exemplary DART-B type diabody that consists of three polypeptide chains and has one binding site for CD3 and one binding site for the cancer antigens CD123, 5T4, or CD19 (see, e.g., Figure 4A).

[0372] [Table 36]

[0373] 1. First Exemplary DART-B Diabody CD123-WT (CD123xCD3 mAb 1) A first exemplary DART-B type diabody (referred to as "CD123-WT") is set forth in SEQ ID NO: 174: JPEG2025186382000075.jpg60153 The polypeptide has a first polypeptide chain having an amino acid sequence of

[0374] Residues 1-113 of the first polypeptide chain of CD123-WT correspond to the VL domain of CD123 mAb 1 (SEQ ID NO: 163). Residues 114-121 (double underlined) of the first polypeptide chain of CD123-WT correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain of CD123-WT correspond to the VH domain of CD3 mAb 1 (SEQ ID NO: 55), with Kabat position 65 (double underlined) being aspartic acid (D). Residues 247-252 (underlined) of the first polypeptide chain of CD123-WT correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 253-280 of the first polypeptide chain of CD123-WT form a heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAALE K; SEQ ID NO: 29). Residues 281-283 of the polypeptide chain of CD123-W correspond to the GGG linker. Residues 284-293 (underlined) of the first polypeptide chain of T correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 294-510 of the first polypeptide chain of CD123-WT correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0375] The second polypeptide chain of CD123-WT is SEQ ID NO: 175: JPEG2025186382000076.jpg36153 It has the amino acid sequence:

[0376] Residues 1-110 of the second polypeptide chain of CD123-WT correspond to the VL domain of CD3 mAb 1 (SEQ ID NO: 56). Residues 111-118 (double underlined) of the second polypeptide chain of CD123-WT correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 119-238 of the second polypeptide chain of CD123-WT correspond to the VH domain of CD123 mAb 1 (SEQ ID NO: 162). Residues 239-244 (underlined) of the second polypeptide chain correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 245-272 of the second polypeptide chain of CD123-WT correspond to the heterodimer-promoting "K-coil" ( K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E; SEQ ID NO: 30).

[0377] The third polypeptide chain of CD123-WT is SEQ ID NO: 176: DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK It has the amino acid sequence:

[0378] Residues 1-10 of the third polypeptide chain of CD123-WT correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 11-227 of the third polypeptide chain of CD123-WT correspond to the IgG1 "hole-bearing" CH2-CH3 domain (SEQ ID NO: 50).

[0379] As will be appreciated, the third polypeptide chain of CD123-WT does not contain any epitope-binding domains and can therefore be adopted by various DAxCD3 binding molecules with such DART-B type structures.

[0380] 2. Second Exemplary DART-B Diabody CD123-M1 (CD123 x CD3 mAb 1 M1) A second exemplary DART-B type diabody is similar to the CD123-WT diabody described above, but contains the VH domain of CD3 mAb 1 M1 and is referred to as "CD123-M1." As noted above, CD3 mAb 1 M1 is a low-affinity variant of CD3 mAb 1 (also referred to as "CD3 mAb 1 Low"). Also as noted above, the VL domain of CD3 mAb 1 M1 has an amino acid sequence identical to that of CD3 mAb 1.

[0381] Thus, a second exemplary DART-B type diabody (CD123-M1) has the following amino acid sequence (SEQ ID NO: 177): JPEG2025186382000077.jpg59153 and a first polypeptide chain having the following structure:

[0382] Residues 1-113 of the first polypeptide chain of CD123-M1 correspond to the VL domain of CD123 mAb 1 (SEQ ID NO: 163). Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M1 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain of CD123-M1 correspond to the VH domain of CD3 mAb 1 M1 (SEQ ID NO: 55), with Kabat position 65 (double underlined) being aspartic acid (D). Residues 247-252 (underlined) of the first polypeptide chain of CD123-M1 correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 253-280 of the first polypeptide chain of CD123-M1 form a heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Residues 281-283 of the first polypeptide chain of CD12 correspond to the GGG linker. Residues 284-293 (underlined) of the first polypeptide chain of CD123-M1 correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 294-510 of the first polypeptide chain of CD123-M1 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0383] Because the VL domain of CD3 mAb 1 M1 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of CD123-M1 is identical to that of the second polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 175). Similarly, the amino acid sequence of the third polypeptide chain of CD123-M1 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0384] 3. Third Exemplary DART-B Diabody CD123-M2 (CD123xCD3 mAb 1 M2) A third exemplary DART-B type diabody is similar to the CD123-M1 diabody described above, but contains the VH domain of CD3 mAb 1 M2 and is referred to as "CD123-M2." As noted above, CD3 mAb 1 M2 has a faster off-rate than CD3 mAb 1 and is therefore also referred to as "CD3 mAb 1 Fast." As also noted above, the VL domain of CD3 mAb 1 M2 has an amino acid sequence identical to that of CD3 mAb 1.

[0385] Thus, a third exemplary DART-B type diabody (CD123-M2) has the following amino acid sequence (SEQ ID NO: 178): JPEG2025186382000078.jpg58148 and a first polypeptide chain having the following structure:

[0386] Residues 1-113 of the first polypeptide chain of CD123-M2 correspond to the VL domain of CD123 mAb 1 (SEQ ID NO: 163). Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M2 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain of CD123-M2 correspond to the VH domain of CD3 mAb 1 M2 (SEQ ID NO: 59), with Kabat position 65 (double underlined) being aspartic acid (D). Residues 247-252 (underlined) of the first polypeptide chain of CD123-M2 correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 253-280 of the first polypeptide chain of CD123-M2 correspond to the heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Residues 281-283 of the first polypeptide chain of CD12 correspond to the GGG linker. Residues 284-293 (underlined) of the first polypeptide chain of CD123-M2 correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 294-510 of the first polypeptide chain of CD123-M2 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48). .

[0387] Because the VL domain of CD3 mAb 1 M2 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of CD123-M2 is identical to that of the second polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 175). Similarly, the amino acid sequence of the third polypeptide chain of CD123-M2 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0388] 4. Fourth Exemplary DART-B Diabody CD123-M18 (CD123xCD3 mAb 1 M18) A fourth exemplary DART-B type diabody is similar to the CD123-M2 diabody described above, but contains the VH domain of CD3 mAb 1 M18, and is referred to as "CD123-M18." As noted above, the VL domain of CD3 mAb 1 M18 has an amino acid sequence identical to that of CD3 mAb 1.

[0389] Thus, a fourth exemplary DART-B type diabody (CD123-M18) has the following amino acid sequence (SEQ ID NO: 179): JPEG2025186382000079.jpg60148 and a first polypeptide chain having the following structure:

[0390] Residues 1 to 113 of the first polypeptide chain of CD123-M18 are the CD123 mAb Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M18 correspond to the VL domain of CD3 mAb 1 M18 (SEQ ID NO: 163). Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M18 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain of CD123-M18 correspond to the VH domain of CD3 mAb 1 M18 (SEQ ID NO: 98), with Kabat position 65 (double underlined) being aspartic acid (D). Residues 247-252 (underlined) of the first polypeptide chain of CD123-M18 correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 253-280 of the first polypeptide chain of CD123-M18 form a heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Residues 281-283 of the first polypeptide chain of D123-M18 correspond to the GGG linker Residues 284-293 (underlined) of the first polypeptide chain of CD123-M18 correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 294-510 of the first polypeptide chain of CD123-M18 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0391] Because the VL domain of CD3 mAb 1 M18 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of CD123-M18 is identical to that of the second polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 175). Similarly, the amino acid sequence of the third polypeptide chain of CD123-M18 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0392] 5. Fifth Exemplary DART-B Diabody CD123-M13 (CD123xCD3 mAb 1 M13) A fifth exemplary DART-B type diabody is similar to the CD123-WT diabody described above, but contains the VH domain of CD3 mAb 1 M13, and is referred to as "CD123-M13." As noted above, the VL domain of CD3 mAb 1 M13 has an amino acid sequence identical to that of CD3 mAb 1.

[0393] Thus, a fifth exemplary DART-B type diabody (CD123-M13) has the following amino acid sequence (SEQ ID NO: 198): JPEG2025186382000080.jpg60148 and a first polypeptide chain having the following structure:

[0394] Residues 1 to 113 of the first polypeptide chain of CD123-M13 are the nucleotides of the CD123 mAb Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M13 correspond to the VL domain of CD3 mAb 1 M13 (SEQ ID NO: 163). Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M13 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain of CD123-M13 correspond to the VH domain of CD3 mAb 1 M13 (SEQ ID NO: 88), with Kabat position 65 (double underlined) being aspartic acid (D). Residues 247-252 (underlined) of the first polypeptide chain of CD123-M13 correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 253-280 of the first polypeptide chain of CD123-M13 correspond to the heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Residues 281-283 of the first polypeptide chain of D123-M13 correspond to the GGG linker Residues 284-293 (underlined) of the first polypeptide chain of CD123-M13 correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 294-510 of the first polypeptide chain of CD123-M13 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0395] Because the VL domain of CD3 mAb 1 M13 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of CD123-M13 is identical to that of the second polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 175). Similarly, the amino acid sequence of the third polypeptide chain of CD123-M13 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0396] 6. Sixth Exemplary DART-B Diabody CD123-M17 (CD123xCD3 mAb 1 M17) A sixth exemplary DART-B type diabody is similar to the CD123-WT diabody described above, but contains the VH domain of CD3 mAb 1 M17, and is referred to as "CD123-M17." As noted above, the VL domain of CD3 mAb 1 M17 has an amino acid sequence identical to that of CD3 mAb 1.

[0397] Thus, a sixth exemplary DART-B type diabody (CD123-M17) has the following amino acid sequence (SEQ ID NO: 199): JPEG2025186382000081.jpg56150 and a first polypeptide chain having the following structure:

[0398] Residues 1 to 113 of the first polypeptide chain of CD123-M17 are the nucleotides of the CD123 mAb Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M17 correspond to the VL domain of CD3 mAb 1 M17 (SEQ ID NO: 163). Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M17 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain of CD123-M17 correspond to the VH domain of CD3 mAb 1 M17 (SEQ ID NO: 96), with Kabat position 65 (double underlined) being aspartic acid (D). Residues 247-252 (underlined) of the first polypeptide chain of CD123-M17 correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 253-280 of the first polypeptide chain of CD123-M17 correspond to the heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Residues 281-283 of the first polypeptide chain of D123-M17 correspond to the GGG linker Residues 284-293 (underlined) of the first polypeptide chain of CD123-M17 correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 294-510 of the first polypeptide chain of CD123-M17 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0399] Because the VL domain of CD3 mAb 1 M17 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of CD123-M17 is identical to that of the second polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 175). Similarly, the amino acid sequence of the third polypeptide chain of CD123-M17 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0400] 7. Seventh Exemplary DART-B Diabody CD123-M19 (CD123xCD3 mAb 1 M19) A seventh exemplary DART-B type diabody is similar to the CD123-WT diabody described above, but contains the VH domain of CD3 mAb 1 M19, and is referred to as "CD123-M19." As noted above, the VL domain of CD3 mAb 1 M19 has an amino acid sequence identical to that of CD3 mAb 1.

[0401] Thus, a seventh exemplary DART-B type diabody (CD123-M19) is Amino acid sequence (SEQ ID NO: 200): JPEG2025186382000082.jpg59150 and a first polypeptide chain having the following structure:

[0402] Residues 1 to 113 of the first polypeptide chain of CD123-M19 are the CD123 mAb Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M19 correspond to the VL domain of CD3 mAb 1 M19 (SEQ ID NO: 163). Residues 114-121 (double underlined) of the first polypeptide chain of CD123-M19 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 122-246 of the first polypeptide chain of CD123-M19 correspond to the VH domain of CD3 mAb 1 M19 (SEQ ID NO: 100), with Kabat position 65 (double underlined) being aspartic acid (D). Residues 247-252 (underlined) of the first polypeptide chain of CD123-M19 correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 253-280 of the first polypeptide chain of CD123-M19 correspond to the heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Residues 281-283 of the first polypeptide chain of CD123-M19 are linked to a GGG linker. Residues 284-293 (underlined) of the first polypeptide chain of CD123-M19 correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 294-510 of the first polypeptide chain of CD123-M19 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0403] Because the VL domain of CD3 mAb 1 M19 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of CD123-M19 is identical to that of the second polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 175). Similarly, the amino acid sequence of the third polypeptide chain of CD123-M19 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0404] 8. Eighth Exemplary DART-B Diabody 5T4-WT (5T4 x CD3 mAb 1) An eighth exemplary DART-B type diabody is similar to the CD123-M18 diabody described above, but contains a 5T4 binding domain in place of the CD123 binding domain of the CD123-M18 diabody. Additionally, this eighth exemplary DART-B type diabody contains the VH domain of CD3 mAb 1. This eighth exemplary DART-B type diabody is referred to as "5T4-WT."

[0405] Thus, an eighth exemplary DART-type B diabody (5T4-WT) has the following amino acid sequence (SEQ ID NO: 180): JPEG2025186382000083.jpg59150 and a first polypeptide chain having the following structure:

[0406] Residues 1-107 of the first polypeptide chain of 5T4-WT correspond to the VL domain of 5T4 mAb 1 (SEQ ID NO: 157). Residues 108-115 (double underlined) of the first polypeptide chain of 5T4-WT correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 116-240 of the first polypeptide chain of 5T4-WT correspond to the VH domain of CD3 mAb 1 (SEQ ID NO: 55), with Kabat position 65 (double underlined) being glycine (G). Residues 241-246 (underlined) of the first polypeptide chain of 5T4-WT correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 247-274 of the first polypeptide chain of 5T4-WT correspond to the heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Residues 278 to 77 of the first polypeptide chain of 5T4-WT correspond to the GGG linker. 287 (underlined) corresponds to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 288-504 of the first polypeptide chain of 5T4-WT correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0407] The second polypeptide chain of 5T4-WT has the following amino acid sequence (SEQ ID NO: 181): JPEG2025186382000084.jpg39150 It has.

[0408] Residues 1-110 of the second polypeptide chain of 5T4-WT correspond to the VL domain of CD3 mAb 1 (SEQ ID NO: 56). Residues 111-118 (double underlined) of the second polypeptide chain of 5T4-WT correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 119-236 of the second polypeptide chain of 5T4-WT correspond to the VH domain of 5T4 mAb 1 (SEQ ID NO: 156). Residues 237-242 (underlined) of the second polypeptide chain of 5T4-WT correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 243-280 of the second polypeptide chain of 5T4-WT correspond to the heterodimer-promoting "K-coil" ( K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E; SEQ ID NO: 30).

[0409] The third polypeptide chain of 5T4-WT has the same amino acid sequence as the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0410] 9. Ninth Exemplary DART-B Diabody 5T4-M1 (5T4xCD3 mAb 1 M1) A ninth exemplary DART-B type diabody is similar to the 5T4-WT diabody described above, but contains the VH domain of CD3 mAb 1 M1 and is referred to as "5T4-M1."

[0411] Thus, a ninth exemplary DART-B type diabody (5T4-M1) has the following amino acid sequence (SEQ ID NO: 182): JPEG2025186382000085.jpg58146 and a first polypeptide chain having the following structure:

[0412] Residues 1-107 of the first polypeptide chain of 5T4-M1 correspond to the VL domain of 5T4 mAb 1 (SEQ ID NO: 157). Residues 108-115 (double underlined) of the first polypeptide chain of 5T4-M1 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 116-240 of the first polypeptide chain of 5T4-M1 correspond to the VH domain of CD3 mAb 1 M1 (SEQ ID NO: 64), with Kabat position 65 (double underlined) being aspartic acid (D). Residues 241-246 (underlined) of the first polypeptide chain of 5T4-M1 correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 247-274 of the first polypeptide chain of 5T4-M1 correspond to the heterodimer-promoting "E-coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Groups 275-277 correspond to the GGG linker. Residues 278-287 (underlined) correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 288-504 of the first polypeptide chain of 5T4-M1 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0413] Because the VL domain of CD3 mAb 1 M1 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of 5T4-M1 is identical to that of the second polypeptide chain of the 5T4-WT diabody (i.e., SEQ ID NO: 181). Similarly, the amino acid sequence of the third polypeptide chain of 5T4-M1 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0414] 10. Tenth Exemplary DART-B Diabody 5T4-M2 (5T4xCD3 mAb 1 M2) A tenth exemplary DART-B type diabody is similar to the 5T4-M1 diabody described above, but contains the VH domain of CD3 mAb 1 M2 and is referred to as "5T4-M2."

[0415] Thus, a tenth exemplary DART-B type diabody (5T4-M2) has the following amino acid sequence (SEQ ID NO: 183): JPEG2025186382000086.jpg58146 and a first polypeptide chain having the following structure:

[0416] Residues 1-107 of the first polypeptide chain of 5T4-M2 correspond to the VL domain of 5T4 mAb 1 (SEQ ID NO: 157). Residues 108-115 (double underlined) of the first polypeptide chain of 5T4-M2 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 116-240 of the first polypeptide chain of 5T4-M2 correspond to the VH domain of CD3 mAb 1 M2 (SEQ ID NO: 66), with Kabat position 65 (double underlined) being aspartic acid (D). Residues 241-246 (underlined) of the first polypeptide chain of 5T4-M2 correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 247-274 of the first polypeptide chain of 5T4-M2 correspond to the heterodimer-promoting "E coil" ( E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Groups 275-277 correspond to the GGG linker. Residues 278-287 (underlined) correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 288-504 of the first polypeptide chain of 5T4-M2 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0417] Because the VL domain of CD3 mAb 1 M2 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of 5T4-M2 is identical to that of the second polypeptide chain of the 5T4-WT diabody (i.e., SEQ ID NO: 181). Similarly, the amino acid sequence of the third polypeptide chain of 5T4-M2 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0418] 11. Eleventh Exemplary DART-B Diabody 5T4-M18 (5T4×CD3 mAb 1 M18) An eleventh exemplary DART-B type diabody is similar to the 5T4-WT diabody described above, but contains the VH domain of CD3 mAb 1 M18 and is referred to as "5T4-M18."

[0419] Thus, an eleventh exemplary DART-B type diabody (5T4-M18) has the following amino acid sequence (SEQ ID NO: 184): JPEG2025186382000087.jpg61146 and a first polypeptide chain having the following structure:

[0420] Residues 1-107 of the first polypeptide chain of 5T4-M18 correspond to the VL domain of 5T4 mAb 1 (SEQ ID NO: 157). Residues 108-115 (double underlined) of the first polypeptide chain of 5T4-M18 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 116-240 of the first polypeptide chain of 5T4-M18 correspond to the VL domain of CD3 mAb 1 (SEQ ID NO: 157). The first polypeptide chain of 5T4-M18 corresponds to the VH domain of M18 (SEQ ID NO: 98), with Kabat position 65 (double underlined) being an aspartic acid (D). Residues 241-246 (underlined) of the first polypeptide chain of 5T4-M18 correspond to linker 2 (GGCGGG; SEQ ID NO: 17). Residues 247-274 of the first polypeptide chain of 5T4-M18 form a heterodimer-promoting "E coil" ( E VAAL E K- E VAALE K- E VAAL E K- E VAAL E K; SEQ ID NO: 29). Residues 275-277 of the peptide chain correspond to the GGG linker. Residues 278-287 (underlined) of the polypeptide chain correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 288-504 of the first polypeptide chain of 5T4-M18 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0421] Because the VL domain of CD3 mAb 1 M18 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of 5T4-M18 is identical to that of the second polypeptide chain of the 5T4-WT diabody (i.e., SEQ ID NO: 181). Similarly, the amino acid sequence of the third polypeptide chain of 5T4-M18 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0422] 12. Twelfth Exemplary DART-B Diabody HIV-WT (HIV x CD3 mAb 1) A twelfth exemplary DART-B diabody is similar to the CD123-WT diabody described above, but contains the HIV-binding domain of the anti-HIV antibody A32 in place of the CD123-WT diabody's CD123-binding domain. This twelfth exemplary DART-B diabody is referred to as "HIV-WT."

[0423] Thus, a twelfth exemplary DART-B diabody (HIV-WT) has the following amino acid sequence (SEQ ID NO: 185): JPEG2025186382000088.jpg59148 and a first polypeptide chain having the following structure:

[0424] Residues 1-110 of the first polypeptide chain of HIV-WT correspond to the VL domain of A32 (SEQ ID NO: 169). Residues 111-118 (double underlined) of the first polypeptide chain of HIV-WT correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 119-243 of the first polypeptide chain of HIV-WT correspond to the VH domain of CD3 mAb 1 (SEQ ID NO: 55), with Kabat position 65 (double underlined) being glycine (G). Residues 244-248 (underlined) of the first polypeptide chain of HIV-WT correspond to linker 2 (ASTKG; SEQ ID NO: 21; underlined). Residues 245-248 of the first polypeptide chain of HIV-WT correspond to linker 2 (ASTKG; SEQ ID NO: 21; underlined). 49-276 is a heterodimer-promoting "E coil" ( E VAA CE K- E VAAL E K- E VAAL E K- E VAAL E K; Residues 277-279 of the first polypeptide chain of HIV-WT correspond to the GGG linker. Residues 280-289 of the first polypeptide chain of HIV-WT correspond to the GGG linker. 9 (underlined) corresponds to the linker DKTHTCPPCP (SEQ ID NO: 40; underlined). Residues 290-506 of the first polypeptide chain of HIV-WT correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0425] The second polypeptide chain of HIV-WT has the following amino acid sequence (SEQ ID NO: 186): JPEG2025186382000089.jpg37148 It has.

[0426] Residues 1-110 of the second polypeptide chain of HIV-WT correspond to the VL domain of CD3 mAb 1 (SEQ ID NO: 56). Residues 111-118 (double underlined) of the second polypeptide chain of HIV-WT correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 119-241 of the second polypeptide chain of HIV-WT correspond to the VH domain of A32 (SEQ ID NO: 209 (i.e., SEQ ID NO: 168 where X is L)). Residues 242-246 (underlined) of the second polypeptide chain of HIV-WT correspond to linker 2 (ASTKG; SEQ ID NO: 21). Residues 247–274 of the second polypeptide chain of HIV-WT correspond to the heterodimer-promoting “K coil” ( K VAA CK E- K VAAL K E- K VAAL K E- K VAAL K E; SEQ ID NO: 32).

[0427] The third polypeptide chain of HIV-WT is the third polypeptide chain of the CD123-WT diabody. It has the same amino acid sequence as the peptide chain (i.e., SEQ ID NO: 176).

[0428] 13. A thirteenth exemplary DART-B diabody HIV-M18 (HIV × CD3 mAb 18) A thirteenth exemplary DART-B diabody is similar to the HIV-WT diabody described above, but contains the VH domain of CD3 mAb 1 M18. This exemplary DART-B diabody is referred to as "HIV-M18."

[0429] Thus, a thirteenth exemplary DART-B diabody (HIV-M18) has the following amino acid sequence (SEQ ID NO: 196): JPEG2025186382000090.jpg60148 and a first polypeptide chain having the following structure:

[0430] Residues 1-110 of the first polypeptide chain of HIV-M18 correspond to the VL domain of A32 (SEQ ID NO: 169). Residues 111-118 (double underlined) of the first polypeptide chain of HIV-M18 correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 119-243 of the first polypeptide chain of HIV-M18 correspond to the VH domain of CD3 mAb 1 M18 (SEQ ID NO: 55), with Kabat position 65 (double underlined) being glycine (G). Residues 244-248 (underlined) of the first polypeptide chain of HIV-M18 correspond to linker 2 (ASTKG; SEQ ID NO: 21; underlined). Residues 249–276 of the peptide chain are heterodimer-promoting “E coil” ( E VAA CE K- E VAAL E K- E VAAL E K- E VAAL E K; SEQ ID NO: 31). The first polypeptide chain of HIV-M18 Residues 277-279 of the HIV-M18 first polypeptide correspond to the GGG linker. Residues 280-289 (underlined) of the first polypeptide chain correspond to the linker DKTHTCPPCP (SEQ ID NO: 40; underlined). Residues 290-506 of the first polypeptide chain of HIV-M18 correspond to the IgG1 "knob-bearing" CH2-CH3 domain (SEQ ID NO: 48).

[0431] Because the VL domain of CD3 mAb 1 M18 is identical to that of CD3 mAb 1, the amino acid sequence of the second polypeptide chain of HIV-M18 is identical to that of the second polypeptide chain of the HIV-WT diabody (i.e., SEQ ID NO: 186). Similarly, the amino acid sequence of the third polypeptide chain of HIV-M18 is identical to that of the third polypeptide chain of the CD123-WT diabody (i.e., SEQ ID NO: 176).

[0432] 14. Fourteenth Exemplary DART-B Diabody CD19-WT (CD19xCD3 mAb 1) A fourteenth exemplary DART-B diabody is similar to the HIV-WT diabody described above, but instead of the A32 binding domain, it contains CD19 mAb 1. This fourteenth exemplary DART-B diabody is referred to as "CD19-WT."

[0433] Thus, a fourteenth exemplary DART-B diabody (CD19-WT) has the following amino acid sequence (SEQ ID NO: 191): JPEG2025186382000091.jpg60148 and a first polypeptide chain having the following structure:

[0434] Residues 1-106 of the first polypeptide chain of CD19-WT correspond to the VL domain of CD19 mAb 1 (SEQ ID NO: 165). Residues 107-114 (double underlined) of the first polypeptide chain of CD19-WT correspond to linker 1 (GGGSGGGG; SEQ ID NO: 16). Residues 115-239 of the first polypeptide chain of CD19-WT correspond to the VH domain of CD3 mAb 1 (SEQ ID NO: 55), with Kabat position 65 (double underlined) being glycine (G). Residues 240-244 (underlined) of the first polypeptide chain of CD19-WT correspond to linker 2 (ASTKG; SEQ ID NO: 21; underlined). Residues 245–272 of the polypeptide chain of α-glucanase (α-glucanase) are heterodimer-promoting “E coil” ( E VAA CE K- E VAAL E K- E VAAL E K- E VAAL E The first polypeptide of CD19-WT corresponds to the first polypeptide of CD19-WT (K; SEQ ID NO: 31). Residues 273-275 of the CD19-W chain correspond to the GGG linker (double underlined). Residues 276-285 (underlined) of the first polypeptide chain of T correspond to the linker DKTHTCPPCP (SEQ ID NO: 40). Residues 286-502 of the first polypeptide chain of CD19-WT correspond to the IgG1 "...

Claims

1. A DAxCD3 binding molecule comprising a CD3 binding domain capable of binding to an epitope of CD3 and a disease antigen binding domain capable of binding to an epitope of a disease antigen, wherein the CD3 binding domain is: (I) (A) a CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:97; H 1 domain; (B) a CDR comprising the amino acid sequence of SEQ ID NO: 58; H 2 domains; (C) a CDR comprising the amino acid sequence of SEQ ID NO:

59. H 3 domains; (D) a CDR comprising the amino acid sequence of SEQ ID NO:

60. L 1 domain; (E) a CDR comprising the amino acid sequence of SEQ ID NO:

61. L 2 domains; and (F) a CDR comprising the amino acid sequence of SEQ ID NO:

62. L 3 domains; or (II) (A) a CDR comprising the amino acid sequence of SEQ ID NO: 57; H 1 domain; (B) a CDR comprising the amino acid sequence of SEQ ID NO: 58; H 2 domains; (C) a CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, and SEQ ID NO:

107. H 3 domains; (D) a CDR comprising the amino acid sequence of SEQ ID NO:

60. L 1 domain; (E) a CDR comprising the amino acid sequence of SEQ ID NO:

61. L 2 domains; and (F) a CDR comprising the amino acid sequence of SEQ ID NO:

62. L 3 domains; or (III) (A) a CDR comprising the amino acid sequence of SEQ ID NO: 57 H 1 domain; (B) a CDR comprising the amino acid sequence of SEQ ID NO: 58; H 2 domains; (C) a CDR comprising the amino acid sequence of SEQ ID NO:

59. H 3 domains; (D) a CDR comprising the amino acid sequence of SEQ ID NO:

60. L 1 domain; (E) a CDR comprising the amino acid sequence of SEQ ID NO:

61. L 2 domains; and (F) a CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 109 and SEQ ID NO:

111. L 3 domains: or (IV) (A) a CDR comprising the amino acid sequence of SEQ ID NO: 57; H 1 domain; (B) a CDR comprising the amino acid sequence of SEQ ID NO: 58; H 2 domains; (C) a CDR comprising the amino acid sequence of SEQ ID NO:

59. H 3 domains; (D) a CDR comprising the amino acid sequence of SEQ ID NO:

60. L 1 domain; (E) a CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 113 and SEQ ID NO:

115. L 2 domains; and (F) a CDR comprising the amino acid sequence of SEQ ID NO:

62. L 3 Domains A DAxCD3 binding molecule comprising:

2. The CD3 binding domain comprises: (I) (A) a VL domain comprising the amino acid sequence of SEQ ID NO: 56; (B) a VH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:98, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, and SEQ ID NO:106; or (II) (A) a VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, and SEQ ID NO: 114; (B) a VH domain comprising the amino acid sequence of SEQ ID NO:

55.

2. The DAxCD3 binding molecule of claim 1, comprising:

3. The DAxCD3 binding molecule may be a bispecific antibody, a bispecific diabody, a bispecific 3. The DAxCD3 binding molecule of claim 1 or 2, which is an scFv, a bispecific TandAb, or a trivalent binding molecule.

4. The DAxCD3 binding molecule of any one of claims 1 to 3, wherein the DAxCD3 binding molecule is capable of binding to two or more disease antigens and / or different cell surface molecules of effector cells.

5. The DAxCD3 binding molecule of any one of claims 1 to 4, wherein the disease antigen is a cancer antigen.

6. The DAxCD3 binding molecule of any one of claims 1 to 4, wherein the disease antigen is a pathogen-associated antigen.

7. The DAxCD3 binding molecule of any one of claims 4 to 6, wherein the different cell surface molecule of the effector cell is CD2, CD8, CD16, TCR, NKp46, or NKG2D.

8. The cancer antigens include cancer antigens: 19.9, 4.2, ADAM-9, AH6, ALCAM, B1, B7-H3, BAGE, β-catenin, blood type ALe. b / Le y , Burkitt lymphoma antigen‐38.13, C14, CA125, carboxypeptidase M, CD5, CD19, CD20, CD22, CD23, CD25, CD27, CD28, CD33, CD36, CD40 / CD154, CD45, CD56, CD46, CD52, CD56, CD79a / CD79b, CD103, CD123, CD317, CDK4, CEA, CEACAM5 / CEACAM6, CO17‐1A, CO‐43, CO‐514, CTA‐1, CTLA‐4, cytokeratin 8, D1.1, D 1 56-22, DR5, E 1 Series, EGFR, ephrin receptor, EphA2, Erb, GAGE, GD2 / GD3 / GM2 ganglioside, GICA19-9, gp100, Gp37, gp75, gpA33, HER2 / neu, HMFG, human papillomavirus-E6 / human papillomavirus-E7, HMW-MAA, I antigen, IL13Rα2, integrin β6, JAM-3, KID3, KID31, KS 1 / 4 pan-carcinoma antigen, L6, L20, LEA, LUCA-2, M1:22:25:8, M18, M39, MAGE, MART, mesothelin, MUC-1, MUM-1, Myl, N-acetylglucosaminyltransferase, neoglycoprotein, NS-10, OFA-1, OFA-2, oncostatin M, p15, p97, PEM, PEMA, PIPA, PSA, PSMA, prostatic acid phosphate, R 24 , ROR1, sphingolipid, SSEA-1, SSEA-3, SSEA-4, sTn, T cell receptor-derived peptide, T 5 A 7 , TAG-72, TL5, TNF-receptor, TNF-γ receptor, TRA-1-85, transferrin receptor, 5T4, TSTA, VEGF, VEGF receptor, VEP8, VEP9, VIM-D5, and Y hapten, Le y 5. The compound according to claim 4, wherein the compound is selected from the group consisting of 8. A DAxCD3 binding molecule according to claim 7.

9. 9. The DAxCD3 binding molecule of claim 8, wherein the disease antigen is B7-H3, CEACAM5 / CEACAM6, EGRF, EphA2, gpA33, HER2 / neu, VEGF, 5T4, IL13Rα2, CD123, CD19, or ROR1.

10. The pathogen-associated antigens include pathogen-associated antigens: herpes simplex virus infected cell protein (ICP) 47, herpes simplex virus gD, Epstein-Barr virus LMP-1, Epstein-Barr virus LMP-2A, Epstein-Barr virus LMP-2B, human immunodeficiency virus gp160, human immunodeficiency virus gp120, human immunodeficiency virus gp41, human papillomavirus E6, human papillomavirus E7, human T-cell leukemia virus gp64, human T-cell leukemia virus gp46, and human T-cell leukemia virus 8. The DAxCD3 binding molecule of claim 6 or 7, wherein the DAxCD3 binding molecule is selected from the group consisting of rus gp21.

11. The DAxCD3 binding molecule comprises a first polypeptide chain and a second polypeptide chain covalently linked to each other: (A) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) Domain 1, comprising: (1) A VL domain (VL) of a monoclonal antibody capable of binding to the epitope of a disease antigen. DA ), and (2) a VH domain (VH) of a monoclonal antibody capable of binding to the epitope of CD3; CD3 ), a subdomain (1B) wherein said Subdomain 1A and said Subdomain 1B are separated from each other by a peptide linker; and (ii) Domain 2, which is a Heterodimer-Promoting Domain Including; (B) the second polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) Domain 1, comprising: (1) The VL domain (VL) of the monoclonal antibody capable of binding to the epitope of CD3 CD3 ), and (2) a VH domain (VH) of the monoclonal antibody capable of binding to the epitope of a disease antigen; DA ), a subdomain (1B) wherein said Subdomain 1A and said Subdomain 1B are separated from each other by a peptide linker; and (ii) Domain 2, which is a Heterodimer-Promoting Domain, wherein the Heterodimer-Promoting Domains of the first polypeptide chain and the second polypeptide chain are different. Including, (a) the VL Domain of the first polypeptide chain and the VH Domain of the second polypeptide chain associate to form the disease antigen-binding domain, and the VH Domain of the first polypeptide chain and the VL Domain of the second polypeptide chain associate to form the CD3-binding domain; or (b) the VL domain of the first polypeptide chain and the VH domain of the second polypeptide chain associate to form the CD3-binding domain, and the VH domain of the first polypeptide chain and the VL domain of the second polypeptide chain associate to form the disease antigen-binding domain. DA×CD3 binding molecule according to any one of claims 1 to 10.

12. (a) the Heterodimer-Promoting Domain of the first polypeptide chain is an E-coil domain and the Heterodimer-Promoting Domain of the second polypeptide chain is a K-coil domain; or (b) the Heterodimer-Promoting Domain of the first polypeptide chain is a K-coil domain and the Heterodimer-Promoting Domain of the second polypeptide chain is an E-coil domain; The DAxCD3 binding molecule of claim 11.

13. The DAxCD3 binding molecule of claim 11 or 12, wherein the first polypeptide chain or the second polypeptide chain further comprises domain 3 comprising the CH2 domain and CH3 domain of an immunoglobulin Fc domain.

14. The DAxCD3 binding molecule comprises a CH2 domain and a CH3 domain of an immunoglobulin Fc domain.

14. The DAxCD3 binding molecule of claim 13, further comprising a third polypeptide chain comprising three domains.

15. The DAxCD3 binding molecule of any one of claims 11 to 14, wherein the DAxCD3 binding molecule further comprises a CD8 binding domain.

16. The DAxCD3 binding molecule comprises: (I) (A) a first polypeptide comprising SEQ ID NO: 179; (B) a second polypeptide comprising SEQ ID NO: 175; and (C) a third polypeptide comprising SEQ ID NO: 176; or (II) (A) a first polypeptide comprising SEQ ID NO: 184; (B) a second polypeptide comprising SEQ ID NO: 181; and (C) a third polypeptide comprising SEQ ID NO: 176; or (III) (A) a first polypeptide comprising SEQ ID NO: 196; (B) a second polypeptide comprising SEQ ID NO: 186; and (C) a third polypeptide comprising SEQ ID NO: 176; or (IV) (A) a first polypeptide comprising SEQ ID NO: 197; (B) a second polypeptide comprising SEQ ID NO: 192; and (C) a third polypeptide comprising SEQ ID NO: 176; or (V) (A) a first polypeptide comprising SEQ ID NO: 193; (B) a second polypeptide comprising SEQ ID NO: 194; and (C) a third polypeptide comprising SEQ ID NO: 176; or (VI) (A) a first polypeptide comprising SEQ ID NO: 179; (B) a second polypeptide comprising SEQ ID NO: 175; (C) a third polypeptide comprising SEQ ID NO: 187; and (D) a fourth polypeptide comprising SEQ ID NO: 188; or (VII) (A) a first polypeptide comprising SEQ ID NO: 184; (B) a second polypeptide comprising SEQ ID NO: 181; (C) a third polypeptide comprising SEQ ID NO: 187; and (D) a fourth polypeptide comprising SEQ ID NO: 188; or (VIII) (A) a first polypeptide comprising SEQ ID NO: 196; (B) a second polypeptide comprising SEQ ID NO: 186; (C) a third polypeptide comprising SEQ ID NO: 187; and (D) a fourth polypeptide comprising SEQ ID NO: 188; or (IX) (A) a first polypeptide comprising SEQ ID NO: 193; (B) a second polypeptide comprising SEQ ID NO: 194; (C) a third polypeptide comprising SEQ ID NO: 187; and (D) a fourth polypeptide comprising SEQ ID NO:

188. The DAxCD3 binding molecule of any one of claims 11 to 15, comprising:

17. A pharmaceutical composition comprising any of the DAxCD3 binding molecules of any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. 18. A method for the treatment of a disease, comprising administering to a subject in need thereof a therapeutically effective amount of a DAxCD3 binding molecule according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17.

19. 19. The method of claim 18, wherein the disease is cancer.

20. The cancers include adrenal gland cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, renal cancer, and non-small cell lung cancer.

20. The method of claim 19, wherein the cancer is selected from the group consisting of hematological cancer, multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal cell carcinoma, testicular cancer, and uterine cancer.

21. 19. The method of claim 18, wherein the disease is a pathogen-associated disease.

22. 22. The method of claim 21, wherein the pathogen-associated antigen is selected from the group consisting of: herpes simplex virus infected cell protein (ICP) 47, herpes simplex virus gD, Epstein-Barr virus LMP-1, Epstein-Barr virus LMP-2A, Epstein-Barr virus LMP-2B, human immunodeficiency virus gp160, human immunodeficiency virus gp120, human immunodeficiency virus gp41, human papillomavirus E6, human papillomavirus E7, human T-cell leukemia virus gp64, human T-cell leukemia virus gp46, and human T-cell leukemia virus gp21.

23. A DAxCD3 binding molecule according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 17 for use in the treatment of a disease.

24. 24. The DAxCD3 binding molecule or pharmaceutical composition of claim 23, wherein the disease is cancer.

25. 25. The DAxCD3 binding molecule or pharmaceutical composition of claim 24, wherein the cancer is selected from the group consisting of adrenal gland cancer, bladder cancer, breast cancer, colorectal cancer, gastric cancer, glioblastoma, kidney cancer, non-small cell lung cancer, blood cancer, multiple myeloma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, renal cell carcinoma, testicular cancer, and uterine cancer.

26. 24. The DAxCD3 binding molecule or pharmaceutical composition of claim 23, wherein the disease is a pathogen-associated disease.

27. 27. The DAxCD3 binding molecule or pharmaceutical composition of claim 26, wherein the pathogen-associated antigen is selected from the group consisting of: herpes simplex virus infected cell protein (ICP) 47, herpes simplex virus gD, Epstein-Barr virus LMP-1, Epstein-Barr virus LMP-2A, Epstein-Barr virus LMP-2B, human immunodeficiency virus gp160, human immunodeficiency virus gp120, human immunodeficiency virus gp41, human papillomavirus E6, human papillomavirus E7, human T-cell leukemia virus gp64, human T-cell leukemia virus gp46, and human T-cell leukemia virus gp21.

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