Binding molecules against CD3 and uses thereof

Abstract

To provide novel CD3 binding molecules, e.g., antibodies and multispecific binding molecules that bind to CD3.SOLUTION: The disclosure provides a CD3 binding molecule that specifically binds to CD3, e.g., monospecific binding molecules that binds to CD3 and a multispecific binding molecule (MBM) that binds to CD3 and a tumor-associated antigen, a conjugate comprising a CD3 binding molecule, and a pharmaceutical composition comprising a CD3 binding molecule and a conjugate. Further provided is a method for activating T cells using the CD3 binding molecule, conjugate and the pharmaceutical composition in a subject, e.g., a subject with cancer or autoimmune disease. The disclosure further provides a recombinant host cell modified to express CD3 binding molecule and a method for generating CD3 binding molecules by culturing the host cell under conditions allowing for expression of CD3 binding molecules.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT application filed on December 4, 2018, PCT / China Patent Application Publication No. This application claims the benefit of priority from patent application No. 2018 / 119074. The entire contents of which are incorporated herein by reference.

[0002] 2. Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format, the entirety of which is as follows: ASCII copy created December 3, 2019. It is named NOV-010WO_SL.txt and is 782,920 bytes in size. do.

[0003] 3. Incorporation by Reference All publications, patents, patent applications, and other references cited in this application are the property of their respective owners. Each publication, patent, patent application or other document is incorporated by reference for all purposes. and incorporated by reference in its entirety for all purposes to the same extent as if individually indicated. Incorporated herein by reference. Between one or more of the references incorporated herein and the teachings of this disclosure In case of conflict, the teachings of the present specification are intended. [Background technology]

[0004] The concept of bispecific and multispecific antibodies is based on the fact that diseases are usually multifactorial and multiple disease factors It arose from the idea that a single antibody could be used to address these issues and increase efficacy. CD3 is a homologous recombinase expressed on T cells in association with the T cell receptor complex (TCR). It is a dimeric or heterodimeric antigen and is required for T cell activation. clusters CD3 on T cells, thereby binding peptide-loaded MHC molecules It has been shown that anti-CD31 receptors induce T cell activation in a manner similar to TCR engagement by CD31 receptors. Anti-CD3 antibodies have been proposed as a therapeutic approach involving T cell activation. It has been used in the treatment of proliferative disorders such as cancer and in the treatment of autoimmune diseases. and bispecific and multispecific antibodies capable of binding to target antigens expressing the target antigen. Therapeutic uses have been proposed, including targeting T cell immune responses to targeting tissues and cells. Currently approved drugs include CD19 / CD3 BiTE and blinatumomab. Bispecific and multispecific antibodies are commercially available. However, bispecific and multispecific antibodies Challenges of intracellular distribution, inhibitory microenvironment, and antigen loss remain. Therefore, there is a need in the art for superior bispecific and multispecific antibodies. Bispecific and multispecific antigen-binding molecules that bind to both the target antigen and the target antigen express the target antigen. This may be useful in therapeutic settings where specific targeting and T cell-mediated killing of targeting cells is desired. . Summary of the Invention [Problem to be solved by the invention]

[0005] New CD3 binding molecules, e.g., antibodies and multispecific binding molecules that bind to CD3, are needed. It is said that... [Means for solving the problem]

[0006] The present disclosure provides CD3 binding molecules that specifically bind to human CD3, e.g., The present invention provides antibodies, antigen-binding fragments thereof, and multispecific molecules that specifically bind to the target antigen.

[0007] In one aspect, the present disclosure provides a CD3 antigen binding domain or antigen binding module ("AB Monospecific CD3 binding molecules (e.g., antibodies and antigen-binding fragments thereof) including Exemplary CD3 binding molecules that may be monospecific are described in Section 7.2 below and in specific It is described in embodiments 1 to 456.

[0008] In another embodiment, the present disclosure provides a multispecific binding molecule comprising a CD3 ABM ("MBM" ), e.g., bispecific and multispecific antibodies. Thus, in one aspect, The present disclosure provides bispecific and In some embodiments, the present disclosure relates to multispecific antibodies. ") and CD3 and / or CD2 or other components of the TCR complex on T cells. Bispecific and multispecific binding molecules are provided.

[0009] In certain embodiments, the MBM is a bispecific binding molecule ("BBM"). M is a first ABM that specifically binds human CD3 ("ABM1" or "CD3 ABM"). ") and a second ABM that specifically binds to a second antigen ("ABM2"), e.g., human T AA (sometimes referred to herein as "TAA ABM"). ABM1, AB The terms M2, CD3 ABM and TAA ABM are used for convenience only; It is not intended to refer to any particular form of BM. This is used to attract CD3+ effector T cells to the TAA+ site, thereby Effector T cells can attack and lyse TAA+ cells and tumors. Features of the MBM are described in Sections 7.5-7.7 below and in specific embodiments 457-536. are.

[0010] In certain embodiments, the MBM is a trispecific binding molecule ("TBM"). M is a first ABM ("ABM1" or "CD3 ABM") that specifically binds to human CD3. M"), a second ABM ("ABM2") that specifically binds to a second antigen, e.g., a human TAA; ") and a third antigen, such as a second human TAA or human CD2, that specifically binds to the third antigen. (1) human CD3, (2) TAA, and (3) CD2 For convenience, TBMs that bind to human CD4+ are referred to herein as "type 1 TBMs." 3, (2) the first TAA (sometimes referred to as "TAA1"), and (3) the second TA For convenience, TBM binding to TAA2 (sometimes referred to as "TAA2") is referred to herein as Both TAA1 and TAA2 are tumor-associated antigens, and are therefore referred to as "type 2 TBM." The designation of the tumor-associated antigens of the present disclosure as TAA1, TAA2, and TAA3 is arbitrary and therefore Unless the context requires otherwise, any disclosure regarding TAA1 shall be applicable to TAA2. can be used, and vice versa.

[0011] In one embodiment, each antigen binding module of the MBM may contain one or more additional antigen binding molecules. Each of the binding modules binds to its respective target at the same time that it binds to its respective target. It is possible to effectively bind.

[0012] In MBM, each ABM (except ABM1, which is immunoglobulin-based) MBMs can be immunoglobulin-based or non-immunoglobulin-based; thus, MBMs can be immunoglobulin-based or non-immunoglobulin-based. The ABM may include a phosphorus-based ABM, a non-immunoglobulin-based ABM, or a combination thereof. Immunoglobulin-based ABMs that may be used in MBM are described below in Sections 7.3.1 and 7.3.2. and specific embodiments 1 to 469. Blinn-based ABM is described in Section 7.3.2 below and in specific embodiments 747-777. Further characteristics of exemplary ABMs that bind to components of the TCR complex are as follows: Further features of exemplary ABMs that bind to CD2 include: These are described below in Section 7.9 and in specific embodiments 746-789. Further features of the exemplary ABM are described in Section 7.10 below, as well as in specific embodiments 592-599. 745 and 790-946.

[0013] The ABM of the MBM (or a portion thereof) may comprise, for example, a short peptide linker or an Fc domain. Methods and Compositions for Linking ABMs to Form MBMs The elements are described below in Section 7.4 and in specific embodiments 947-1155.

[0014] The MBM has at least two ABMs (i.e., the MBM is at least bivalent) ) can have more than two ABMs. For example, an MBM can have three ABMs (i.e., trivalent), four ABMs (i.e., tetravalent), five ABMs (i.e., pentavalent) In some embodiments, the ABM may have six ABMs (i.e., six valences). In the present invention, the MBM contains at least one ABM, CD3, that can bind to a TAA. At least one ABM capable of binding to another antigen and at least one ABM capable of binding to another antigen. Exemplary bivalent, trivalent, tetravalent, pentavalent and hexavalent MBM forms include: As described in Sections 7.5-7.7 below and in specific embodiments 477-536 and 554-590 It has been done.

[0015] The present disclosure provides CD3 binding molecules (e.g., MBMs) (either single nucleic acid or multiple nucleic acids). and a nucleic acid encoding a CD3 binding molecule (e.g., MBM) that expresses the nucleic acid and the CD3 binding molecule. Further provided are recombinant host cells and cell lines engineered to produce the nucleic acid. Cells and cell lines are described below in Section 7.11 and in specific embodiments 1439-1441. It is being done.

[0016] The present disclosure further provides drug conjugates comprising CD3 binding molecules (e.g., MBM). Such conjugates may have a non-immunoglobulin domain in part of the ABM. Nevertheless, for convenience, we will refer to these compounds as "antibody drug conjugates" or "ADCs." Examples of ADCs are described in Section 7.12 below and in specific embodiments 1225-126. 2.

[0017] Pharmaceutical compositions comprising a CD3 binding molecule (e.g., MBM) and an ADC are also provided. Examples of compositions are described below in Section 7.14 and in specific embodiment 1263.

[0018] CD3 binding molecules, e.g., for treating proliferative conditions (e.g., cancer) in which TAAs are expressed. Further provided herein are methods of using the ADCs (e.g., MBM), ADCs, and pharmaceutical compositions. Exemplary methods are described below in Section 7.15 and in specific embodiments 1264-1437. It is listed.

[0019] The present disclosure provides methods for combining CD3 binding molecules (e.g., MBM), ADCs, and pharmaceutical compositions with other drugs and Further provided are methods of use in combination with therapeutic regimens. Exemplary Agents, Therapies and Combinations Methods of treatment are described below in Section 7.16 and in specific embodiment 1438. [Brief explanation of the drawings]

[0020] [Figure 1-1] Figure 1: Exemplary BBM Configurations. Figure 1A shows the components of the exemplary BBM configurations shown in Figures 1B-1AH. Not all regions connecting the different domains of each chain are shown (e.g., the linker connecting the VH and VL domains of an scFv, the hinge connecting the CH2 and CH3 domains of an Fc domain, etc. are omitted). Figures 1B-1F show bivalent BBMs; Figures 1G-1Z show trivalent BBMs; and Figures 1AA-1AH show tetravalent BBMs. In BBMs, mutant CD58 domains can replace Fab and / or scFv in any of the configurations shown. [Figure 1-2] (As mentioned above.) [Figure 1-3] (As mentioned above.) [Figure 1-4] (As mentioned above.) [Figure 1-5] (As mentioned above.) [Figure 1-6] (As mentioned above.) [Figure 1-7] (As mentioned above.) [Figure 2-1] Figure 2: Figures 2A-2V are exemplary TBM configurations. Figure 2A shows the components of the exemplary TBM configurations shown in Figures 2B-2V. Not all regions connecting the different domains of each chain are shown (e.g., the linker connecting the VH and VL domains of the scFv, the hinge connecting the CH2 and CH3 domains of the Fc, etc. are omitted). Figures 2B-2P show trivalent TBMs, Figures 2Q-2S show tetravalent TBMs, Figure 2T show pentavalent TBMs, and Figures 2U-2V show hexavalent TBMs. In TBMs, mutant CD58 domains can replace Fab and / or scFv in any of the configurations shown. [Figure 2-2] (As mentioned above.) [Figure 2-3] (As mentioned above.) [Figure 2-4] (As mentioned above.) [Figure 2-5] (As mentioned above.) [Figure 2-6] (As mentioned above.) [Figure 3-1] Figure 3: Exemplary MBM formats are shown. Figure 3A shows a conventional IgG format, Figure 3B shows a BITE format, Figure 3C shows a bispecific format, Figure 3D shows a trispecific format, and Figure 3E shows a tetraspecific format. [Figure 3-2] (As mentioned above.) [Figure 3-3] (As mentioned above.) [Figure 3-4] (As mentioned above.) [Figure 3-5] (As mentioned above.) [Figure 4-1] Figure 4: Figures 4A-4D are surface plasmon resonance (SPR / Biacore) measurements showing the Kd of CD3. Figure 4A: NOV292; Figure 4B: sp34; Figure 4C: NOV123; Figure 4D: sp1c. [Figure 4-2] (As mentioned above.) [Figure 4-3] (As mentioned above.) [Figure 4-4] (As mentioned above.) [Figure 5] Binding of anti-CD3 antibodies to cells transfected with human CD3 is shown. [Figure 6] Binding of anti-CD3 antibodies to cells transfected with human CD3 is shown. [Figure 7] Binding of anti-CD3 antibodies to cells transfected with human CD3 is shown. [Figure 8] Binding of anti-CD3 antibodies to cells transfected with cynomolgus monkey (cyno) CD3 is shown. [Figure 9] Binding of anti-CD3 antibodies to cells transfected with cynomolgus monkey (cyno) CD3 is shown. [Figure 10]Binding of anti-CD3 antibodies to cells transfected with cynomolgus monkey (cyno) CD3 is shown. [Figure 11] 1 shows agonistic activation of CD3 by bispecific antibodies in a JNL cell model. [Figure 12] 1 shows agonistic activation of CD3 by bispecific antibodies in a JNL cell model. [Figure 13] 1 shows agonistic activation of CD3 by bispecific antibodies in a JNL cell model. [Figure 14] 1 shows agonistic activation of CD3 by bispecific antibodies in a JNL cell model. [Figure 15] 1 shows agonistic activation of CD3 by bispecific antibodies in a JNL cell model. [Figure 16] Figure 1 shows the ability of anti-CD19 / anti-CD3 bispecific antibodies to lyse target cells in a redirected T-cell cytotoxicity (RTCC) assay. [Figure 17] Figure 1 shows the ability of anti-CD19 / anti-CD3 bispecific antibodies to lyse target cells in a redirected T-cell cytotoxicity (RTCC) assay. [Figure 18] Figure 1 shows the ability of anti-CD19 / anti-CD3 bispecific antibodies to lyse target cells in an RTCC assay. "OKT3" and "sp34" refer to positive control bispecific antibodies. [Figure 19] Figure 1 shows the affinity of anti-CD19 / anti-CD3 bispecific antibodies for CD3+ T cells. "OKT3" and "sp34" refer to positive control bispecific antibodies. [Figure 20] Figure 1 shows the ability of anti-CD19 / anti-CD3 bispecific antibodies to lyse target cells in an RTCC assay. "OKT3," "sp34," and "H2C" refer to positive control bispecific antibodies. [Figure 21] Figure 1 shows the ability of anti-CD19 / anti-CD3 bispecific antibodies to lyse target cells in an RTCC assay. "OKT3," "sp34," and "H2C" refer to positive control bispecific antibodies. [Figure 22]Figure 1 shows the ability of anti-CD19 / anti-CD3 bispecific antibodies to lyse target cells in an RTCC assay. "OKT3" and "sp34" refer to positive control bispecific antibodies. [Figure 23] Figure 1 shows the affinity of anti-CD19 / anti-CD3 bispecific antibodies for CD3+ T cells. "OKT3" and "sp34" refer to positive control bispecific antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0021] 7.1.Definition As used herein, the following terms are intended to have the following meanings: .

[0022] Antigen binding module: As used herein, "antigen binding module" or " The term "ABM" refers to a molecule that has the ability to bind non-covalently, reversibly, and specifically to an antigen. ABM refers to the portion of the MBM of the present disclosure that is immunoglobulin-based or non-immunoglobulin-based. As used herein, "ABM1" and "CD3 AB" are used interchangeably. The terms "ABM" (and the like) refer to ABMs that specifically bind to CD3, and "ABM2" and The term "TAA ABM" (and the like) refers to an ABM that specifically binds to a tumor-associated antigen. Terms such as ABM1 and ABM2 are used for convenience only and do not imply any specificity of MBM. It is not intended to represent a specific form.

[0023] Antibody: The term "antibody" as used herein refers to a non-covalently, reversibly A polypeptide of the immunoglobulin family capable of efficiently and specifically binding to an antigen. For example, naturally occurring "antibodies" of the IgG type are composed of disulfide bonds. It contains at least two heavy (H) chains and two light (L) chains interconnected by bond bonds. Each heavy chain is a tetramer. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light The light chain constant region is composed of one domain (referred to herein as CL). The VH and VL regions are called framework regions (FR). They contain hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions. Each VH and VL can be further subdivided into regions in the following order: FR1, CDR1, FR2 , CDR2, FR3, CDR3, FR4 arranged from the amino terminus to the carboxy terminus It consists of three CDRs and four FRs. The variable regions of the heavy and light chains interact with antigens. The constant region of an antibody contains a binding domain that binds to various cells of the immune system (e.g., immune cells). to host tissues or factors, including the first component of the classical complement system (Clq) and the first component of the classical complement system (C1q). The term "antibody" includes, but is not limited to, monoclonal antibodies capable of mediating the binding of immunoglobulins. Clonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or polyclonal antibodies Polyspecific and anti-idiotypic (anti-Id) antibodies (e.g., anti-Id antibodies to the antibodies of the present disclosure) Antibodies include antibodies of any isotype / class (e.g., IgG, IgE , IgM, IgD, IgA and IgY) or subclass (e.g., IgG1, IgG2, The antibodies may be of the following types: IgG3, IgG4, IgA1 and IgA2.

[0024] Both the light and heavy chains are divided into regions of structural and functional homology. The term "variable" is used functionally. In this context, light (VL) and heavy (VH) It will be appreciated that the variable domains of both chains determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) are involved in secretion, They confer important biological properties such as transplacental mobility, Fc receptor binding, and complement fixation. Therefore, the numbering of the constant region domains is more distal from the antigen binding site or amino terminus of the antibody. The N-terminus is the variable region and the C-terminus is the constant region; The CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0025] Antibody fragment: As used herein, an "antibody fragment" of an antibody The term "antibody" refers to one or more portions of an antibody. In certain embodiments, these portions are part of an antibody. In certain other embodiments, these portions are part of the contact domains of the In the context of the present invention, the term "antigen-binding fragment," "antigen-binding fragment thereof," "antigen-binding portion," etc. The ability to bind antigen non-covalently, reversibly, and specifically, sometimes referred to as Examples of binding fragments include, but are not limited to, However, single chain Fv (scFv), Fab fragments, VL, VH, CL and CH1 domains monovalent fragments consisting of F(ab)2 fragments, disulfides in the hinge region; A bivalent fragment containing two Fab fragments linked by a peptide bridge; an Fd fragment consisting of the CH1 domain and the VL and VH domains of a single arm of an antibody; Fv fragments consisting of the VH domain; dAb fragments (Ward et al. al., (1989) Nature 341:544-546); and isolated complement The term "antibody fragment" therefore includes the CDRs. Proteolytic fragments of antibodies (e.g., Fab and F(ab)2 fragments) and and modified proteins comprising one or more portions of an antibody (e.g., scFv).

[0026] Antibody fragments include single domain antibodies, maxibodies, minibodies, intrabodies, bibodies, Also in bispecific antibodies, trispecific antibodies, tetraspecific antibodies, v-NAR and bis-scFv (e.g., Hollinger and Hudson, 2005, Na (See Nature Biotechnology 23:1126-1136). Antibody fragments are based on polypeptides such as fibronectin type III (Fn3). and can be grafted into a scaffold (US Pat. No. 6,233,199 describes a fibronectin polypeptide monobody). See Patent No. 6,703,199).

[0027] An antibody fragment comprises a complementary light chain polypeptide (e.g., VL-VC-VL-VC). A pair of tandem Fv segments (e.g., VH) that together form a pair of antigen-binding regions -CH1-VH-CH1) (Zapata et al. al., 1995, Protein Eng. 8:1057-1062; and U.S. Pat. 5,641,870).

[0028] Antigen-binding domain: The term "antigen-binding domain" refers to a non-covalently, reversibly acting An antigen-binding domain refers to a portion of a molecule that has the ability to specifically bind to an antigen. Exemplary antigen-binding domains are antigen-binding fragments that retain the ability to non-covalently, reversibly, and specifically bind antigens; The present invention includes fragments and portions of both immunoglobulin and non-immunoglobulin based scaffolds. As used herein, the term "antigen-binding domain" refers to a domain that non-covalently binds to an antigen. Antibody fragments that retain the ability to reversibly and specifically bind antigen are included.

[0029] Half antibody: The term "half antibody" refers to an antibody that contains at least one ABM or ABM chain, e.g., For example, disulfide bridges or molecular interactions (e.g., knob-in-hole interactions between Fc heterodimers) ABM or another molecule containing an ABM chain can be bound to it by a knob-in-hole interaction. A half antibody refers to a molecule that can associate with one polypeptide chain or two or more polypeptides. In a preferred embodiment, the polypeptide chains are composed of two polypeptide chains (e.g., two polypeptide chains of a Fab). In this case, the half antibody comprises an Fc region.

[0030] An example of a half antibody is a molecule that contains the heavy and light chains of an antibody (e.g., an IgG antibody). Another example is a first polypeptide comprising a VL domain and a CL domain, and a VH domain a second polypeptide comprising a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain; and a peptide, wherein the VL and VH domains form an ABM. Yet another example of a half antibody includes an scFv domain, a CH2 domain, and a CH3 domain. It is a polypeptide.

[0031] A half antibody can contain two or more ABMs, for example, a half antibody can contain (in order from N-terminus to C-terminus) 2) scFv domain, CH2 domain, CH3 domain and another scFv domain nothing.

[0032] A half antibody forms a complete ABM when associated with another ABM chain in another half antibody. It may also include a BM chain.

[0033] Thus, an MBM may contain one, more typically two or even three or more half antibodies. , a half antibody may comprise one or more ABMs or ABM chains.

[0034] In some MBMs, the first half antibody associates with a second half antibody, e.g., In other MBMs, the first half antibody is heterodimerized, e.g., by a disulfide bridge. or by chemical cross-linking to the second half antibody. One half antibody has both covalent and non-covalent interactions, e.g., disulfide bridges and knob- It associates with the second half antibody through in-hole interactions.

[0035] The term "half antibody" is intended for descriptive purposes only and does not refer to a specific form or preparation. The "first" half antibody, the "second" half antibody, the "left" half antibody, The description of half antibodies as "right" half antibodies is for convenience and illustrative purposes only.

[0036] Complementarity Determining Region: "Complementarity Determining Region" or "CDR" as used herein The term refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, each heavy chain variable region generally has three CDRs (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H and CDR-H2 and CDR-H3) and three CDRs in each light chain variable region (CDR-L1, The exact amino acid sequence boundaries of a given CDR are abat et al., 1991, “Sequences of Proteins of Immunological Interest”,5th Ed.Public Health Service,National Institutes of H health, Bethesda, MD ("Kabat" numbering scheme), Al-La zikani et al., 1997, JMB 273:927-948 (“Chot hia numbering scheme) and ImMunoGenTics (IMGT) numbering scheme (L efranc,1999,The Immunologist 7:132-136(1 999);Lefranc et al.,2003,Dev.Comp.Immuno l.27:55-77 ("IMGT" numbering scheme) , can be determined using any of several well-known schemes. For example, in the classical form The CDR amino acid residues in the heavy chain variable domain (VH) are 31 to 33 by the Kabat method. 5 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) The CDR amino acid residues in the light chain variable domain (VL) are numbered 24 to 34 (CDR -L1), 50 to 56 (CDR-L2) and 89 to 97 (CDR-L3) According to the Chothia method, the CDR amino acids in VH are 26 to 32 (CDR-H1), Numbered 52–56 (CDR-H2) and 95–102 (CDR-H3); in VL The amino acid residues of CDR-L1 are 26-32, those of CDR-L2 are 50-52, and those of CDR-L3 are 91-92. 96 (CDR-L3). Both Kabat and Chothia CDRs Combining the definitions, the CDRs are amino acid residues 26 to 35 (CD R-H1), 50-65 (CDR-H2) and 95-102 (CDR-H3) and human Amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 8 in VL The CDR amino acid residues in VH are 9 to 97 (CDR-L3). Approximately 26-35 (CDR-H1), 51-57 (CDR-H2) and 93-102 (CDR -H3), and the CDR amino acid residues in the VL are approximately 27 to 32 (CDR-L1). , numbered 50-52 (CDR-L2) and 89-97 (CDR-L3) ("K The IMGT method numbers the CDR regions of an antibody according to the program IM This can be determined using GT / Domain Gap Align.

[0037] Single-chain Fv or scFv: As used herein, "single-chain Fv" or "scFv" The term "Fv" refers to an antibody fragment comprising the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide The peptides comprise the VH and VH domains, which enable the scFv to form the desired structure for antigen binding. The scFv further comprises a polypeptide linker between the VL domain and the VL domain. lueckthun in The Pharmacology of Monoclo nal Antibodies,vol.113,Rosenburg and Moo re eds., (1994) Springer-Verlag, New York, p. Please refer to p.269-315.

[0038] Bispecific antibody: The term "bispecific antibody" as used herein means Typically, it is a small antibody with two antigen-binding sites formed by the pairing of scFv chains. Each scFv is a light chain variable domain (VL) in the same polypeptide chain. ) (VH-VL, where VH is linked to VL) (VH and VL are either N-terminal or C-terminal to each other) a linker that enables VH and VL to pair to form an antigen-binding domain Unlike typical scFvs, which are separated, bispecific antibodies typically have VH and VH on the same chain. and VL domains, thereby The VL and VL domains pair with the complementary domains of another chain to form two antigen-binding sites. Bispecific antibodies are described, for example, in EP 404,097; WO 93 / 1 1161; and Hollinger et al., 1993, Proc. More fully described in Natl Acad Sci USA 90:6444-6448 It has been done.

[0039] Fv: The term "Fv" refers to an immunoglobulin that contains a complete target recognition and binding site. This refers to the smallest antibody fragment that can be derived from this region. This region consists of a single tightly non-covalently bound In this form, the antibody consists of a dimer of one light chain variable domain and one light chain variable domain (VH-VL dimer). The three CDRs of each variable domain interact to form a target binding site on the surface of the VH-VL dimer. In most cases, six CDRs confer target binding specificity to an antibody. However, in some cases, a single variable domain (or only three CDRs) specific for a target Even a single Fv half containing the target may have the ability to recognize and bind to the target. Reference to a VH-VL dimer in this specification is not intended to denote any particular form. For example, but not limited to, VH and VL can be combined in any manner described herein. The half antibodies can be formed by the addition of the nucleotides, or each can be present on a separate half antibody and can be separated into separate half antibodies. When antibodies assemble together they form an antigen-binding domain, e.g., the MBMs of the present disclosure When present on a single polypeptide chain (e.g., scFv), The VH can be N-terminal or C-terminal to the VL.

[0040] Multispecific binding molecule: The term "multispecific binding molecule" or "MBM" refers to at least one It also refers to a molecule comprising two antigen-binding domains, wherein at least one of the antigen-binding domains One is CD3, and at least one antigen-binding domain is specific for a TAA. Each binding domain can be independently formed into an antibody fragment (e.g., scFv, Fab, nano). antibodies), ligands or non-antibody-derived binding agents (e.g., fibronectin, Fynomer, Representative MBMs are shown in Figures 3A-3E. , two, three, four or even more polypeptide chains.

[0041] VH: The term "VH" refers to, but is not limited to, Fv, scFv, dsFv, or Fab. The term "variable region" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of the antibody.

[0042] VL: The term "VL" refers to, but is not limited to, Fv, scFv, dsFv, or Fab. The term "variable region" refers to the variable region of an immunoglobulin light chain, including the light chain of an immunoglobulin.

[0043] Operably linked: The term "operably linked" refers to two or more peptides or refers to a functional relationship between polypeptide domains or nucleic acid (e.g., DNA) segments. The term "operably linked" in reference to a fusion protein or other polypeptide The term refers to two or more amino acid segments linked to form a functional polypeptide. For example, in the context of the MBMs of the present disclosure, a separate ABM (or chain of ABMs) The polypeptide chains of the MBMs of the present disclosure may be linked via a peptide linker sequence. With respect to nucleic acids encoding proteins, "operably linked" means that two nucleic acids are linked together in a manner that allows the two nucleic acids to The amino acid sequences encoded by the nucleic acids are joined so that they remain in frame. In the context of transcriptional regulation, the term refers to the function of a transcriptional regulatory sequence on a transcribed sequence. For example, a promoter or enhancer sequence may be used in a manner that is compatible with the host cell or is operative for a coding sequence if it stimulates or regulates transcription of the coding sequence in another expression system. is linked to.

[0044] Associated: In relation to MBM, the term "associated" refers to two or more polypeptides. In particular, the term "associated" refers to the functional relationship between two or more polypeptide chains. The markers produce functional MBMs in which ABM1, ABM2, etc. can bind to their respective targets. to form a bond, for example, non-covalently or via one or more disulfides, by molecular interactions. Covalently associated with one another by crosslinking or chemical crosslinking. Examples of associations that may exist in MBMs include (but are not limited to) Fc in the Fc domain Interdomain association (homodimeric association or, more preferably, as described in Section 7.4.1.5) heterodimeric association), the association between the VH and VL regions in Fab or Fv and the association between the VH and VL regions in Fab The association between CH1 and CL is mentioned.

[0045] ABM chain: An individual ABM is expressed as a single polypeptide chain (e.g., in the case of an scFv). may be present or by association of two or more polypeptide chains (e.g., in the case of Fab) As used herein, the term "ABM chain" refers to a single polynucleotide. The term "ABM chain" refers to all or part of the ABM present on a peptide chain. are intended for convenience and illustrative purposes only and do not imply any particular form or method of manufacture. Not yet.

[0046] Host cell or recombinant host cell: The term "host cell" or "recombinant host cell" refers to a refers to cells that have been genetically modified, for example, by the introduction of heterologous nucleic acid. It is understood that the term "cell" is intended to refer not only to the particular subject cell but also to the progeny of such a cell. It should be understood that certain modifications may occur in subsequent generations, either due to mutation or environmental influences. Such progeny may or may not be identical to the parent cell, since they may exist in different cultures. However, they are still included within the scope of the term "host cell" as used herein. The host cell may be, for example, transiently expressing an extrachromosomal heterologous expression vector or, for example, The MBMs of the present disclosure can stably harbor heterologous nucleic acids by integration of the heterologous nucleic acids into the cell genome. For expression purposes, the host cell is preferably a monkey kidney cell (COS, e.g., COS -1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21 ), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human liver Cellular cancer cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby Bovine kidney (MDBK), myeloma and lymphoma cells or their derivatives and / or modifications and cell lines of mammalian origin or mammalian-like characteristics, such as mutants. For example, glycan profile modified and / or site-specific integrated site derivatives. do.

[0047] Sequence Identity: The term percent "identity" refers to the percentage of two or more nucleic acid or polypeptide sequences. In the context of a sequence, this refers to two or more sequences that are the same. When measured using one of the rhythms or by manual alignment and visual inspection , compared and aligned for maximum matches over a comparison window or specified region. If so, a specified percentage of amino acid residues or nucleotides that are the same (e.g., a specified 60% identity over a specified region or, if not specified, over the entire sequence, optionally Selectively 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 7 9%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 Two sequences are "substantially identical" if they share at least 9% identity (9% identity or less). Identity is measured over at least about 50 nucleotides (or in the case of a peptide or polypeptide). over a region of length (in this case, at least about 10 amino acids) or more preferably 100 ~500 or 1000 or more nucleotides (or 20, 50, 20 The nucleotide sequence is present over a region of length (0 or more amino acids).

[0048] For sequence comparison, typically one sequence acts as a reference sequence, When using a sequence comparison algorithm, test and reference sequences are compared. The coordinates of the subarray are specified and the array algorithm program is executed as needed. Program parameters are specified. Default program parameters can be used, or Alternative parameters can be designated. The sequence comparison algorithm then runs Based on the data, the percent sequence identity of the test sequence relative to the reference sequence is calculated. Methods for aligning sequences for comparison are well known in the art. The optimal alignment is determined, for example, by Smith and Waterman, 1970, A Need by the local homology algorithm of dv.Appl.Math.2:482c leman and Wunsch, 1970, J. Mol. Biol. 48:443. The homology alignment algorithm, Pearson and Lipman, 88, Proc. Nat'l. Acad. Sci. USA 85:2444 The search method used these algorithms (the Wisconsin Genetics GAP, BESTFIT, FASTA and TF in the Software Package ASTA(Genetics Computer Group,575 Science (Dr., Madison, WI)) computer-implemented or manual algorithms. ment and visual inspection (e.g., Brent et al., 2003, Curren (See Protocols in Molecular Biology) This can be done by:

[0049] Two examples of suitable algorithms for determining percent sequence identity and sequence similarity are , BLAST, and BLAST 2.0 algorithms, which are Alts chul et al.,1977,Nuc.Acids Res.25:3389-3 402; and Altschul et al., 1990, J. Mol. Biol. 21 5:403-410. Software for performing BLAST analysis is available from Applied Mathematics, vol. National Center for Biotechnology Information This information is publicly available from the National Institute of Standards and Technology (NIS).

[0050] The percent identity between two amino acid sequences is calculated using the method of Meyers and Miller, Using the algorithm from 1988, Comput. Appl. Biosci. 4:11-17 The algorithm uses the PAM120 residue weight table, 12 gaps, and The ALIGN program (vers) was used with a group length penalty of 1 and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences is Needleman and Wunsch, 1970, J. Mol. Biol. 48:444-453) algorithm, which , Blossom 62 matrix or PAM250 matrix and 1 Gap weights of 6, 14, 12, 10, 8, 6, or 4 and lengths of 1, 2, 3, 4, 5, or 6 The weights are incorporated into the GAP program in the GCG software package. .

[0051] Conservative sequence modification: The term "conservative sequence modification" refers to a modification of an MBM or a component thereof (e.g., Amino acids that do not substantially affect or alter the binding properties of the ABM or Fc region Such conservative modifications include amino acid substitutions, additions, and deletions. Standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, can be used. Conservative amino acid substitutions can be introduced into the MBMs of the present disclosure by conventional techniques. is replaced by an amino acid residue having a similar side chain. Families of acid residues have been defined in the art. These families include are amino acids with basic side chains (e.g., lysine, arginine, histidine), Amino acids with uncharged polar side chains (e.g., aspartic acid, glutamic acid) amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, thyroxine) amino acids with nonpolar side chains (e.g., alanine, cysteine, tryptophan); , valine, leucine, isoleucine, proline, phenylalanine, methionine), β- Amino acids with branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains Chain-containing amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine) Thus, one or more amino acid residues in the MBMs of the present disclosure may be located on the same side. Other amino acid residues from the chain family may be substituted, and the modified MBM may be, for example, Test for binding to molecules and / or effective heterodimerization and / or effector function It can be done.

[0052] Mutation or modification: As used herein, in reference to a polypeptide, The terms "mutation" and "modification" may include substitution, addition, or deletion of one or more amino acids. .

[0053] Antibody numbering system: As used herein, numbered amino acid residues in an antibody domain References to EU numbering systems are based on the EU numbering system unless otherwise specified. delman et al.,1969,Proc.Nat'l Acad.Sci.U SA 63:78-85, originally devised by Kabat et al., 1991 ,in Sequences of Proteins of Immunologic al Interest, US Department of Health and This is described in detail in Human Services, NIH, USA.

[0054] dsFv: The term "dsFv" refers to a disulfide-stabilized Fv fragment. In dsFv, VH and VL are linked by an interdomain disulfide bond. To generate a molecule such as are each mutated to cysteine, which then form stable interchain disulfide bonds Typically, position 44 in VH and position 100 in VL are mutated to cysteine. Brinkmann,2010,Antibody Engineering 181- 189,DOI:10.1007 / 978-3-642-01147-4_14 The term dsFv is known in the art. and VL are linked by an interchain disulfide bond rather than a linker peptide) or scdsFv (VH and VL are linked by a linker and an interchain disulfide bond) The term "antibody" encompasses both the α- and β-terminally substituted molecules.

[0055] Tandem of VH domains: As used herein, a "VH domain (or VH The term "tandem of VH domains" refers to a series of VH domains consisting of multiple identical VH domains of an antibody. Each VH domain, except for the last one at the end of the tandem, is connected to the linker - has its C-terminus linked to the N-terminus of another VH domain. has at least two VH domains, and in certain embodiments of the MBMs of the present disclosure, three , 4, 5, 6, 7, 8, 9 or 10 VH domains. Combinations with or without linkers that allow them to be produced as a single polypeptide chain Using a recombination method (e.g., as described in Section 7.4.3), each V The first VH domain in tandem can be generated by combining nucleic acids encoding the VH domains. The main N-terminus is defined as the N-terminus of the tandem, while the final VH domain of the tandem The C-terminus of the in is defined as the C-terminus of the tandem.

[0056] Tandem of VL domains: As used herein, a "VL domain (or VL The term "tandem of VL domains" refers to a series of VL domains consisting of multiple identical VL domains of an antibody. Each VL domain, except for the last one at the end of the tandem, is connected to the linker The VL has its C-terminus linked to the N-terminus of another VL, with or without a VL. In certain embodiments of the MBMs of the present disclosure, the MBMs may have at least two VL domains, and in certain embodiments of the MBMs of the present disclosure, the MBMs may have at least three, four, five, or five VL domains. , 6, 7, 8, 9 or 10 VL domains. Recombinant methods with or without linkers that allow the polypeptide chains to be produced 3. Isolate each VL domain in the desired order using a method (e.g., as described in Section 7.4.3). The first VL domain of the tandem can be produced by combining the coding nucleic acid of the first VL domain. The N-terminus is defined as the N-terminus of the tandem, while the C of the last VL domain of the tandem The termini are defined as the C-termini of the tandem.

[0057] Monovalent: The term "monovalent" as used herein in reference to antigen-binding molecules , refers to an antigen-binding molecule that has a single antigen-binding domain.

[0058] Bivalent: The term "bivalent" as used herein in reference to an antigen-binding molecule refers to an MBM having two antigen-binding domains, where one antigen-binding domain is C D3. The antigen-binding domains can be the same or different. Therefore, bivalent antigen binding The combined molecule can be monospecific or bispecific. An example of a bivalent MBM of the present disclosure is shown in Figure 3C. Schematically shown.

[0059] Trivalent: The term "trivalent" as used herein in reference to antigen-binding molecules refers to an antigen-binding molecule having three antigen-binding domains. Trivalent MBM refers to an antigen-binding molecule having three antigen-binding domains, including CD3, TAA The trivalent MBMs of the present disclosure each bind to a different antigen. An example of a trivalent MBM of the present disclosure is shown generally in Figure 3D. Schematically shown.

[0060] Tetravalent: The term "tetravalent" as used herein in reference to MBM refers to four The MBM of the present disclosure is tetravalent and binds to CD3 The tetravalent MBM of the present disclosure specifically binds to a TAA and at least one other antigen. Generally, two antigen-binding domains that bind to the same antigen (preferably a TAA) and one that binds to CD3. An example of a tetravalent MBM of the present disclosure is shown in Figure 3E. is shown diagrammatically in

[0061] Pentavalent: The term "pentavalent" as used herein in reference to MBM refers to five The MBM of the present disclosure is a pentaspecific antigen-binding molecule having an antigen-binding domain of , CD3, TAA, and three other antigens. A BM generally comprises (a) two pairs of antigen-binding domains, each of which binds to the same antigen, and a third (b) a single antigen-binding domain that binds to an antigen, or (b) three antigen-binding domains that bind to the same antigen. It has either one of two antigen-binding domains that bind to a different antigen and another that binds to a different antigen.

[0062] Hexavalent: The term "hexavalent" as used herein in reference to MBM refers to six The MBM of the present disclosure refers to an antigen-binding molecule having an antigen-binding domain of CD3, TAA, and and at least one other antigen. The hexavalent MBMs of the present disclosure generally Each has three pairs of antigen-binding domains that bind to the same antigen, but in different forms (e.g., T Three antigen-binding domains that bind to AA and at least one that binds to CD3 three antigen-binding domains that bind to a domain or TAA and at least one that binds to CD3 two antigen-binding domains) are within the scope of this disclosure.

[0063] Specific (or selective) binding: "specifically (or selectively)" binding to an antigen or epitope The term "bind" refers to the binding of a homologous antigen or enzyme to a heterogeneous population of proteins and other biological substances. The term "binding reaction" refers to a binding reaction that determines the presence of a target. It can be, but need not be, mediated by a 7th-generation ligand, e.g., a DARPin. The ABMs of this disclosure may also be mediated by any type of ABM described in Section 3. , typically 5 × 10 -2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 M not yet Full, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Less than M or 10 -9 Dissociation rate constant (KD) less than M ) (koff / kon) and its ability to bind to non-specific antigens (e.g., HSA). Binds to a target antigen with an affinity at least two times higher than the affinity of the target antigen. The term "antigen" does not exclude cross-species reactivity. For example, an antibody that "specifically binds" to an antigen from one species may be An antigen-binding module (e.g., an antigen-binding fragment of an antibody) that is capable of binding to one or more other species Therefore, such cross-species cross-reactivity can be achieved by "specifically binding" to the corresponding antigen in the The sex itself does not change the classification of an antigen binding module as a "specific" binder. In embodiments, an antigen binding module of the disclosure that specifically binds to a human antigen (e.g., ABM1, ABM2, etc.) are expressed in one or more non-human mammalian species, e.g., primate species (e.g., cynomolgus monkeys). Macaque (Macaca fascicularis), Rhesus macaque (Macaca mulata) atta) and pig-tailed macaques (Macaca nemestrina), , but not limited to) or rodent species such as the house mouse (Mus musculus In another embodiment, the antigen-binding module of the present disclosure has cross-species cross-reactivity with (e.g., ABM1, ABM2, etc.) do not have cross-species reactivity.

[0064] Monoclonal antibody: As used herein, the term "monoclonal antibody" refers to a The term refers to antibodies, antibody fragments, molecules (including MBMs) derived from the same genetic source. It refers to a polypeptide comprising:

[0065] Humanized: The term "humanized" forms of non-human (e.g., murine) antibodies refers to the use of non-human immunoglobulins. A humanized antibody is a chimeric antibody that contains minimal sequence derived from a human globulin. Residues from the recipient hypervariable region are selected to provide a polypeptide with the desired specificity, affinity, and potency. derived from the hypervariable regions of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate In some cases, the target antibody is a human immunoglobulin (recipient antibody) in which the target amino acid residues have been replaced by the target amino acid residues. , in which framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. Generally, humanization An antibody comprises substantially all of at least one, and typically two, variable domains, wherein: All or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and the FR All or substantially all of the antibody is human immunoglobulin lo sequence. Optionally, at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin For further details, see Jones et al., 1986, Nature 321:522-525;Riechmann et al.,1988 , Nature 332:323-329; and Presta, 1992, Curr. O See p. Struct. Biol. 2:593-596. References cited in: Vaswani and Hamilton, 1998, An n.Allergy,Asthma&Immunol.1:105-115;Harri s,1995,Biochem.Soc.Transactions 23:1035- 1038;Hurle and Gross,1994,Curr.Op.Biotec See also h.5:428-433.

[0066] Human antibody: As used herein, the term "human antibody" refers to a human antibody in the framework Also included are antibodies having variable regions in which both the CDR and CDR regions are derived from sequences of human origin. Additionally, if the antibody contains a constant region, the constant region may be derived from such human sequences, e.g., human germline sequences. Human germline sequences or mutants of the human germline sequence or, for example, Knappik et al. As described in J Mol Biol 296, 57-86, 2000, The antibody-containing consensus framework sequence derived from framework sequence analysis also The structure and location of immunoglobulin variable domains, e.g., CDRs, are determined according to well-known numbering schemes. numbering scheme, e.g., Kabat numbering scheme, Chothia numbering scheme, or Kab It can be defined using a combination of at and Chothia (e.g., Lazikani e t al.,1997,J.Mol.Bio.273:927 948;Kabat e et al., 1991, Sequences of Proteins of Immu. nological interest,5th edit.,NIH Publica tion no.91-3242 USDepartment of Health and Human Services;Chothia et al.,1987, J.Mol.Biol.196:901-917;Chothia et al.,19 89, Nature 342:877-883).

[0067] Human antibodies may contain amino acid residues that are not encoded by human sequences (e.g., amino acids derived from in vitro Introduced by random or site-specific mutation or in vivo somatic mutation However, the present invention may contain mutations that enhance the stability or production of the polypeptides. However, the term "human antibody" as used herein refers to antibodies derived from other mammals, such as mice. CDR sequences derived from the germline of a mammalian species are grafted onto human framework sequences. The term "antibodies" is not intended to include antibodies.

[0068] Chimeric antibody: The term "chimeric antibody" (or antigen-binding fragment thereof) refers to: (a) The constant region or a part thereof is a class of antigen-binding site (variable region) that is different or modified. The chimeric antibody may also contain other antibody-specific constant regions, such as the nucleotide sequence, effector function, and / or species constant region, or a whole antibody that confers new properties to the chimeric antibody. They can be modified to be linked to different molecules, such as enzymes, toxins, hormones, growth factors, drugs, etc. (b) the variable region or a portion thereof is different or modified; Antibody molecules (or their antigen binding domains) that have been modified, substituted, or replaced with variable regions that have antigen specificity. For example, mouse antibodies derive their constant regions from human immunoglobulins. The substitution with a human constant region can modify the The murine antibody has reduced antigenicity in humans compared to the original murine antibody, while retaining its anti- It is possible to retain the specificity of the original when recognizing it.

[0069] Effector function: The term "effector function" usually refers to the binding of an effector molecule. by binding through a domain of the antibody other than the antigen-binding domain, which is mediated by binding Effector functions refer to the activities of antibody molecules mediated by the antibody. Complement activation involves the activation of complement-mediated effector functions mediated by the binding of complement factors. It is important in the opsonization and lysis of vacuolar pathogens. Complement activation also stimulates the inflammatory response. Effector functions are mediated by Fc receptors (FcRs) and may also be involved in autoimmune hypersensitivity. Effector functions also include binding of the constant domain of an antibody to an Fc receptor (FcR) Binding of antibodies to Fc receptors on the cell surface can sometimes result in the formation of antibodies that are coated on the cell surface. Phagocytosis and destruction of infected particles, clearance of immune complexes, and antibody-coated particles by killer cells Lysis of target cells (called antibody-dependent cell-mediated cytotoxicity or ADCC), inflammatory mediators, It plays many important and diverse roles in the regulation of receptor release, placental transit, and immunoglobulin production. The effector functions of antibodies are expressed through binding to antigens such as Fc receptors or complement components. By altering, e.g., increasing or decreasing, the affinity of the antibody for the effector molecule. Binding affinity can generally be altered by modifying the effector molecule binding site. In this case, the region of interest is located and at least a portion of this region is subjected to a suitable It is suitable to modify the antibody by the following method. The mutation need not substantially alter overall binding affinity, but may counteract effector mechanisms. It is also conceivable that the geometry of the interaction can be altered to nullify the binding. Effector functions can also be involved in the binding of effector molecules but not directly. It is further believed that the target function can be altered by modifying sites that are otherwise involved in the performance of target function. It is also possible that

[0070] Recognize: The term "recognize" as used herein refers to the ability of an individual to recognize an epitope. ABM refers to an ABM that finds a target and interacts with (e.g., binds to) it.

[0071] Epitope: An epitope or antigenic determinant is a molecule that binds to an antibody or other antigen described herein. The portion of an antigen recognized by a binding domain. An epitope may be linear or conformational. It is possible.

[0072] Nucleic Acid: The term "nucleic acid" is used interchangeably herein with the term "polynucleotide." and deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form. The term refers to nucleotides and their polymers, including synthetic, natural, and non-natural nucleotides. A known nucleotide that has similar binding properties to the reference nucleic acid and is metabolized similarly to the reference nucleotide. These include nucleic acids containing nucleotide analogs or modified backbone residues or linkages. Examples of thiol groups include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphatides, and the like. Sulfonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptides -nucleic acids (PNA).

[0073] Unless otherwise indicated, a particular nucleic acid sequence refers to the nucleic acid sequence, not just the sequence explicitly indicated. Conservatively modified variants of a sequence (e.g., degenerate codon substitutions) and complementary sequences are also implicitly encompassed. Specifically, as detailed below, degenerate codon substitutions are made by using one or more selected ( or all) codons with a substitution of mixed base and / or deoxyinosine residues at the third position. This can be achieved by generating sequences (Batzer et al., (1991) Nucleic Acid Res.19:5081;Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossol ini et al.,(1994)Mol.Cell.Probes 8:91-98 ).

[0074] Vector: The term "vector" refers to a vector that transports another polynucleotide to which it is linked. A vector is intended to refer to a polynucleotide molecule capable of carrying out the steps of: is a "plasmid," which is a vector into which additional DNA segments can be ligated. Another type of vector is a viral vector. wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial bacterial vectors with an origin of replication and episomal mammalian vectors). Other vectors (e.g. (e.g., non-episomal mammalian vectors) are vectors that, upon introduction into a host cell, are integrated into the genome of the host cell. In addition, certain vectors can be integrated into the host genome, thereby replicating along with the host genome. They are capable of inducing the expression of genes to which they are operably linked. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA techniques are in the form of plasmids. Since plasmids are the most commonly used form of vector, In the specification, the terms "plasmid" and "vector" may be used interchangeably. However, the present disclosure also provides viral vectors (e.g., replication-deficient retroviruses) that perform equivalent functions. such other forms of expression vectors (e.g., HIV, adenoviruses and adeno-associated viruses) is intended to include.

[0075] Combination Sequence: With reference to the Table (including its subsections), the term "combination sequence" refers to the "ABM" refers to an ABM having a complete set of CDRs, a VH-VL pair, or an scFv listed in the table.

[0076] VH-VL or VH-VL pair: refers to a VH-VL pair, either on the same polypeptide chain or on different polypeptide chains. Regardless of which polypeptide chains are involved, the terms "VH-VL" and "VH-VL pair" refer to are used for convenience and are not intended to indicate any particular orientation unless the context requires otherwise. Therefore, scFs containing "VH-VL" or "VH-VL pairs" are not intended to be synonymous with "VH-VL" or "VH-VL pairs." v can be VH and VL in any orientation, e.g., VH N-terminus to VL or VL N-terminus to VH. and a VL domain.

[0077] Polypeptides and proteins: The terms "polypeptide" and "protein" refer to The term is used interchangeably herein to refer to a polymer of amino acid residues. An amino acid in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acid It also applies to polymers and to natural and non-natural amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof. do.

[0078] Subject: The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates. Animals, e.g., mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles Unless otherwise specified, the term "patient" or "subject" refers to a mammal or non-mammal. Used interchangeably herein.

[0079] Cancer: The term "cancer" refers to the uncontrolled (often rapid) growth of abnormal cells. Cancer cells spread to the rest of the body either locally or through the bloodstream and lymphatic system. It may spread to other sites. Examples of various cancers are described herein, including but not limited to: , breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer Cancer, adrenal cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive cancer, colon cancer Cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer , gastric cancer, testicular cancer, thymic cancer, thyroid cancer, upper aerodigestive cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma Any TAA-positive cancer of any of the aforementioned types, for example, leukemia, lung cancer, etc. do.

[0080] Tumor: The term "tumor" is used interchangeably herein with the term "cancer." For example, both terms encompass solid and liquid, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" refers to pre-malignant as well as malignant cancers and This includes tumors.

[0081] Tumor-associated antigen: The term "tumor-associated antigen" or "TAA" refers to a molecule that is present on the surface of a cancer cell. It is expressed either as a whole or as a fragment (e.g., MHC / peptide) and acts on cancer cells. Molecules (typically proteins, carbohydrates, lipids) that are useful for preferential targeting of drugs In one embodiment, the TAA refers to a normal cell and Markers expressed by both cancer cells and cells, e.g., cell lineage markers, e.g., B cell markers In one embodiment, the TAA is CD19, which is expressed in cancer cells compared to normal cells. Overexpressed (e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or more overexpression compared to normal cells) In one embodiment, the TAA is a cell surface molecule that is expressed on cancer cells, e.g., Inappropriate sequences within a molecule containing deletions, additions, or mutations compared to the molecule expressed in normal cells In one embodiment, the TAA is a cell surface molecule that is selectively synthesized on the cell surface of a cancer cell. expressed entirely or as fragments (e.g., MHC / peptide) on the surface only , are not synthesized or expressed on the surface of normal cells. Hence, the term "TAA" , sometimes known in the art as tumor-specific antigens ("TSAs"), It includes cell-specific antigens.

[0082] Treat, Treatment, Treating: As used herein, "treat," "cure," The terms "therapy" and "treating" refer to a therapeutic agent resulting from the administration of one or more MBMs of the present disclosure. , a reduction or amelioration of the progression, severity and / or duration of a proliferative disorder or a reduction or amelioration of one of the proliferative disorders In certain embodiments, the term "improvement" refers to the improvement of one or more symptoms (preferably one or more identifiable symptoms). In this context, the terms "treat," "treatment," and "treating" are not necessarily used by patients. at least one measurable physical symptom of a proliferative disorder, such as tumor growth, that is not identified by In other embodiments, "treat," "treatment," and "treating" refer to an improvement in a patient's condition. The term "physical" refers to, for example, the stabilization of discernible symptoms, e.g., physical The stabilization of parameters may result in the inhibition of the progression of proliferative disorders, either physiologically or both. In other embodiments, the terms "treat," "treatment," and "treating" are used interchangeably. The term refers to a reduction or stabilization of tumor size or cancerous cell number.

[0083] 7.2.CD3 binding molecules In one aspect, the present disclosure includes monospecific and multispecific molecules that bind to human CD3. In one embodiment, the CD3 binding molecule is a monospecific For example, a monospecific binding molecule may be an antibody or an antigen-binding fragment thereof (e.g., For example, antibody fragments, scFv, dsFv, Fv, Fab, scFab, (Fab In another embodiment, the antibody may be a CD3′ antibody or a single domain antibody (SDAB). The binding molecule may be a multispecific (e.g., bispecific) CD3 binding molecule (e.g., bispecific antibody body).

[0084] In one embodiment, the CD3 binding molecule is a chimeric or humanized monoclonal antibody. Chimeric and / or humanized antibodies are antibodies produced in non-human subjects or derived from non-human antibody genes. and modifying the antibody to minimize an immune response by a human patient to antibodies derived from expression of the antibody. A chimeric antibody contains a non-human animal antibody variable region and a human antibody constant region. Such antibodies retain the epitope binding specificity of the original monoclonal antibody but are suitable for human administration. may be less immunogenic when administered, making them more likely to be tolerated by patients For example, one or more of the variable regions of the light chain of a mouse antibody (e.g., a mouse monoclonal antibody) and / or one or all (e.g., one, two, or three) of the variable regions of the heavy chain. For example, one, two, or three) may each be, but are not limited to, an IgG1 human constant region. Chimeric monoclonal antibodies can bind to human constant regions that are not expressed in human antibodies. They can be produced by recombinant DNA technology. For example, the constant region of a non-human antibody molecule can be cloned. The coding gene can be replaced with a gene encoding a human constant region (Robi nson et al., PCT / US86 / 02269; Akira, et al. al., European Patent Application No. 184,187; or Taniguchi, M., European (See U.S. Patent Application No. 171,496.) In addition, to generate chimeric antibodies, Other suitable techniques that can be used for this purpose are described, for example, in U.S. Pat. No. 4,816,567. 4,978,775; 4,975,369; and No. 4,816,397.

[0085] Chimeric or humanized antibodies and antigen-binding fragments thereof of the present disclosure may be derived from mouse monoclonal antibodies. The sequences encoding the heavy and light immunoglobulin chains can be prepared based on the sequences of the human antibody. The DNA was obtained from the mouse hybridoma of interest and purified using standard molecular biology techniques. can be modified to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to produce a chimeric antibody, murine variable regions can be fused to human antibodies using known methods. It can be linked to a constant region (see, e.g., U.S. Patent No. 6,229,493 to Cabilly et al.). (See, for example, US Pat. No. 4,816,567.) Known methods for producing humanized antibodies are available. Methods can be used to insert the murine CDR regions into a human framework. For example, Winter in U.S. Pat. No. 5,225,539 and U.S. Pat. No. 1; No. 5,585,089; No. 5,693,762 and See Queen et al., US Pat. No. 6,180,370.

[0086] Humanized antibodies include, but are not limited to, CDR grafting (see, e.g., European Patent No. 239,400 Specification; International Publication No. 91 / 09967; U.S. Patent No. 5,225,539 See US Pat. Nos. 5,530,101 and 5,585,089. veneering or resurfacing (see, for example, European Patent No. 592,106 Padlan, 1991, Molecular Immunology,28(4 / 5):489-498;Studnicka et al. al.,1994,Protein Engineering,7(6):805-81 4; and Roguska et al., 1994, PNAS, 91:969-973 chain shuffling (see, e.g., U.S. Pat. No. 5,565,332); See, for example, U.S. Patent Application Publication No. 2005 / 0042664. Publication No. 2005 / 0048617, U.S. Pat. No. 6,407,200 No. 13, U.S. Pat. No. 5,766,886, WO 9317105 Nflett, Tan et al., J. Immunol., 169:1119-25( 2002), Caldas et al., Protein Eng., 13(5):3 53-60(2000), Morea et al.,Methods,20(3):2 67-79(2000), Baca et al., J. Biol. Chem., 272 (16):10678-84(1997), Roguska et al., Prote in Eng.,9(10):895-904(1996), Couto et al. ,Cancer Res.,55(23 Supp):5973s-5977s(199 5), Couto et al., Cancer Res., 55(8):1717-2 2(1995), Sandhu JS, Gene, 150(2):409-10(19 94) and Pedersen et al., J. Mol. Biol., 235(3): 959-73 (1994) Often, framework residues in the framework regions can be selected to enhance antigen binding. To alter, e.g., improve, the conformation of a CDR, the corresponding residue from the CDR donor antibody is substituted. These framework substitutions, e.g., conservative substitutions, can be made using known methods, e.g., those that affect antigen binding. Modeling the interactions between CDR and framework residues to identify important framework residues by sequence comparison to identify unusual framework residues at specific positions and (See, e.g., Queen et al., U.S. Pat. No. 5,585,089.) and Riechmann et al., 1988, Nature, 332:3 23).

[0087] As provided herein, a humanized antibody or antibody fragment may comprise a humanized antibody or antibody fragment having a framework amino acid sequence similar to that of a humanized antibody or antibody fragment. Non-human immunoglobulin molecules and frameworks derived entirely or predominantly from the human germline For humanization of antibodies or antibody fragments, the antibody may contain one or more CDRs from the target region. Several techniques are well known for translating rodent CDRs or CDR sequences into corresponding sequences of human antibodies. CDR grafting (European Patent No. 239,400; International Publication No. Patent Publication No. 91 / 09967; and U.S. Patent No. 4,816,567; Specification No. 6,331,415; Specification No. 5,225,539; Specification No. 5,530,1 01; 5,585,089; 6,548,640) According to the method of Winter and coworkers (Jones et al., Nature ,321:522-525(1986);Riechmann et al.,Natu re,332:323-327(1988);Verhoeyen et al.,Sc ience, 239:1534-1536 (1988) In such humanized antibodies and antibody fragments, substantially less than one In this case, an intact human variable domain is replaced by the corresponding sequence from a non-human species. A modified antibody often has some CDR residues and possibly some framework (FR) residues that are Human antibodies in which residues from analogous sites in rodent antibodies are substituted. Humanization of body fragments is also used for veneering or resurfacing (European Patent No. 592,1 06; European Patent No. 519,596; Padlan, 1991, Mole cular Immunology,28(4 / 5):489-498;Studnic ka et al., Protein Engineering, 7(6):805-8 14(1994); and Roguska et al., PNAS, 91:969-97 3 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332 ) can also be achieved.

[0088] The choice of human variable domains, both light and heavy, to use in making the humanized antibodies depends on the antigen The so-called "best fit" method allows the identification of the variable domains of rodent antibodies. The sequence of the gene is screened against the entire library of known human variable domain sequences. Next, the human sequence that is closest to the rodent sequence is the human framework (F) of the humanized antibody. R) (Sims et al., J. Immunol., 151:22 96(1993);Chothia et al., J.Mol.Biol.,196: Another method involves the isolation of whole antibody components of specific subgroups of light or heavy chains. Use a specific framework derived from the census sequence. The same framework can be used for several can be used for different humanized antibodies (e.g., Nicholson et al. Mol. I mmun.34(16-17):1157-1165(1997);Carter et al.,Proc.Natl.Acad.Sci.USA,89:4285(1992 ); Presta et al., J. Immunol., 151:2623 (1993 In some embodiments, the framework regions of the heavy chain variable region, e.g., For example, all four framework regions are derived from the VH4_4-59 germline sequence. In some embodiments, the framework regions may be, for example, identical to the amino acids of the corresponding murine sequence. These may include one, two, three, four, or five modifications, e.g., substitutions, e.g., conservative substitutions. In one embodiment, the framework regions of the light chain variable region, e.g., all four frames, The network region is derived from the VK3_1.25 germline sequence. The framework regions may be, for example, one, two, three or more amino acids from the corresponding murine sequence. The amino acid sequence may include one, four or five modifications, eg substitutions, eg conservative substitutions.

[0089] In certain embodiments, the CD3 binding molecule is selected from the group consisting of a specific germline heavy chain immunoglobulin gene. heavy chain variable region from a gene and / or light chain from a specific germline light chain immunoglobulin gene For example, such antibodies may be "products" of particular germline sequences. Human antibodies include or are derived from heavy or light chain variable regions. It can be "the product of" or "derived from" human germline immunoglobulin sequences. Human antibodies are those that derive their amino acid sequence from the amino acid sequence of a human germline immunoglobulin. and (using the methods outlined herein) the sequences closest to those of human antibodies ( By selecting a human germline immunoglobulin sequence (i.e., the highest percent identity), can be identified as "products" of specific human germline immunoglobulin sequences. Human antibodies are those produced or "derived" from antibodies that have undergone, for example, naturally occurring somatic mutations or deliberate mutations. May contain amino acid differences relative to the germline sequence due to the introduction of site-directed mutations However, humanized antibodies typically contain amino acid sequences that are identical to those of human germline antibodies. At least 90% identical to the amino acid sequence encoded by the globulin gene, compared to the germline immunoglobulin amino acid sequence of the species (e.g., mouse germline sequence) If so, the antibody contains amino acid residues that identify it as derived from a human sequence. In this case, a humanized antibody is one that is identical in amino acid sequence to a human antibody derived from a germline immunoglobulin gene. at least 95, 96, 97, 98, or 99% or less of the amino acid sequence encoded by may be at least 96%, 97%, 98% or 99% identical. Typically, Humanized antibodies derived from human germline sequences are derived from human germline immunoglobulin genes. The amino acid sequence encoded by the nucleotide sequence of the present invention is different from that of the nucleotide sequence of the present invention by 10 to 20 amino acids or less (as used herein, before the introduction of any skew, pI and ablation mutations that may be present; i.e., (Before the introduction of these mutations, the number of mutations is generally small.) In certain cases, humanized antibodies are 5 or fewer amino acids from the amino acid sequence encoded by the cellular immunoglobulin gene , 3, 2, or 1 amino acid difference or less (again, as used herein). before the introduction of any distortion, pI and ablation mutations in the Before introduction, the number of mutants is generally low).

[0090] In one embodiment, the parent antibody is affinity matured. Structure-based methods include, for example, , used for humanization and affinity maturation, as described in USSN 11 / 004,590. Examples of such methods include, but are not limited to, Wu et al., 1999, J. Mol. Biology ol.294:151-162;Baca et al.,1997,J.Biol.C hem.272(16):10678-10684;Rosok et al.,199 6,J.Biol.Chem.271(37):22611-22618;Rader et al.,1998,Proc.Natl.Acad.Sci.USA 95:89 10-8915;Krauss et al.,2003,Protein Engine Selection-based methods, including those described in [Publication ID No. 16(10):753-759]. Methods can be used to humanize and / or affinity mature antibody variable regions. Methods for condensation include, but are not limited to, those described in USSN 09 / 810,510; Tan et al. ,2002,J.Immunol.169:1119-1125;De Pascali s et al., 2002, J. Immunol. 169:3076-3084 This may involve grafting of only a portion of the CDRs, including methods such as those described above.

[0091] In one embodiment, the CD3 binding molecule comprises an ABM that is a Fab. The immunoglobulins are produced by the proteolytic cleavage or assembly of immunoglobulin molecules using enzymes such as papain. The Fab domain is typically linked to a VL domain. It contains a CH1 domain linked to a VH domain paired with a CL domain. In globulins, the VH domain is paired with the VL domain to form the Fv region, and The H1 domain is paired with the CL domain to further stabilize the binding module. Disulfide bonds between the constant domains may further stabilize the Fab domain.

[0092] In one embodiment, the CD3 binding molecule comprises an ABM that is an scFab. In this embodiment, the antibody domains and linker in the scFab fragment are arranged in the N-terminus to C-terminus one of the following sequences end-to-end: a) VH-CH1-linker-VL-CL or b) VL In some cases, the VL-CL-linker-VH -CH1 is used.

[0093] In another embodiment, the antibody domains and linker in the scFab fragment are In one of the following orders from the terminal to the C-terminus: a) VH-CL-linker-VL-CH1 or b) VL-CH1-linker-VH-CL.

[0094] Optionally, in the scFab fragment, a CL-domain and a CH1 domain In addition to the natural disulfide bond between the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL), The domain (VL) consists of the following positions: i) heavy chain variable domain position 44 and light chain variable domain position 1 00, ii) position 105 in the heavy chain variable domain and position 43 in the light chain variable domain, or iii) Chain variable domain position 101 and light chain variable domain position 100 (EU index of Kabat) Disulfide stabilization can also be achieved by the introduction of a disulfide bond between the amino acid residues (numbered by [number]).

[0095] This additional disulfide stabilization of the scFab fragments allows for the synthesis of single-chain Fab fragments. This is achieved by the introduction of a disulfide bond between the variable domains VH and VL of the fragment. Techniques for introducing non-native disulfide bridges for the stabilization of single-chain Fvs include, for example: , International Publication No. 94 / 029350, Rajagopal et al., 1997,Prot.Engin.10:1453-59;Kobayashi et. al.,1998,Nuclear Medicine&Biology,25:387 -393; and Schmidt, et al., 1999, Oncogene 18:1 In one embodiment, the possibility of scFab fragments is described in The optional disulfide bond between the variable domains is at position 44 of the heavy chain variable domain and the light chain variable domain. In one embodiment, the variable domain of the scFab fragment is between positions 100 and 110. The optional disulfide bond between heavy chain variable domain position 105 and light chain variable domain position 4 It is between third and fourth place (numbering according to Kabat's EU index).

[0096] In one embodiment, the CD3 binding molecule comprises an ABM that is an scFv. An antibody fragment comprises the VH and VL domains of an antibody in a single polypeptide chain. It can be expressed as a single polypeptide chain and has the specificity of the intact antibody from which it is derived. Generally, the scFv polypeptide is designed to retain the desired structure for target binding. Furthermore, a polypeptide linker between the VH and VL domains that allows the formation of a VH structure is also provided. Examples of linkers suitable for linking the VH and VL chains of scFVs are described in Section 7.4.3. Any of the ABM linkers identified in It is Reka.

[0097] Unless otherwise specified, as used herein, scFv refers to, e.g., a polypeptide and may have the VL and VH variable regions in either order with respect to the N- and C-termini of the peptide, The scFv may comprise VL-linker-VH or VH-linker-VL.

[0098] To generate scFv-encoding nucleic acids, VH- and VL-encoding DNA fragments are 7.4.3, encoding a linker, e.g., any of the linkers described in Section 7.4.3 (amino acid A separate fragment encoding the sequence (Gly4-Ser) (SEQ ID NO: 47) VL and VH domains are movably linked together, with the VL and VH domains connected by a flexible linker. and VL sequences can be expressed as a contiguous single chain protein (e.g. Bird et al., 1988, Science 242:423-426; uston et al.,1988,Proc.Natl.Acad.Sci.USA 85:5879-5883; McCafferty et al., 1990, Nat. See ure 348:552-554).

[0099] CD3 binding molecules include Fv, dsFv, (Fab')2, and single domain antibodies (SDAB). , VH or VL domains or camelid VHH domains (also called nanobodies) ) may also include ABM.

[0100] CD3-binding molecules consist of a single VH or VL domain that exhibits sufficient affinity for CD3. In one embodiment, the single domain antibody may comprise a single domain antibody composed of Camelidae VHH domains (e.g., Riechmann, 1999, Journa l of Immunological Methods 231:25-38;International (See brochure No. 94 / 04678).

[0101] Tables 1A-1J-2 (collectively "Table 1") show exemplary CD3 binding molecules that may be included in a CD3 binding molecule. List the sequences of the 3-linked sequences.

[0102] [Table 1]

[0103] [Table 2]

[0104] [Table 3]

[0105] [Table 4]

[0106] [Table 5]

[0107] [Table 6]

[0108] [Table 7]

[0109] [Table 8]

[0110] [Table 9]

[0111] [Table 10]

[0112]

Table 11

[0113]

Table 12

[0114]

Table 13

[0115]

Table 14

[0116]

Table 15

[0117] Table 16

[0118]

Table 17

[0119]

Table 18

[0120]

Table 19

[0121] Table 20

[0122] Table 21

[0123] Table 22

[0124] Table 23

[0125] Table 24

[0126] Table 25

[0127] Table 26

[0128] Table 27

[0129] Table 28

[0130] Table 29

[0131]

Table 30

[0132] Table 31

[0133] Table 32

[0134] Table 33

[0135] Table 34

[0136] Table 35

[0137] Table 36

[0138] Table 37

[0139] Table 38

[0140] Table 39

[0141] Table 40

[0142] Table 41

[0143] Table 42

[0144] Table 43

[0145] Table 44

[0146] Table 45

[0147] Table 46

[0148] Table 47

[0149] Table 48

[0150] Table 49

[0151]

Table 50

[0152] Table 51

[0153] Table 52

[0154] Table 53

[0155] Table 54

[0156] Table 55

[0157] Table 56

[0158] Table 57

[0159] Table 58

[0160] Table 59

[0161] Table 60

[0162] Table 61

[0163] Table 62

[0164] Table 63

[0165] Table 64

[0166] Table 65

[0167] Table 66

[0168] Table 67

[0169] Table 68

[0170] Table 69

[0171] Table 70

[0172] Table 71

[0173] Table 72

[0174] Table 73

[0175] [Table 74]

[0176] [Table 75]

[0177] [Table 76]

[0178] [Table 77]

[0179] [Table 78]

[0180] [Table 79]

[0181] [Table 80]

[0182] [Table 81]

[0183] [Table 82]

[0184] Tables 1A-1C provide CD3 binding molecules based on the CDR sequences of exemplary CD3 binding molecules described herein. The Group C1 CDR sequences in Table 1A are exemplary CD3 The binding molecules NOV292, NOV589, NOV567 and other binding agents containing "sp11a" in their names Exemplary CD3 binding molecules including Kabat CDR sequences, Chothia CDR sequences, I Based on the MGT CDR sequences and combinations thereof. Group C2 CDRs in Table 1B The sequences are shown for exemplary CD3 binding molecules NOV453, NOV229, NOV580, NOV2 The Kabat CDR sequences of exemplary CD3 binding molecules, including 21 and "sp9a" in the binder name, are Based on sequences, Chothia CDR sequences, IMGT CDR sequences, and combinations thereof The Group C3 CDR sequences in Table 1C are those of the exemplary CD3 binding molecule NOV123, s Kabat CDR sequences of p10b, NOV110 and NOV832, Chothia The sequences are based on the CDR sequences, the IMGT CDR sequences and combinations thereof.

[0185] The specific CDR sequences of the exemplary CD3 binding molecules described in the Examples are listed in Tables 1B-1 through 1H. Exemplary VH and VL sequences are listed in Tables 1J-1 and 1J-2, respectively. are listed in.

[0186] In one embodiment, the CD3 binding molecule is a CD3 binding molecule selected from the group consisting of CD3 binding molecules listed in Table 1A, Table 1B, or Table 1C. The heavy chain CDRs have the amino acid sequence of any one of the DR consensus sequences. In embodiments, the present disclosure provides a heavy chain CDR selected from those set forth in Table 1A, Table 1B, or Table 1C. The heavy chain CDRs may be selected from one, two, three or more heavy chain CDRs (or alternatively, The present invention provides a CD3 binding molecule comprising:

[0187] In one embodiment, the CD3 binding molecule is a CD3 binding molecule selected from the group consisting of CD3 binding molecules listed in Table 1A, Table 1B, or Table 1C. The light chain CDRs have the amino acid sequence of any one of the DR consensus sequences. In embodiments, the present disclosure provides a light chain CDR selected from those set forth in Table 1A, Table 1B, or Table 1C. and / or a combination of one, two, three or more light chain CDRs selected from the group consisting of: The present invention provides a CD3 binding molecule comprising:

[0188] In one embodiment, the CD3 binding molecule comprises a CDR-H1 sequence as set forth in Table 1A, CDR-H2 sequence, CDR-H3 sequence, CDR-L1 sequence, CDR-L2 sequence and CDR- Contains the L3 sequence.

[0189] In one embodiment, the amino acid designated X1 in Table 1A is T. In some embodiments, the amino acid designated X1 in Table 1A is A. In one embodiment, the amino acid designated X2 in Table 1A is S. The amino acid designated X2 in Table 1A is R. In one embodiment, the amino acid designated X2 in Table 1A is R. The amino acid designated X3 in Table 1A is N. The amino acid designated X3 in Table 1A is Y. In one embodiment, the amino acid designated X4 in Table 1A is Q. In one embodiment, the amino acid designated X4 in Table 1A is In one embodiment, the amino acid designated X5 in Table 1A is M. In one embodiment, the amino acid designated X5 in Table 1A is L. In some embodiments, the amino acid designated X6 in Table 1A is K. In one embodiment, the amino acid designated X6 in Table 1A is R. The amino acid designated X7 in Table 1A is S. In one embodiment, the amino acid designated X7 in Table 1A is S. The amino acid designated X7 in Table 1A is K. 55 The amino acid designated X in Table 1A is F. In one embodiment, 55 and The amino acid represented is Y. In one embodiment, X in Table 1A 55 It is shown that In one embodiment, the amino acid designated X8 in Table 1A is S. In one embodiment, the amino acid designated X8 in Table 1A is Y In one embodiment, the amino acid designated X8 in Table 1A is S. In one embodiment, the amino acid designated X8 in Table 1A is T. In some embodiments, the amino acid designated X9 in Table 1A is W. In one embodiment, the amino acid designated X9 in Table 1A is Y. The amino acid designated X9 in Table 1A is S. The amino acid designated X9 in Table 1A is T. In one embodiment, the amino acid designated X1 in Table 1A is T. The amino acids designated X in Table 1A are H. 10 and indicated The amino acid selected from X in Table 1A is Y. 11 It is shown as The amino acid is S. In one embodiment, X in Table 1A 11 Amino acid The acid is G. In some embodiments, X in Table 1A 12 The amino acid indicated is In one embodiment, X in Table 1A is I. 12 The amino acid indicated is L. In one embodiment, X in Table 1A 13 The amino acid indicated is V. In some embodiments, X in Table 1A 13 The amino acid indicated is G. In the form of X in Table 1A 14 In some embodiments, the amino acid indicated as In Table 1A, X 14 In one embodiment, the amino acid indicated as , X in Table 1A 15 In one embodiment, the amino acid shown in Table 1 is D. X in A 15 The amino acid indicated as follows is E. In one embodiment, the amino acid indicated as follows in Table 1A is E. IteX 15 The amino acid designated X in Table 1A is L. 16 The amino acid designated X in Table 1A is G. 16 and The amino acid shown is N. In one embodiment, X in Table 1A 16 It is shown that The amino acid in Table 1A is E. 17 The net shown as The acid is R. In some embodiments, X in Table 1A 17 The amino acid indicated is , S. In one embodiment, X in Table 1A 18 The amino acid shown is V. In some embodiments, X in Table 1A 18 The amino acid indicated is T. In one embodiment, X in Table 1A 19 The amino acid indicated as is N. In an embodiment, X in Table 1A 19The amino acid indicated as follows is T. In Table 1A, X 20 The amino acid indicated as is R. Therefore, in Table 1A, X 20 In one embodiment, the amino acid shown in Table X in 1A 21 The amino acid indicated in Table 1A is F. Place X 21 The amino acid indicated in Table 1A is E. X 22 The amino acid designated X in Table 1A is S. In one embodiment, 22 The amino acid designated X in Table 1A is Y. 23 and indicated The amino acid selected from the group consisting of X and S in Table 1A is S. 23 A shown as The amino acid is Y. In one embodiment, X in Table 1A 24 Amino acids indicated as is S. In one embodiment, X in Table 1A 24 The amino acid indicated as A In one embodiment, in Table 1A, X 25 The amino acid indicated is H. In one embodiment, X in Table 1A 25 The amino acid indicated as is T. In an embodiment, X in Table 1A 26 The amino acid indicated as follows is F. In this state, X in Table 1A 26 The amino acid indicated as follows is Y. In Table 1A, X 27 is W. In one embodiment, In Table 1A, X 27 The amino acid indicated is Y.

[0190] In one embodiment, the CD3 binding molecule comprises the CDR-H1 sequence C1-1. In some embodiments, the CD3 binding molecule comprises the CDR-H1 sequence C1-2. In one embodiment, the CD3 binding molecule comprises the CDR-H1 sequence C1-3. The CD3 binding molecule comprises the CDR-H1 sequence C1-4.

[0191] In one embodiment, the CD3 binding molecule comprises the CDR-H2 sequence C1-5. In some embodiments, the CD3 binding molecule comprises the CDR-H2 sequence C1-6. wherein the CD3 binding molecule comprises the CDR-H2 sequence C1-7.

[0192] In one embodiment, the CD3 binding molecule comprises the CDR-H3 sequence C1-8. In some embodiments, the CD3 binding molecule comprises the CDR-H3 sequence C1-9. In one embodiment, the CD3 binding molecule comprises the CDR-H3 sequence C1-10. , the CD3 binding molecule comprises the CDR-H3 sequence C1-11.

[0193] In one embodiment, the CD3 binding molecule comprises the CDR-L1 sequence C1-12. In an embodiment, the CD3 binding molecule comprises the CDR-L1 sequence C1-13. In one embodiment, the CD3 binding molecule comprises the CDR-L1 sequence C1-14. In one embodiment, the CD3 binding molecule comprises the CDR-L1 sequence C1-15. The CD3 binding molecule comprises the CDR-L1 sequence C1-16. The binding molecule comprises the CDR-L1 sequence C1-17.

[0194] In one embodiment, the CD3 binding molecule comprises the CDR-L2 sequence C1-18. In an embodiment, the CD3 binding molecule comprises the CDR-L2 sequence C1-19.

[0195] In one embodiment, the CD3 binding molecule comprises the CDR-L3 sequence C1-20. In an embodiment, the CD3 binding molecule comprises the CDR-L3 sequence C1-21. In one embodiment, the CD3 binding molecule comprises the CDR-L3 sequence C1-22. In this case, the CD3 binding molecule comprises the CDR-L3 sequence C1-23.

[0196] In one embodiment, the CD3 binding molecule comprises a CDR-H1 sequence as set forth in Table 1B, CDR-H2 sequence, CDR-H3 sequence, CDR-L1 sequence, CDR-L2 sequence and CDR- Contains the L3 sequence.

[0197] In some embodiments, X in Table 1B 28 The amino acid indicated as is V. In an embodiment, X in Table 1B 28 The amino acid designated as is I. In this state, X in Table 1B 29 The amino acid indicated as follows is F. In Table 1B, X 29 is Y. In one embodiment, In Table 1B, X 30 In one embodiment, the amino acid shown in Table 1B is N. In X 30 In one embodiment, the amino acid indicated in Table 1B is S. TeX 31 In one embodiment, the amino acid indicated as X3 in Table 1B is A. The amino acid designated X in Table 1B is S. 32and indicated The amino acid selected from the group consisting of X and X in Table 1B is T. 32 It is shown as The amino acid is K. In one embodiment, X in Table 1B 33 Amino acid The acid is T. In some embodiments, X in Table 1B 33 The amino acid indicated is In some embodiments, X in Table 1B is 34 The amino acid indicated is S. In one embodiment, X in Table 1B 34 The amino acids indicated as R are In some embodiments, X in Table 1B 35 The amino acids indicated as ' are N'. In the form of X in Table 1B 35 The amino acid indicated as follows is G. In Table 1B, X 36 The amino acid indicated as follows is S. In one embodiment , X in Table 1B 36 In one embodiment, the amino acid shown in Table 1 is A. X in B 37 In one embodiment, the amino acid in Table 1B is A. IteX 37 The amino acid designated X in Table 1B is T. 37 The amino acid designated X in Table 1B is S. 38 and The amino acid shown is N. In one embodiment, X in Table 1B 38 It is shown that The amino acid in Table 1B is D. 39 The net shown as In some embodiments, the X acid in Table 1B is N. 39 The amino acid indicated is , K. In some embodiments, X in Table 1B 40 The amino acid shown is D. In some embodiments, X in Table 1B 40 The amino acid indicated is N. In some embodiments, X in Table 1B 41 The amino acid indicated as is H. In an embodiment, X in Table 1B 41 The amino acid indicated as is N. In Table 1B, X 42 The amino acid indicated as follows is Q. Therefore, in Table 1B, X 42 The amino acid shown in Table 1 is E. X in 1B 43 In one embodiment, an amino acid shown in Table 1B is R. Place X 43 The amino acid indicated in Table 1B is S. X 43 The amino acid designated as C is G. In one embodiment, the CD3 binding molecule In one embodiment, the CD3 binding molecule comprises the CDR-H1 sequence C2-1. Contains 1 sequence C2-2.

[0198] In one embodiment, the CD3 binding molecule comprises the CDR-H1 sequence C2-3. In embodiments, the CD3 binding molecule comprises the CDR-H1 sequence C2-4.

[0199] In one embodiment, the CD3 binding molecule comprises the CDR-H2 sequence C2-5. In some embodiments, the CD3 binding molecule comprises the CDR-H2 sequence C2-6. In the present invention, the CD3 binding molecule comprises the CDR-H2 sequence C2-7.

[0200] In one embodiment, the CD3 binding molecule comprises the CDR-H3 sequence C2-8. In embodiments, the CD3 binding molecule comprises the CDR-H3 sequence C2-9.

[0201] In certain embodiments, the CD3 binding molecule comprises the CDR-L1 sequence C2-10. In an embodiment, the CD3 binding molecule comprises the CDR-L1 sequence C2-11. In one embodiment, the CD3 binding molecule comprises the CDR-L1 sequence C2-12.

[0202] In one embodiment, the CD3 binding molecule comprises the CDR-L2 sequence C2-13. In an embodiment, the CD3 binding molecule comprises the CDR-L2 sequence C2-14. In one embodiment, the CD3 binding molecule comprises the CDR-L2 sequence C2-15.

[0203] In one embodiment, the CD3 binding molecule comprises the CDR-L3 sequence C2-16. In an embodiment, the CD3 binding molecule comprises the CDR-L3 sequence C2-17.

[0204] In one embodiment, the CD3 binding molecule comprises a CDR-H1 sequence as set forth in Table 1C, CDR-H2 sequence, CDR-H3 sequence, CDR-L1 sequence, CDR-L2 sequence and CDR- Contains the L3 sequence.

[0205] In some embodiments, X in Table 1C 44 The amino acid indicated as is G. In an embodiment, X in Table 1C 44 The amino acid indicated as follows is A. In this state, X in Table 1C 45 The amino acid indicated as is H. In Table 1C, X 45 In one embodiment, the amino acid indicated as: is N. In Table 1C, X 46 The amino acid indicated as follows is D. In one embodiment, the amino acid shown in Table 1C In X 46 The amino acid indicated as follows in Table 1C is G. TeX 47 In one embodiment, the amino acid indicated as X4 in Table 1C is A. The amino acid designated X in Table 1C is G. 48 and indicated In one embodiment, the amino acid represented by X in Table 1C is N. 48 It is shown as The amino acid is K. In one embodiment, the amino acid X in Table 1C 49 Amino acid The acid is V. In some embodiments, X in Table 1C 49 The amino acid indicated is In one embodiment, X in Table 1C is 50 The amino acid indicated is N. In one embodiment, X in Table 1C 50 The amino acid indicated is V. In some embodiments, X in Table 1C 51 The amino acid indicated as is A. In the form of X in Table 1C 51 The amino acid designated as is V. In Table 1C, X 52 The amino acid designated as is Y. In one embodiment , X in Table 1C 52 The amino acid indicated as follows is F. In one embodiment, the amino acid shown in Table 1 X in C 53 The amino acid indicated as ' is I. In one embodiment, the amino acid indicated as ' in Table 1C is I. IteX 53 The amino acid designated X in Table 1C is V. 54The amino acid designated X in Table 1C is I. 54 and The amino acid shown is H.

[0206] In one embodiment, the CD3 binding molecule comprises the CDR-H1 sequence C3-1. In some embodiments, the CD3 binding molecule comprises the CDR-H1 sequence C3-2. In one embodiment, the CD3 binding molecule comprises the CDR-H1 sequence C3-3. The CD3 binding molecule comprises the CDR-H1 sequence C3-4.

[0207] In one embodiment, the CD3 binding molecule comprises the CDR-H2 sequence C3-5. In some embodiments, the CD3 binding molecule comprises the CDR-H2 sequence C3-6. In the present invention, the CD3 binding molecule comprises the CDR-H2 sequence C3-7.

[0208] In one embodiment, the CD3 binding molecule comprises the CDR-H3 sequence C3-8. In embodiments, the CD3 binding molecule comprises the CDR-H3 sequence C3-9.

[0209] In one embodiment, the CD3 binding molecule comprises the CDR-L1 sequence C3-10. In an embodiment, the CD3 binding molecule comprises the CDR-L1 sequence C3-11. In one embodiment, the CD3 binding molecule comprises the CDR-L1 sequence C3-12.

[0210] In one embodiment, the CD3 binding molecule comprises the CDR-L2 sequence C3-13. In an embodiment, the CD3 binding molecule comprises the CDR-L2 sequence C3-14.

[0211] In one embodiment, the CD3 binding molecule comprises the CDR-L3 sequence C3-15. In an embodiment, the CD3 binding molecule comprises the CDR-L3 sequence C3-16.

[0212] In one embodiment, the CD3 binding molecule comprises a CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H17, CDR-H18 CDR-H2 and CDR-H3 sequences and corresponding CDR-L1 sequences listed in Table 1D-2, It contains the CDR-L2 and CDR-L3 sequences.

[0213] In one embodiment, the CD3 binding molecule comprises a CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H17, CDR-H18 CDR-H2 and CDR-H3 sequences and corresponding CDR-L1 sequences listed in Table 1E-2, It contains the CDR-L2 and CDR-L3 sequences.

[0214] In one embodiment, the CD3 binding molecule comprises a CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H17, CDR-H18 CDR-H2 and CDR-H3 sequences and corresponding CDR-L1 sequences listed in Table 1F-2, It contains the CDR-L2 and CDR-L3 sequences.

[0215] In one embodiment, the CD3 binding molecule comprises a CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H17, CDR-H18 CDR-H2 and CDR-H3 sequences and corresponding CDR-L1 sequences listed in Table 1G-2; It contains the CDR-L2 and CDR-L3 sequences.

[0216] In one embodiment, the CD3 binding molecule comprises a CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H17, CDR-H18 CDR-H2 and CDR-H3 sequences and corresponding CDR-L1 sequences listed in Table 1H-2, It contains the CDR-L2 and CDR-L3 sequences.

[0217] In one embodiment, the CD3 binding molecule comprises a CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H17, CDR-H18 CDR-H2 and CDR-H3 sequences and corresponding CDR-L1 sequences listed in Table 1I-2, It contains the CDR-L2 and CDR-L3 sequences.

[0218] In certain embodiments, the CD3 binding molecule is selected from the group consisting of those listed in Table 1B-1, Table 1C-1, Table 1D-1, Table 1E-1, Table 1F-1, Table 1G-1, Table 1H-1, Table 1I-1, Table 1J ... any of the CDRs listed in Table 1E-1, Table 1F-1, Table 1G-1, Table 1H-1 or Table 1I-1 In certain embodiments, the present disclosure provides a heavy chain CDR having any one of the amino acid sequences. Table 1B-1, Table 1C-1, Table 1D-1, Table 1E-1, Table 1F-1, Table 1G-1, Table 1 H-1 and one, two, three or more heavy chain CDRs selected from those set forth in Table 1I-1 provides a CD3 binding molecule comprising (or alternatively consisting of) more than .

[0219] In certain embodiments, the CD3 binding molecule is selected from the group consisting of those listed in Table 1B-2, Table 1C-2, Table 1D-2, Table any of the CDRs listed in Table 1E-2, Table 1F-2, Table 1G-2, Table 1H-2 or Table 1I-2 In certain embodiments, the present disclosure provides a light chain CDR having any one of the amino acid sequences. Table 1B-2, Table 1C-2, Table 1D-2, Table 1E-2, Table 1F-2, Table 1G-2, Table 1 H-2 and one, two, three or more light chain CDRs selected from those set forth in Table 1I-2 and a CD3 binding molecule comprising (or alternatively consisting of) more than one light chain CDR. .

[0220] Other CD3 binding molecules have mutated but still contain the CDR sequences and CD3 sequences listed in Table 1. At least 80, 85, 90, 95, 96, 97, 98 or 99 percent identity in the R region In one embodiment, such a CD3 binding molecule comprises amino acids having the same identity. When compared with the CDR sequences listed in Table 1, the CDR regions may have 1, 2, 3, 4 or includes variant amino acid sequences in which five or fewer amino acids are mutated.

[0221] In one embodiment, the CD3 binding molecule comprises any VH and / or VH sequence listed in Table 1. Other CD3 binding domains include VH and / or VL domains having the amino acid sequence of the L domain. The molecule may comprise at least 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, VH and VH sequences containing amino acid sequences with 5, 96, 97, 98 or 99 percent identity In one embodiment, the CD3 binding molecule comprises a VL domain and / or a VL domain. Substantially the same therapeutic activity as compared to the VH and / or VL domains shown in the sequences provided above. VH and / or VH sequences having 1, 2, 3, 4 or 5 or fewer amino acids mutated while retaining the VH sequence. contains a VL domain.

[0222] VH and VL sequences (amino acid sequences and nucleotide sequences encoding the amino acid sequences) It can be "mix and matched" to generate other CD3-binding molecules. Such "mix-and-match" CD3 binding molecules are well known in the art. can be tested using a combination assay (e.g., the FACS assay described in the Examples). When chains are mixed and matched, the VH sequence from a particular VH / VL pairing is , should be replaced with a structurally similar VH sequence. The sequence should be replaced with a structurally similar VL sequence.

[0223] Thus, in one embodiment, the present disclosure provides a method for the production of a medicament comprising administering to a mammalian subject any of the VH sequences set forth in Table 1-J1. a heavy chain variable region (VH) comprising an amino acid sequence selected from any one of the following: The present invention provides a CD3 binding molecule having a light chain variable region (VL) comprising an amino acid sequence as defined above.

[0224] The CD3 binding molecule may be a heterologous protein or polypeptide (or a fragment thereof, e.g. , at least 10, at least 20, at least 30, at least 40, at least 50 , at least 60, at least 70, at least 80, at least 90 or at least 1 fused or chemically conjugated (covalently and non-covalently) to a polypeptide of 00 amino acids For example, a CD3 binding molecule can bind to a detectable protein. The nucleic acid molecule can be fused directly or indirectly to a protein, such as an enzyme or a fluorescent protein. Fusing or conjugating proteins, polypeptides, or peptides to antibodies or antibody fragments Methods for fusing proteins or polypeptides to the CD3 binding molecules of the present disclosure are known. or conjugates thereof. Specification No. 603, Specification No. 5,622,929, Specification No. 5,359,046, Nos. 5,349,053, 5,447,851 and 5,112 ,946; European Patent Nos. 307,434 and 367,166; International Patent Nos. Publication No. 96 / 04388 and Publication No. 91 / 06570; As hkenazi et al.,1991,Proc.Natl.Acad.Sci.U SA 88:10535-10539;Zheng et al.,1995,J.Im munol.154:5590-5600; and Vil et al., 1992, Pr. See oc. Natl. Acad. Sci. USA 89:11337-11341 sea ​​bream.

[0225] Additional CD3-binding molecules have been developed using gene shuffling, motif shuffling, and exon shuffling. Shuffling and / or codon shuffling (collectively referred to as "DNA shuffling") DNA shuffling can be used to generate The disclosed molecules or fragments thereof (e.g., those with higher affinity and lower dissociation rates) Generally, the activity of a molecule (or a fragment thereof) can be altered. ,605,793 specification, 5,811,238 specification, 5,830,721 Nos. 5,834,252 and 5,837,458;Pa tten et al.,1997,Curr.Opinion Biotechnol .8:724-33;Harayama,1998,Trends Biotechno l.16(2):76-82;Hansson et al.,1999,J.Mol. Biol. 287:265-76; and Lorenzo and Blasco, 199 8, Biotechniques 24(2):308-313. The CD3 binding molecules or fragments thereof described herein may be prepared by error-prone methods prior to recombination. subject to random mutagenesis by PCR, random nucleotide insertion, or other methods. The fragments of the CD3 binding molecules described herein can be modified by The encoding polynucleotide may contain one or more components, motifs, segments, or fragments of one or more heterologous molecules. The recombinant fragments can be composed of sections, parts, domains, fragments, etc.

[0226] Additionally, CD3 binding molecules can be fused to marker sequences, such as peptides, to facilitate purification. In one embodiment, the marker amino acid sequence can be, inter alia, QIAGEN, Inc., 9259 Eton Avenue, Chatswor th,CA,91311) ) tag . such as a hexa-histidine peptide , Many of these are commercially available. See, for example, Gentz ​​et al., 1989, Proc. Atl. Acad. Sci. USA 86:821-824, The histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification Examples include, but are not limited to, epitopes derived from influenza hemagglutinin proteins. The corresponding hemagglutinin ("HA") tag (Wilson et al., 1984 Cel l 37:767) and the "flag" tag.

[0227] 7.3. Antigen-binding modules Typically, one or more ABMs of an MBM comprise an immunoglobulin-based antigen-binding domain, For example, antibody fragment or derivative sequences are included. These antibody fragments and derivatives typically contains the CDRs of an antibody and includes larger fragments and derivatives thereof, such as F It may include ab, scFab, Fv and scFv.

[0228] Immunoglobulin-based ABMs can be used to improve the properties of MBMs, including e.g. The VH and / or VL may include modifications to framework residues within the VH and / or VL. Framework modifications can be made to reduce the immunogenicity of MBM. One approach to making framework modifications is to replace one or more framework residues of the ABM with a corresponding Such residues are "backmutated" to the germline sequence. The target gene can be identified by comparing the target gene sequence to the germline sequence from which the ABM is derived. To "match" the framework region sequences to the desired germline configuration, residues should be For example, site-directed mutagenesis can be used to "backmutate" a gene to its corresponding germline sequence. MBMs with such "backmutated" ABMs are encompassed by the present disclosure. It is intended to be.

[0229] Another type of framework modification is a modification of one or more CDs within the framework region or in addition One or more residues within the R region are mutated to remove T cell epitopes, thereby This involves reducing the immunogenic potential of MBM. This approach is also known as "deimmunization." and U.S. Patent Application Publication No. 20030153043 to Carr et al. It is written.

[0230] The ABM can also be modified to have altered glycosylation, e.g., Such carbohydrates may be useful in increasing the affinity of MBM for one or more of the antigens. Compound modifications can be made, for example, by altering one or more sites of glycosylation within the ABM sequence. For example, removal of one or more variable region framework glycosylation sites can be performed. one or more amino acid substitutions that result in a loss of glycosylation at that site. Such aglycosylation can be carried out on the antigen. Such an approach may increase the affinity of MBM for . Nos. 5,714,350 and 6,350,861.

[0231] 7.3.1. Immunoglobulin-based modules 7.3.1.1.Fab In certain embodiments, the ABM is a Fab domain. by proteolytic cleavage of immunoglobulin molecules using enzymes such as α-glucan or by recombinant expression. Fab domains typically consist of a CL domain joined to a VL domain. It comprises a CH1 domain linked to a VH domain paired with

[0232] In wild-type immunoglobulins, the VH domain is paired with the VL domain to form the Fv region. The CH1 domain is paired with the CL domain to further stabilize the binding module. The disulfide bond between the two constant domains may further stabilize the Fab domain. do.

[0233] In MBM, Fab heterodimerization techniques are used to separate Fab domains belonging to the same ABM. This allows for proper association of Fab domains and minimizes aberrant pairing of Fab domains belonging to different ABMs. For example, the Fab heterodimerization strategy shown in Table 2 below can be used. It can be used.

[0234] [Table 83]

[0235] Thus, in certain embodiments, the proper association between the two polypeptides of a Fab is , for example, Fab as described in WO 2009 / 080251 by swapping the VL and VH domains with each other, or by swapping the CH1 and CL domains with each other. This is facilitated by exchanging

[0236] A suitable Fab pairing may involve one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CH2 domain. amino acid modifications into the CL domain of the Fab and / or one or more amino acid modifications into the V by introducing one or more amino acid modifications into the H domain and into the VL domain Fab components preferentially pair with each other over other Fab components. Thus, the amino acids that are modified are typically part of the VH:VL and CH1:CL boundaries. do.

[0237] In one embodiment, one or more amino acid modifications are indicated by the Kabat numbering of the residues. As shown, the conserved frames of the variable (VH, VL) and constant (CH1, CL) domains Almagro, 2008, Frontiers in Bioscience 13:1619-1633, Kabat, Chothia and and framework residue definitions based on the IMGT numbering scheme are provided.

[0238] In one embodiment, the VH and CH1 and / or VL and CL domains are introduced The modifications are complementary to each other. Complementarity at the heavy and light chain interface is due to steric and hydrophobic contacts. This can be achieved based on electrostatic / charge interactions or a combination of various interactions. Complementarity between surfaces allows for lock-and-key mating, knob-into-hole, protrusion and cavity , donors and acceptors, etc., which are all described in the literature as This suggests the nature of the structural and chemical match between the interacting surfaces.

[0239] In one embodiment, the one or more modifications introduced are novel modifications spanning the boundaries of the Fab components. In one embodiment, one or more of the modifications introduced are in the Fab structure. Introducing new salt bridges across the boundaries of the constituent elements. Exemplary substitutions are described in WO 2014 / 013494. 150973 brochure and WO 2014 / 082179 brochure It is listed.

[0240] In one embodiment, the Fab domain comprises a 192E substitution in the CH1 domain and a C It contains 114A and 137K substitutions in the L domain, which are located between the CH1 and CL domains. Introducing salt bridges (Golay et al., 2016, J Immunol 196 :3199-211).

[0241] In one embodiment, the Fab domain comprises 143Q and 188V in the CH1 domain. substitutions in the CH1 and CL domains, as well as 113T and 176V substitutions in the CL domain. It plays a role in alternating hydrophobic and polar contact regions between domains (Golay et al. l., 2016, J Immunol 196:3199-211).

[0242] In one embodiment, the Fab domains are directly linked to facilitate proper assembly of the Fab domains. In order to introduce cross-Fab boundaries, some or all of the VH, CH1, VL, and CL domains (Lewis et al., 2014 Nature Biol. (Echnology 32:191-198). In one embodiment, 39K, 62E The modification was introduced into the VH domain, and H172A and F174G modifications were introduced into the CH1 domain. The 1R, 38D, and (36F) modifications were introduced into the VL domain, and L135Y, S17 In another embodiment, a 6W modification is introduced into the CL domain. A 38R modification is introduced into the VL domain.

[0243] The Fab domain replaces the native CH1:CL disulfide bond with an engineered disulfide bond The Fab fragments may also be modified to increase the efficiency of Fab component pairing. The disulfide bonds are 126C in the CH1 domain and 121C in the CL domain. It can be introduced by introducing (Mazor et al., 2015, MAbs 7:377-89).

[0244] The Fab domain binds the CH1 and CL domains to another domain that facilitates proper assembly. It can also be modified by substitution at the base. For example, Wu et al., 2015 ,MAbs 7:364-76, which uses the CH1 domain of the α T cell receptor as a constant domain. and replacing the CL domain of the T cell receptor with the β domain, and The introduction of the D modification into the VL domain and the 39K modification into the VH domain resulted in a VL and combining these domain substitutions with additional charge-charge interactions between the VH domains. It is stated that:

[0245] The ABM can comprise a single chain Fab fragment, which is an antibody heavy chain variable domain. variable domain (VH), antibody constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain It is a polypeptide consisting of a constant domain (CL) and a linker. The antibody domains and linkers are arranged in one of the following orders from N-terminus to C-terminus: a) V H-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) V d) VL-CH1-linker-VH-CL or d) VL-CH1-linker-VH-CL The linker is a polymer of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab domain may be a polypeptide between the CL domain and the CH1 domain. It is stabilized by the native disulfide bonds of

[0246] In one embodiment, the antibody domains and linker in the single chain Fab fragment are In one of the following orders from the terminal to the C-terminus: a) VH-CH1-linker-VL-CL or b) VL-CL-linker-VH-CH1, more preferably VL-CL-linker- It has VH-CH1.

[0247] In another embodiment, the antibody domains and linker in a single-chain Fab fragment are In one of the following orders from N-terminus to C-terminus: a) VH-CL-linker-VL-CH 1 or b) VL-CH1-linker-VH-CL.

[0248] Optionally, in the single chain Fab fragment, the CL-domain and the CH1 domain In addition to the natural disulfide bonds between the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (LCH), The variable domains (VL) are located at the following positions: i) position 44 in the heavy chain variable domain and position 54 in the light chain variable domain ii) position 105 in the heavy chain variable domain and position 43 in the light chain variable domain, or iii) Heavy chain variable domain position 101 and light chain variable domain position 100 (Kabat EU index) Disulfide stabilization can also be achieved by the introduction of disulfide bonds between the amino acids (numbered by the amino acid sequence).

[0249] This additional disulfide stabilization of the single-chain Fab fragment provides This is achieved by introducing a disulfide bond between the variable domains VH and VL of the fragment. Techniques for introducing non-native disulfide bridges for the stabilization of single-chain Fvs are available, e.g. For example, see International Publication No. 94 / 029350, Rajagopal et al. ,1997,Prot.Engin.10:1453-59;Kobayashi et. al.,1998,Nuclear Medicine&Biology,25:38 7-393; and Schmidt, et al., 1999, Oncogene 18: 1711-1721. In one embodiment, a single chain Fab fragment The optional disulfide bond between the variable domains of the heavy chain and the light chain is at position 44 of the variable domain. In one embodiment, the variable region of the single chain Fab fragment is located between domains 100 and 100. An optional interdomain disulfide bond is located at position 105 of the heavy chain variable domain and at position 106 of the light chain variable domain. It is between 43rd and 44th place (numbering according to Kabat's EU index).

[0250] 7.3.1.2.scFv Single-chain Fv or "scFv" antibody fragments contain the V of an antibody in a single polypeptide chain. It contains H and VL domains and can be expressed as a single polypeptide chain, scFv polypeptides retain the specificity of the intact antibody from which they are derived. V between the VH and VL domains, which enables them to form the desired structure for target binding. The scFv further comprises a polypeptide linker. Examples of linkers are the ABM linkers identified in Section 7.4.3, e.g., L1-L54. The linker is either one of the linkers shown as

[0251] Unless otherwise specified, as used herein, scFv refers to, e.g., a polypeptide and may have the VL and VH variable regions in either order with respect to the N- and C-termini of the peptide, The scFv may comprise VL-linker-VH or VH-linker-VL.

[0252] To generate scFv-encoding nucleic acids, VH- and VL-encoding DNA fragments are 7.4.3, encoding an ABM linker, e.g., any of the ABM linkers described in Section 7.4.3 operably linked to another fragment encoding a nucleotide sequence (e.g., Gly4-Ser) The VH and VL domains are linked together by a flexible linker. The sequence can be expressed as a continuous single-chain protein (e.g., Bir d et al.,1988,Science 242:423-426;Huston et al.,1988,Proc.Natl.Acad.Sci.USA 85:5 879-5883;McCafferty et al.,1990,Nature 3 48:552-554).

[0253] 7.3.1.3. Other Immunoglobulin-Based Modules MBMs can be Fab or scFv, e.g., Fv, dsFv, (Fab'), single domain antibody (SDAB), VH or VL domain or camelid VHH ABMs may also include immunoglobulin forms other than domains (also called nanobodies). do.

[0254] ABMs consist of a single VH or VL domain that exhibits sufficient affinity for the target. In certain embodiments, the single domain antibody may be a camelid-derived single domain antibody. family VHH domains (e.g., Riechmann, 1999, Journal of f Immunological Methods 231:25-38; International Publication No. 9 (See brochure no. 4 / 04678).

[0255] 7.3.2. Non-immunoglobulin-based modules In certain embodiments, one or more of the ABMs are non-antibody scaffold proteins (e.g., engineered antibodies). Deoxyribonucleic acid repeat proteins (DARPins), avimers (abbreviation for avidity multimers) ), anticalins / lipocalins, centirins, Kunitz domains, adnexins, affinities Phosphorin, affitin (also known as nonphytin), knottin, pronectin, and ba (including but not limited to catabodies, duocalins and finomers), ligands , receptors, cytokines or chemokines.

[0256] Non-immunoglobulin scaffolds that can be used in MBM include those described by Mintz and C rea,2013,Bioprocess International 11(2): Tables 3 and 4 of pp. 40-48; Vazquez-Lombardi et al., 2015 ,Drug Discovery Today 20(10):1271-83 Figure 1, Table 1 and Figure I; Skrlec et al., 2015, Trends in Biot Those listed in Table 1 and Box 2 of Chemology 33(7):408-18 Mintz and Crea, 2013, Bioprocess In ternational 11(2):40-48, Tables 3 and 4; Vazquez-Lo mbardi et al.,2015,Drug Discovery Today 20(10):1271-83, Figure 1, Table 1 and Figure I; Skrlec et al.,2 015,Trends in Biotechnology 33(7):408-18 The contents of Table 1 and Box 2 of the Scaffold Disclosure (collectively the "Scaffold Disclosure") are incorporated herein by reference. In certain embodiments, the disclosure of scaffolds is inconsistent with that disclosed for adnexins. In another embodiment, the disclosure of scaffolds provides that they are The disclosure regarding Bimmer is incorporated by reference. The disclosures of the fields are incorporated by reference for what they disclose regarding affibodies. In yet another embodiment, the scaffold disclosures are those disclosed for anticalins. In yet another embodiment, the scaffold disclosure is is incorporated by reference for its disclosure regarding DARPins. In the form, the disclosure of scaffolds is about what they disclose about Kunitz domains. In yet another embodiment, the disclosure of scaffolds provides that they are knotty In yet another embodiment, the present invention is directed to a method for treating a foot, the method comprising: The disclosures of the Field are incorporated by reference for what they disclose regarding Pronectin. In yet another embodiment, the scaffolds disclosed are those disclosed in terms of nanophytin. In yet another embodiment, the scaffold disclosure is is incorporated by reference for its disclosure regarding affilins. In this embodiment, the disclosures of scaffolds are incorporated by reference for what they disclose with respect to Adnectins. In yet another embodiment, the scaffold disclosure provides that they are useful in the context of ABM. In yet another embodiment, the scaffold disclosed are incorporated by reference for what they disclose regarding Adhiron. In embodiments, the disclosure of scaffolds is similar to that disclosed for Affimers. In yet another embodiment, the scaffold disclosure provides that they are alpha The disclosure regarding the body is incorporated by reference. Therefore, the disclosure of scaffolds is not intended to be a substitute for what they disclose with respect to armadillo repeat proteins. In yet another embodiment, the scaffold disclosure provides that they are The disclosure regarding mer / tetranectin is incorporated by reference. In embodiments, the disclosure of scaffolds relates to the obody / OB-fold. In yet another embodiment, the disclosure of the scaffold comprises: They are incorporated by reference for what they disclose regarding sentin. In the embodiments, the disclosure of scaffolds is in accordance with what they disclose regarding repebodies. In yet another embodiment, the scaffold disclosure provides that they are anticalins. In yet another embodiment, the present invention is directed to a method for treating a foot, the method comprising: The disclosures of Field are incorporated by reference for what they disclose regarding atrimers. In yet another embodiment, the scaffold disclosures relate to bicyclic peptides. In yet another embodiment, the scaffold disclosure is is incorporated by reference for its disclosure regarding cys-knots. In embodiments, the disclosure of scaffolds includes scaffolds that are Fn3 scaffolds (adnectins, centrilin, protease inhibitors, etc.). The disclosures relating to the IL-16 nucleotide sequences (including Ionectin and Tn3) are incorporated by reference.

[0257] In one embodiment, the ABM is a designed ankyrin repeat protein ("DAR"). DARPins can be highly specific and high-affinity target protein binding proteins. They are typically recombinant antibodies. The ankyrin protein is derived from a naturally occurring ankyrin protein, and at least three of these proteins They usually consist of four or five repeating motifs. Their molecular weights are RPins are approximately 14 or 18 kDa (kilodaltons), respectively. Examples of DARPins are See, for example, U.S. Patent No. 7,417,130. Multispecific binding molecules comprising antibody- and immunoglobulin-based binding modules are described, for example, in It is disclosed in US Patent Application Publication No. 2015 / 0030596 A1.

[0258] In another embodiment, the ABM can be an affibody. Affibodies are known in the art. It is well known in the art that staphylococcal protein A Based on a 58 amino acid residue protein domain derived from one of the IgG binding domains It refers to an affinity protein that binds to the target molecule.

[0259] In another embodiment, the ABM can be an anticalin. Anticalins are known in the art. is well known in the art and refers to another antibody mimetic technology, in which the binding specificity is Derived from pocalin, anticalins are dual-targeting proteins called duocalins. It can still be formatted.

[0260] In another embodiment, the ABM can be a Versabody. Versabodies are known in the art. are well known in the art and refer to other antibody mimetic technologies. The resulting disulfide-rich scaffold replaces the hydrophobic core of the protein, forming a high disulfide density scaffold. It is a low molecular weight protein of 3 to 5 kDa that contains cysteine ​​residues.

[0261] Other non-immunoglobulin ABMs include "A" domain oligomers (also known as avimers). known) (see, for example, U.S. Patent Application Publication No. 2005 / 0164301; See Patent Publications 2005 / 0048512 and 2004 / 017576. ), Fn3-based protein scaffolds (e.g., U.S. Patent Application Publication No. 2003 / 017 0753), VASP polypeptide, avian pancreatic polypeptide (a PP), tetranectin (CTLD3-based), affilirin (γB-crystallin) phospho / ubiquitin-based), knottin, SH3 domain, PDZ domain, ten Damistat, neocarzinostatin, protein A domain, lipocalin, transferrin In one embodiment, ABs useful in constructing MBMs include the alpha- and alpha-binding domains. M is fibronectin, as exemplified in WO 2011 / 130324. Includes base scaffolding.

[0262] Additionally, in certain embodiments, the ABM is a ligand binding domain of a receptor or a ligand For example, when the TAA is an EGF receptor, ABM3 contains the receptor binding domain: It can contain a portion of EGF that binds to EGFR, and if the TAA is a PDGF receptor , ABM3 may contain a portion of the PDGF receptor that binds PDGF, etc. In this embodiment, ABM1 is a CD2 ligand, particularly as described in Section 7.9.2. The binding domains of each of the many ligand / receptor pairs are known in the art. are well known in the art and therefore can be easily selected and adapted for use in MBMs. Cut.

[0263] Connectors The CD3 binding molecule (e.g., MBM) may, in some cases, be a fusion protein without a linker, e.g., As a protein, an ABM or a pair of ABM chains (e.g., VH- of Fab) directly linked to each other It is contemplated that the CD3 binding molecule may comprise a C H1 or V L-CL component. (e.g., MBM) contains connector moieties that link individual ABMs or ABM chains. The use of a catalyzer moiety can, for example, increase the flexibility of the ABM within a CD3 binding molecule (e.g., MBM). ABMs may be used to improve target binding by reducing steric hindrance, e.g. For example, Fc domains (each Fc domain represents a pair of associated Fc regions) and / or AB The use of Fc domains is typically used to identify the antigen of choice. Binding requires the use of the hinge region as a connector of the ABM or ABM chains. Thus, the term "connector" includes, but is not limited to, an Fc region, an Fc domain, It includes the hinge region and the ABM linker.

[0264] Fc domain (formed by pairing of two Fc domains), hinge region and ABM link Examples of anchors are given in Sections 7.4.1, 7.4.2 and 7.4.3, respectively.

[0265] Fc Domain The CD3 binding molecule (e.g., MBM) may comprise an Fc domain derived from any suitable species. In one embodiment, the Fc domain is derived from a human Fc domain.

[0266] The Fc domain is responsible for IgA (including subclasses IgA1 and IgA2), IgD, and IgE , IgG (including subclasses IgG1, IgG2, IgG3, and IgG4) and IgM In one embodiment, the Fc domain may be derived from any suitable class of antibody, including: It is derived from IgG1, IgG2, IgG3 or IgG4. In one embodiment, the Fc domain In one embodiment, the Fc domain is derived from IgG1. In another embodiment, the Fc domain is derived from IgG4. do.

[0267] The Fc domain contains two polypeptide chains, each called a heavy chain Fc region. The heavy chain Fc regions of the IgG1 and IgG2a dimerize to generate the Fc domain. The Fc regions of the antibodies may be the same or different from each other. In a native antibody, the Fc region is typically For purposes of generating typologically identical but multispecific binding molecules, e.g., MBMs, Fc The region is designed to allow heterodimerization, as described in Section 7.4.1.5 below. can differ advantageously for this purpose.

[0268] Typically, each heavy chain Fc region comprises or consists of two or three heavy chain constant domains. become.

[0269] In natural antibodies, the heavy chain Fc regions of IgA, IgD, and IgG consist of two heavy chain constant domains. The heavy chain Fc region of IgE and IgM consists of three heavy chains (CH2 and CH3). The Fc domain is composed of the Fc chain constant domains (CH2, CH3, and CH4). dimerize to produce

[0270] In the present disclosure, the heavy chain Fc region may be a region of one or more different classes of antibodies, e.g., one, two, or more. or may comprise heavy chain constant domains from three different classes.

[0271] In one embodiment, the heavy chain Fc region comprises the CH2 and CH3 domains derived from IgG1. Includes.

[0272] In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG2. Includes.

[0273] In one embodiment, the heavy chain Fc region comprises the CH2 and CH3 domains derived from IgG3. Includes.

[0274] In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG4. Includes.

[0275] In one embodiment, the heavy chain Fc region comprises a CH4 domain derived from IgM. The M CH4 domain is typically located C-terminal to the CH3 domain.

[0276] In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG. and a CH4 domain derived from IgM.

[0277] Used to generate heavy chain Fc regions for the CD3 binding molecules (e.g., MBM) of the present disclosure The heavy chain constant domains for the synthesis of ribozymes may comprise variants of the naturally occurring constant domains described above. It will be understood that such variants may have one or more amino acids different from the wild-type constant domain. In one example, the heavy chain Fc region of the present disclosure may contain amino acid changes from the wild-type constant domain. The variant constant domain comprises at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that the domain may be longer or shorter. The constant domain is at least 60% identical or similar to the wild-type constant domain In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 75% identical or similar to In another example, the variant constant domain is at least 80% identical or In another example, the variant constant domain is at least 90% identical or In another example, the variant constant domain is at least 95% identical. Exemplary Fc variants are described in Sections 7.4.1.1-7.4.1.5. It is written.

[0278] IgM and IgA are naturally occurring in humans as covalently linked multimers of a common H2L2 antibody unit. IgM exists as a pentamer when incorporating a J chain, or as a pentamer when incorporating a J chain. IgA exists as a hexamer when the IgG is absent. IgA exists in both monomeric and dimeric forms. The heavy chains of IgM and IgA consist of a single chain connected to a C-terminal constant domain known as the tail. The tail has an 8 amino acid extension. The tail forms disulfide bonds between the heavy chains in the polymer. The tail contains a cysteine ​​residue that is thought to play an important role in polymerization. In certain embodiments, the CD3 binding molecules of the present disclosure (e.g., For example, MBM) does not include the tail.

[0279] The Fc domain incorporated into the CD3 binding molecules (e.g., MBM) of the present disclosure is a protein Functional properties of the protein, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cell proliferation. The CD3 binding molecules may contain one or more modifications that alter their cytotoxicity. (e.g., one or more chemical moieties may be attached to the CD3 binding molecule) or and to alter one or more functional properties of the CD3 binding molecule. It can be modified to

[0280] The effector functions of antibody molecules are determined, for example, by the binding of the C1 component of complement to the antibody. Complement activation includes the opsonization of pathogens and complement-mediated effector functions. and direct lysis. In addition, it recruits phagocytes to the site of complement activation. The effector function is Fc receptor agonist. Fc receptor (FcR)-mediated effector functions, which involve the binding of antibodies to Fc receptors (FcRs). This can be triggered by binding of the constant domain of an Fc receptor on the surface of an effector cell. Antigen-antibody complex-mediated cross-linking leads to the phagocytosis and destruction of antibody-coated particles, the clearance of immune complexes, and the Lysis of antibody-coated target cells by killer cells (antibody-dependent cell-mediated lysis) Antibody-mediated cytotoxicity (ADCC), release of inflammatory mediators, placental passage and immunoglobulins It triggers many important and diverse biological responses, including the regulation of phosphorus production.

[0281] The Fc region contains at least one amino acid residue that modifies effector function. It can be modified by substituting different amino acid residues. For example, one or more amino acids The Fc region has a different affinity for effector ligands. The effector ligand for which affinity is modified can be substituted with an amino acid residue. For example, the Fc receptor or the C1 component of complement. Also, Winter et al., U.S. Pat. Nos. 5,624,821 and 5,648,222. The modified Fc region may also alter C1q binding and / or Complement-dependent cytotoxicity (CDC) can be reduced or eliminated. For example, see U.S. Patent No. 6,194,551 to Idusogie et al. The modified Fc region may also alter the ability of the Fc region to fix complement. This approach can be used, for example, Bodmer et al., PCT Publication WO 94 / 29351 Jefferis et al., 2009, MAbs, 1:332-338 Thus, as described, allotypic amino acid residues include, but are not limited to, I The constant regions of the heavy chains of the gG1, IgG2, and IgG3 subclasses and the light chain of the kappa isotype Examples of the constant region include:

[0282] The Fc region is used to "silence" effector functions, e.g., antibody-dependent CD3 mediates cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP) The binding molecule may also be modified to reduce or eliminate its ability to bind. This can be achieved by introducing mutations in the region. Described in the art: LALA and N297A (Strohl, 2009, Curr. Opin. Biotechnol. 20(6):685-691); and D2 65A(Baudino et al.,2008,J.Immunol.181:66 64-69; Strohl, supra). An example of a silent Fc IgG1 antibody is IgG1 The so-called LALA mutation, which contains the L234A and L235A mutations in the Fc amino acid sequence Another example of a silent IgG1 antibody contains a D265A mutation. The rent IgG1 antibody contains D265A and P329A in the IgG1 Fc amino acid sequence. Another silent IgG1 antibody is the N29 7A mutation, which is aglycosylated / aglycosylated This results in a cosylated antibody.

[0283] The Fc region, for example, increases the affinity of the CD3 binding molecule for activating Fcγ receptors. or to reduce the affinity of the CD3 binding molecule for inhibitory Fcγ receptors. By modifying one or more amino acid residues, antibody-dependent cellular cytotoxicity (ADCC) and / or or the ability of CD3-binding molecules containing Fc regions to mediate antibody-dependent cellular phagocytosis (ADCP) Human activating Fcγ receptors include FcγRIa, FcγRIa, FcγRIb, FcγRIc ... human inhibitory Fcγ receptors include FcγRIIa, FcγRIIIa, and FcγRIIIb; An example of such a receptor is FcγRIIb. This technique is described, for example, in the publication by Presta et al. It is described in PCT Publication WO 00 / 42072. Binding in human IgG1 for γRl, FcγRII, FcγRIII, and FcRn The site has been mapped and mutants with improved binding have been described (Sh ields et al.,J.Biol.Chem.276:6591-6604,2 (See 001) F of monoclonal antibodies, such as improved ADCC / ADCP function. Optimization of c-mediated effector function has been described (Strohl, 2009, Cu rrent Opinion in Biotechnology 20:685-69 1). Mutations that may improve ADCC / ADCP function include G23 6A, S239D, F243L, P247I, D280H, K290S, R292P, S 298A, S298D, S298V, Y300L, V305I, A330L, I332E , E333A, K334A, A339D, A339Q, A339T and P396L (all The positions are according to EU numbering).

[0284] The Fc region typically contains an active domain that does not recognize the parent CD3 binding molecule, such as FcαRI. Modifying one or more amino acids to increase the affinity of the CD3 binding molecule for the activated receptor By combining the CD3 binding molecules, the ability of the CD3 binding molecules to mediate ADCC and / or ADCP is improved. This technique has been described, for example, by Borrok et al., 201 5, mAbs. 7(4):743-751.

[0285] Thus, in certain embodiments, the CD3 binding molecules of the present disclosure include, but are not limited to, F Binding to Fc-receptors such as cRn or leukocyte receptors (e.g., see Section 7.4.1.1 ), complement binding (e.g., as described in Section 7.4.1.2), ), modified disulfide bond structures (e.g., as described in Section 7.4.1.3), or Modified glycosylation patterns (e.g., as described in Section 7.4.1.4) The Fc domain may comprise an Fc domain with altered effector function. Enables heterodimerization, which is the preferential pairing of non-identical over identical Fc regions This allows for modifications that improve the manufacturability of asymmetric CD3 binding molecules (e.g., MBMs). Heterodimerization can also be achieved by modifying the Fc region to include different ABMs with different sequences. CD3 binding molecules (e.g., MBM) linked to each other by an Fc domain containing Examples of heterodimerization techniques are provided in Section 7.4.1.5 (and its subsections). This is illustrated in the example below.

[0286] Any of the modifications described in Sections 7.4.1.1 to 7.4.1.5 may be used in any suitable manner. may be combined in a manner to achieve desired functional properties and / or combined with other modifications. It will be appreciated that the properties of the CD3 binding molecule (eg, MBM) may be altered by such techniques.

[0287] 7.4.1.1. Fc Domains with Altered FcR Binding The Fc domain of a CD3-binding molecule (e.g., MBM) is similar to the corresponding native immunoglobulin. In comparison, the antibody may exhibit altered binding to one or more Fc-receptors (FcRs). Binding to specific Fc-receptors can be increased or decreased. In one embodiment, the Fc domain The antibody contains one or more modifications that alter its Fc-receptor binding profile.

[0288] The human cells are selected from FcαR, FcεR, FcγR, FcRn and glycan receptors. Some cells express soluble (extracellular domain) FcRs. It is also possible to express FcR (for review, see Fridman et al. , 1993, J Leukocyte Biology 54:504-512). Fc γRs are further differentiated by their IgG binding affinity (high / low) and biological effect (activation / inhibition). Human FcγRI is widely considered to be the only "high affinity" receptor. While all others are considered to be of intermediate to low affinity, FcγRIIb is the It is the only receptor with "inhibitory" functionality due to its intracellular ITIM motif, while all other receptors These are either "activated" by the ITAM motif or paired with the common FcγR-γ chain. FcγRIIIb is active, but it is thought to bind to cells via a GPI anchor. Overall, humans have six "canonical" FcγRs: cγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, Fc In addition to these sequences, the γRIIIb gene is also expressed across these families. Numerous sequence or allotypic variants exist. Some of these have important functional consequences. It has been shown that β-glucanase receptors have the same nucleotide sequence as β-glucanase receptors and are therefore considered to be their own receptor subtype. Examples include FcγRIIa H134R , FcγRIIb I190T , Fc γRIIIa F158V and FcγRIIIb NA1 , FcγRIIIb NA2 , Fcγ RIII SH Each receptor sequence includes IgG: IgG1, IgG2, IgG3, and It has been shown that the antibodies have different affinities for the four subclasses of IgG1 and IgG2 (Bruh ns, 1993, Blood 113:3716-3725). Other species have FcγR The murine system is the best studied to date, with four Fcγ R, including FcγRI, FcγRIIb, FcγRIII, and FcγRIV (Bruhns , 2012, Blood 119:5640-5649). Human FcγRI on cells is Usually, its affinity for IgG1 / IgG3 / IgG4 (approximately 10 -8 M) and serum The concentration of these IgGs (approximately 10 mg / ml) led to the formation of monomeric IgG under normal serum conditions. Therefore, they are considered to be "occupied" by carrying FcγRI on their surface. Cells with this antigen are surrogately "screened" for their antigen environment by the bound multispecific IgG. It is thought that "scanning" or "sampling" of IgG subclasses is possible. Lower affinity (approximately 10 -5 ~10 -7 Other receptors with a range of M are usually Therefore, low affinity receptors are considered to be "unoccupied" by antibody-mediated immunity. Intrinsically sensitive to detection and activation by the complex. Increased Fc density results in increased functional affinity of binding avidity to low affinity FcγRs. This has been demonstrated in vitro using several methods (Shields et al., 2001, J Biol Chem 276(9):6591-6604 ;Lux et al.,2013,J Immunol 190:4315-4323 ) which is the primary mode of action for the use of anti-RhD antibodies to treat ITP in humans. It has also been suggested that this is one of the factors (Crow, 2008, Transfusion Medicine edicine Reviews 22:103-116).

[0289] Many cell types express multiple types of FcγR, and therefore, FcγR-bearing cells The binding of IgG or antibody immune complexes to cells can have multiple and complex outcomes depending on the biological context. Most simply, cells can receive either activating, inhibitory, or mixed signals. This includes phagocytosis (e.g., macrophages and neutrophils), antigen processing (e.g., dendritic cells), decreased IgG production (e.g., B cells) or degranulation (e.g., neutrophils) Inhibitory signals from FcγRIIb can lead to events such as activation signals. There is data supporting that it may be superior to the naru one (Proulx, 2010, Clinical Immunology 135:422-429).

[0290] FcRn plays an important role in maintaining the long half-life of IgG in serum in adults and children. The receptor binds to IgG in acidified vesicles (pH < 6.5) and releases IgG molecules. It protects the molecule from degradation and then releases it at the higher pH of 7.4 in the blood.

[0291] FcRn differs from leukocyte Fc receptors, instead sharing structural similarity with MHC class I molecules. It consists of a β2-protein complex non-covalently linked to a membrane-associated chain containing three extracellular domains. It is a heterodimer composed of microglobulin chains. These chains contain carbohydrate chains. The main one, together with β2-microglobulin, is between the CH2 and CH3 domains of Fc. The interaction occurs at pH < 6.5, where the histidine residues on IgG are positively charged. At higher pH, His residues lose their positive charge. This leads to loss of FcRn-IgG interaction, weakening the FcRn-IgG interaction and dissociation of IgG.

[0292] In one embodiment, the CD3 binding molecule (e.g., MBM) binds to human FcRn. Contains the Fc domain.

[0293] In one embodiment, the Fc domain also contains a histidine at position 310, preferably at position 435. and one (e.g., one or two) Fc region containing these histidine residues. are important for human FcRn binding. The histidine residues in the nucleotide sequence are natural residues, i.e., positions 310 and 435 are modified. Alternatively, one or both of these histidine residues may be present as a result of modification. .

[0294] CD3 binding molecules (e.g., MBMs) contain one or more FcRs that alter Fc binding to FcRn. The altered binding may be increased binding or decreased binding.

[0295] In one embodiment, the CD3 binding molecule (e.g., MBM) comprises at least one (optionally The Fc region of the immunoglobulin (and optionally both) has a higher affinity and affinity than the corresponding native immunoglobulin. The antibody comprises an Fc domain containing one or more modifications that allow it to bind to FcRn at the nucleotide level.

[0296] In one embodiment, the Fc region replaces the threonine residue at position 250 with a glutamine residue (T250Q).

[0297] In one embodiment, the Fc region modifies the methionine residue at position 252 to a tyrosine residue ( M252Y).

[0298] In one embodiment, the Fc region mutates the serine residue at position 254 to a threonine residue (S 254T).

[0299] In one embodiment, the Fc region replaces the threonine residue at position 256 with a glutamic acid residue. The compound is modified by substituting it with the group (T256E).

[0300] In one embodiment, the Fc region replaces the threonine residue at position 307 with an alanine residue ( T307A).

[0301] In one embodiment, the Fc region mutates the threonine residue at position 307 with a proline residue ( It is modified by substituting with T307P.

[0302] In one embodiment, the Fc region mutates the valine residue at position 308 to a cysteine ​​residue (V 308C).

[0303] In one embodiment, the Fc region replaces the valine residue at position 308 with a phenylalanine residue. It is modified by substituting with the group (V308F).

[0304] In one embodiment, the Fc region is modified by substituting a valine residue at position 308 with a proline residue (V3 08P).

[0305] In one embodiment, the Fc region mutates the glutamine residue at position 311 with an alanine residue ( Q311A).

[0306] In one embodiment, the Fc region replaces the glutamine residue at position 311 with an arginine residue (Q311R).

[0307] In one embodiment, the Fc region comprises a methionine residue at position 428 replaced by a leucine residue ( M428L).

[0308] In one embodiment, the Fc region comprises a histidine residue at position 433 replaced by a lysine residue (H 433K).

[0309] In one embodiment, the Fc region comprises an Fc region in which the asparagine residue at position 434 is replaced by a phenylalanine residue. It is modified by substituting the nin residue (N434F).

[0310] In one embodiment, the Fc region replaces the asparagine residue at position 434 with a tyrosine residue (N434Y).

[0311] In one embodiment, the Fc region comprises an Fc amino acid sequence in which the methionine residue at position 252 is replaced with a tyrosine residue. The serine residue at position 254 is replaced with a threonine residue, and the threonine residue at position 256 is replaced with a threonine residue. by substituting the α-glutamic acid residues (M252Y / S254T / T256E) be qualified.

[0312] In one embodiment, the Fc region comprises a valine residue at position 308 replaced with a proline residue, and and replacing the asparagine residue at position 434 with a tyrosine residue (V308P / N434Y). It is modified by

[0313] In one embodiment, the Fc region comprises an Fc amino acid sequence in which the methionine residue at position 252 is replaced with a tyrosine residue. , the serine residue at position 254 is replaced by a threonine residue, and the threonine residue at position 256 is replaced by a threonine residue. A glutamic acid residue was replaced with a lysine residue at position 433 and a histidine residue at position 434. The asparagine residue in / H433K / N434F).

[0314] It is understood that any of the modifications listed above may be combined to alter FcRn binding. It will be understood.

[0315] In one embodiment, the CD3 binding molecule (e.g., MBM) comprises one or both Fc regions. However, the Fc domain binds to the Fc domain with lower affinity and avidity than the corresponding native immunoglobulin. It comprises an Fc domain that includes one or more modifications that allow it to bind to Rn.

[0316] In one embodiment, the Fc region comprises histidine at positions 310 and / or 435. Any amino acid residue other than

[0317] CD3 binding molecules (e.g., MBM) have one or both Fc regions that bind to FcγRIIb. The Fc domain may contain one or more modifications that increase its binding to FcγRIIb. is the only inhibitory receptor in humans and the only Fc receptor found on B cells.

[0318] In one embodiment, the Fc region replaces the proline residue at position 238 with an aspartic acid residue. The compound is modified by substituting it with the group (P238D).

[0319] In one embodiment, the Fc region replaces the glutamic acid residue at position 258 with an alanine residue (E258A).

[0320] In one embodiment, the Fc region is modified by substituting the serine residue at position 267 with an alanine residue (S2 67A).

[0321] In one embodiment, the Fc region comprises a nucleotide sequence in which the serine residue at position 267 is replaced with a glutamic acid residue ( S267E).

[0322] In one embodiment, the Fc region mutates the leucine residue at position 328 with a phenylalanine It is modified by substituting with residue (L328F).

[0323] In one embodiment, the Fc region replaces the glutamic acid residue at position 258 with an alanine residue and the serine residue at position 267 is replaced with an alanine residue (E258A / S267A). It is modified by

[0324] In one embodiment, the Fc region comprises an Fc amino acid sequence in which the serine residue at position 267 is replaced with a glutamic acid residue. and the leucine residue at position 328 is replaced with a phenylalanine residue (S267E / L328F )

[0325] Any of the modifications listed above may be combined to increase FcγRIIb binding It will be understood that.

[0326] In one embodiment, a CD3 binding antibody comprising an Fc domain that exhibits reduced binding to FcγR. A molecule (e.g., MBM) is provided.

[0327] In one embodiment, the CD3 binding molecule (e.g., MBM) comprises one or both Fc regions. but comprises an Fc domain that contains one or more modifications that reduce Fc binding to FcγR.

[0328] The Fc domain may be derived from IgG1.

[0329] In one embodiment, the Fc region mutates the leucine residue at position 234 with an alanine residue (L 234A).

[0330] In one embodiment, the Fc region mutates the leucine residue at position 235 with an alanine residue (L 235A).

[0331] In one embodiment, the Fc region replaces the glycine residue at position 236 with an arginine residue ( G236R).

[0332] In one embodiment, the Fc region replaces the asparagine residue at position 297 with an alanine residue (N297A) or glutamine residue (N297Q).

[0333] In one embodiment, the Fc region is modified by substituting the serine residue at position 298 with an alanine residue (S2 98A).

[0334] In one embodiment, the Fc region mutates the leucine residue at position 328 with an arginine residue ( L328R).

[0335] In one embodiment, the Fc region comprises an Fc amino acid sequence comprising the leucine residue at position 234 replaced by an alanine residue; and replacing the leucine residue at position 235 with an alanine residue (L234A / L235A). It is modified by

[0336] In embodiments, the Fc region replaces the phenylalanine residue at position 234 with an alanine residue. and the leucine residue at position 235 was replaced with an alanine residue (F234A / L235A). It is modified by substitution.

[0337] In one embodiment, the Fc region comprises an Fc amino acid sequence in which the glycine residue at position 236 is replaced with an arginine residue. and replacing the leucine residue at position 328 with an arginine residue (G236R / L328R). It is modified by converting

[0338] It has been found that any of the modifications listed above may be combined to reduce FcγR binding. It will be understood.

[0339] In one embodiment, the CD3 binding molecules (e.g., MBM) of the present disclosure comprise one or both The Fc region is capable of binding to FcγRIIIa without affecting Fc binding to FcγRII. The Fc domain comprises one or more modifications that reduce binding.

[0340] In one embodiment, the Fc region is modified by substituting the serine residue at position 239 with an alanine residue (S2 39A).

[0341] In one embodiment, the Fc region replaces the glutamic acid residue at position 269 with an alanine residue (E269A).

[0342] In one embodiment, the Fc region replaces the glutamic acid residue at position 293 with an alanine residue (E293A).

[0343] In one embodiment, the Fc region comprises a tyrosine residue at position 296 replaced by a phenylalanine It is modified by substituting with residue (Y296F).

[0344] In one embodiment, the Fc region is modified by substituting a valine residue at position 303 with an alanine residue (V3 03A).

[0345] In one embodiment, the Fc region mutates the alanine residue at position 327 with a glycine residue (A 327G).

[0346] In one embodiment, the Fc region is modified by substituting the lysine residue at position 338 with an alanine residue (K3 38A).

[0347] In one embodiment, the Fc region is modified by replacing the aspartic acid residue at position 376 with an alanine residue. The compound is modified by substituting it with the group (D376A).

[0348] Any of the modifications listed above may be combined to reduce FcγRIIIa binding. It will be understood that

[0349] Fc region mutants with reduced FcR binding are called "FcγR ablation mutants" and "Fc These mutants are called "γR-silencing mutants" or "Fc knockout (FcKO or KO) mutants." For some therapeutic applications, more than one mechanism of action may be used to avoid further Any of the above or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, It is desirable to reduce or eliminate the normal binding of the Fc domain to FcγRIIIa). Thus, for example, in many embodiments, the use of MBMs that specifically bind monovalently to CD3 is In this application, FcγRIIIa binding is inhibited to eliminate or significantly reduce ADCC activity. In one embodiment, the Fc of the MBMs described herein is preferably cleaved. At least one of the regions contains one or more Fcγ receptor ablation variants. In some embodiments, both of the Fc regions comprise one or more Fcγ receptor ablation mutants. These ablation mutants are shown in Table 3, each independently and optionally consisting of: G236R / L328R, E233P / L234V / L235A / G236del / S2 39K, E233P / L234V / L235A / G236del / S267K, E233 P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G and E233P / L 234V / L235A / G236del ("del" means deletion, e.g., G236 del refers to deletion of glycine at position 236) Variants may be included or excluded in some embodiments that utilize them. The ablation mutants used ablate FcγR binding but generally ablate FcRn binding. Note that there is no

[0350] [Table 84]

[0351] In one embodiment, the CD3 binding molecule (e.g., MBM) of the present disclosure comprises a first Fc region In one embodiment, the first Fc region and / or the second Fc region The Fc region contains the following mutations: E233P, L234V, L235A, G236del, and and S267K.

[0352] The Fc domain of human IgG1 is the most highly functional for binding to Fcγ receptors, and therefore forms heterodimers. When the constant domain (or Fc domain) in the antibody framework is IgG1, abrasion A mutant form can be used.

[0353] Alternatively or in addition to ablation mutants of the IgG1 background, glycosylation a mutation at position 297, e.g., a substitution of an asparagine residue at position 297 with an alanine residue (N297A ) or glutamine residue (N297Q), e.g., improves binding to FcγRIIIa. Human IgG2 and IgG4 naturally exhibit reduced binding to Fcγ receptors. Therefore, these scaffolds can be used with or without ablation mutations.

[0354] 7.4.1.2. Fc Domain with Altered Complement Binding CD3 binding molecules (e.g., MBM) are molecules in which one or both Fc regions are involved in Fc binding to complement. The modified complement binding may include an Fc domain containing one or more modifications that alter the binding of complement to the Fc domain. The binding may be increased or decreased.

[0355] In one embodiment, the Fc region contains one or more modifications that reduce its binding to C1q. The initiation of the classical complement pathway involves the binding of hexamers to the CH2 domain of antigen-binding IgG and IgM. It starts with the binding of C1q protein.

[0356] In one embodiment, the CD3 binding molecule (e.g., MBM) comprises one or both Fc regions. but comprises an Fc domain that contains one or more modifications that reduce Fc binding to C1q.

[0357] In one embodiment, the Fc region mutates the leucine residue at position 234 with an alanine residue (L 234A).

[0358] In one embodiment, the Fc region mutates the leucine residue at position 235 with an alanine residue (L 235A).

[0359] In one embodiment, the Fc region replaces the leucine residue at position 235 with a glutamic acid residue (L235E).

[0360] In one embodiment, the Fc region mutates the glycine residue at position 237 with an alanine residue (G 237A).

[0361] In one embodiment, the Fc region is modified by substituting the lysine residue at position 322 with an alanine residue (K3 22A).

[0362] In one embodiment, the Fc region mutates the proline residue at position 331 to an alanine residue (P 331A).

[0363] In one embodiment, the Fc region is modified by substituting the proline residue at position 331 with a serine residue (P3 31S).

[0364] In one embodiment, the CD3 binding molecule (e.g., MBM) comprises an Fc IgG4 naturally has a lower complement activation profile than IgG1 Thus, in one embodiment, CD 3. Binding molecules (e.g., MBM) contain an IgG4 Fc domain and increase FcγR binding It contains one or more modifications that cause

[0365] It is understood that any of the modifications listed above may be combined to reduce C1q binding. It will be understood.

[0366] 7.4.1.3. Fc Domains with Engineered Disulfide Structures CD3 binding molecules (e.g., MBM) may be modified to generate and / or remove cysteine ​​residues. The cysteine ​​residues may be present in the polypeptide monoclonal antibody. Fc-based multispecific binding is achieved by forming disulfide bridges between individual pairs of mers. Therefore, the number and structure of cysteine ​​residues play an important role in the spontaneous assembly of polymer molecules. and / or modifying the structure of the CD3 binding molecule (e.g., MBM) by altering the position of the Thus, it is possible to produce proteins with improved therapeutic properties.

[0367] The CD3 binding molecules (e.g., MBM) of the present disclosure may comprise one or both Fc regions, preferably Both Fc regions may comprise an Fc domain containing a cysteine ​​residue at position 309. In some embodiments, the cysteine ​​residue at position 309 is generated by modification, e.g., in IgG In the case of the Fc domain derived from IgG1, the leucine residue at position 309 is replaced by a cysteine ​​residue (L In the case of the Fc domain derived from IgG2, the variance at position 309 is The amino acid residue is replaced with a cysteine ​​residue (V309C).

[0368] In one embodiment, the Fc region mutates the valine residue at position 308 to a cysteine ​​residue (V 308C).

[0369] In one embodiment, the two disulfide bonds in the hinge region are Removed by mutating CPPC (SEQ ID NO: 9) to SPPS (SEQ ID NO: 14) will be done.

[0370] 7.4.1.4. Fc Domains with Altered Glycosylation In certain embodiments, the immunoglobulins contain fewer glycosylation sites than the corresponding immunoglobulin. CD3 binding molecules (e.g., MBM) with the above manufacturability are provided. Proteins have simpler post-translational glycosylation patterns and are therefore simpler. , and are cheaper to manufacture.

[0371] In one embodiment, the glycosylation site in the CH2 domain is at position 297 The asparagine residue is replaced with an alanine residue (N297A) or a glutamine residue (N297Q). In addition to improved manufacturability, these glycosyl radicals are The variants also reduce FcγR binding as described herein above.

[0372] In certain embodiments, hypofucosylated antibodies or antibodies having reduced amounts of fucosyl residues are modified types of glycosylation, such as antibodies with increased bisecting GlcNac structures. Such modified glycosylation patterns can be used to generate CD3 binding molecules having: These carbohydrate modifications have been demonstrated to improve the ADCC ability of antibodies. For example, expressing a CD3 binding molecule in a host cell with altered glycosylation machinery can Cells with altered glycosylation machinery are well known in the art. and a CD3-binding molecule is internally expressed, thereby resulting in modified glycosylation. The host cells can be used to produce CD3 binding molecules with silyl groups. In European Patent No. 1,176,195, fucosyltransferases were used to A cell line has been described that has a functionally disrupted FUT8 gene encoding Antibodies expressed in such cell lines tend to exhibit hypofucosylation. In the PCT publication WO 03 / 035835 pamphlet by TA, fucose is have a reduced ability to bind sn(297)-linked carbohydrates in the appropriate host cells A mutant CHO cell line, Lecl3 cells, has been described that also results in hypofucosylation of expressed antibodies (Shields et al., 2002, J. Biol. Chem. 27 7:26733-26740). Umana et al., PCT Publication No. Brochure No. 99 / 54342 describes glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnT III)) and in the modified cell lines Antibodies expressed in this manner have increased bisecting GlcNa chains, resulting in increased ADCC activity of the antibody. The c structure is shown (Umana et al., Nat. Biotech. 17:176-180, 1999).

[0373] Fc Heterodimerization Many multispecific molecule formats differ from natural immunoglobulins in that they contain non-identical antigen-binding domains. (or a portion thereof, e.g., VH or VH-CH1 of a Fab), Insufficient Fc regions to form an Fc domain. Unwanted heterodimerization has always been an obstacle to increasing the yield of desired multispecific molecules. Various techniques available in the art are available, e.g. See, for example, European Patent Application Publication No. 1870459A1; U.S. Patent No. 5,582,996 No. 5,731,168; U.S. Pat. No. 5,910,573 Details; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441 ;U.S. Patent No. 7,183,076;U.S. Patent Application Publication No. 2006204493A No. 1 specification; and PCT publication number WO 2009 / 089004A1 pamphlet As disclosed in, Fc regions that may be present in the CD3 binding molecules (e.g., MBM) of the present disclosure. It can be used to promote dimerization of the domains.

[0374] The present disclosure provides Fc heterodimers, i.e., Fc domains comprising heterologous, non-identical Fc regions. The present invention provides a CD3 binding molecule (e.g., MBM) containing a heterodimer. operably linked to the ABM (or portion thereof, e.g., VH or VH-CH1 of the Fab) Fc regions that promote dimerization of Fc regions and are operably linked to the same ABM or portion thereof Typically, each Fc region in an Fc heterodimer is associated with the CH3 domain of an antibody. The CH3 domain may be any isotype, clone, or combination thereof, as described in the previous section. class or subclass, preferably IgG (IgG1, IgG2, IgG3 and IgG4) It is derived from the constant region of an antibody of the class.

[0375] Typically, MBMs contain CH1 domain, CH2 domain, human IL-1 domain, and human IL-2 domain in addition to the CH3 domain. The VH domain, VL domain, CDR and / or antigen binding domain described herein In one embodiment, two helical fragments are combined to form a single antibody fragment. The polypeptide is two heavy chains that form a bispecific or multispecific molecule. Heterodimerization of two different heavy chains in the α-domain produces the desired antibody or antibody-like molecule. While this results in a decrease in the yield of the desired antibody or molecule, homodimerization of identical heavy chains reduces the yield of the desired antibody or molecule. In an exemplary embodiment, two or more heteropolypeptide chains comprise a CH3 domain. and comprising two chains forming a molecule of any of the above-described multispecific molecule formats of this disclosure. In one embodiment, the two heteropolypeptide chains comprising the CH3 domain are unmodified. Modifications that favor heterodimeric association of the polypeptides relative to the unmodified chains are included. Various examples are shown in Table 4 below and in sections 7.4.1.5.1 to 7.4.1.5.8. .

[0376] [Table 85]

[0377] [Table 86]

[0378] [Table 87]

[0379] [Table 88]

[0380] [Table 89]

[0381] [Table 90]

[0382] [Table 91]

[0383] [Table 92]

[0384] 7.4.1.5.1. Stereovariants CD3 binding molecules (e.g., MBM) bind to one or more of the constant domains of the Fc domain. For example, the polypeptide may include one or more, e.g., multiple, modifications to the CH3 domain. Thus, the CD3 binding molecules (e.g., MBM) of the present disclosure may contain antibody heavy chain constant domains, such as C In one example, the CD4 domain comprises two polypeptides each comprising an H2 or CH3 domain. 3. Two heavy chain constant domains of a binding molecule (e.g., MBM), e.g., CH2 or CH3 domains The amino acid sequence contains one or more modifications that allow for heterodimeric association between the two chains. In one embodiment, the one or more modifications are located on the CH2 domains of the two heavy chains. The one or more modifications may be made to at least two polypeptides of a CD3 binding molecule (e.g., MBM). It is located on the CH3 domain of the ribosomal protein.

[0385] One mechanism of Fc heterodimerization is commonly referred to as "knob-and-hole" or "knob-into" These terms are used, for example, in Ridgway et al., 96,Protein Engineering 9(7):617;Atwell e t al.,1997,J.Mol.Biol.270:26;U.S. Patent No. 8,216, 805, which favors the formation of Fc heterodimers over Fc homodimers. Knob-in-hole mutations are amino acid mutations that result in beneficial steric effects. It can be combined with other techniques to improve the efficiency.

[0386] In one embodiment, a first CD3 binding molecule (e.g., MBM) comprising a heavy chain constant domain One or more modifications to the polypeptide can generate a "knob," which can be used to identify the CD3-binding molecule. One or more modifications to the second polypeptide of the molecule (e.g., MBM) create a "hole." and a polypeptide heterocycle of a CD3 binding molecule (e.g., MBM) containing a heavy chain constant domain. Dimerization allows the "knob" to associate with the "hole" (e.g., interact, e.g., The CH2 domain of the first polypeptide interacts with the CH2 domain of the second polypeptide. or the CH3 domain of the first polypeptide is fused to the CH3 domain of the second polypeptide. As used herein, the term "no The "antibody" refers to the first polypeptide of a CD3 binding molecule (e.g., MBM) that contains a heavy chain constant domain. protrude from the boundary of the domain, thus stabilizing the heteromultimer, thereby forming e.g. CD3 binding containing heavy chain constant domains to favor heteromultimer formation over The complementary "hole" at the interface of the molecule (e.g., MBM) with the second polypeptide is located. A knob refers to at least one amino acid side chain that can be positioned. or can be introduced synthetically (e.g., by modifying the nucleic acid encoding the boundary). Preferred import residues for forming the knob are generally natural amino acid residues, Preferably, arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (T). Most preferred are tryptophan and tyrosine. In a preferred embodiment, the original residues for the formation of the protrusion have small side chain volumes, e.g. For example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine is.

[0387] The "hole" is the second portion of the CD3 binding molecule (e.g., MBM) that contains the heavy chain constant domain. CD3 binding molecules that are recessed from the polypeptide boundary and therefore contain the heavy chain constant domain the corresponding knobs on the adjacent interaction surface of the first polypeptide (e.g., MBM) A hole can be present in the original boundary, or They may be introduced synthetically (e.g., by modifying the nucleic acid encoding the boundary). Preferred import residues for the formation of the nucleotide are usually naturally occurring amino acid residues, preferably is selected from alanine (A), serine (S), threonine (T) and valine (V). Most preferred is serine, alanine or threonine. The original residue for hole formation has a large side chain capacity, e.g., tyrosine, arginine, phenylalanine or tryptophan.

[0388] In a preferred embodiment, the first CH3 domain comprises residues 366, 405, or 407 modified in order to produce either a "knob" or a "hole" (as described above) The second CH3 domain, which heterodimerizes with the first CH3 domain, is located at residue 366 is modified in the first CH3 domain, residue 407, residue 405 are When modified in the CH3 domain, residue 394 or residue 407 is in the first CH3 domain. and modified in the first CH3 domain to form a "hole" complementary to the "knob" or "hole" of the first CH3 domain. When generating the "knob" or "knob", it is modified at residue 366.

[0389] In another preferred embodiment, the first CH3 domain is modified at residue 366 The second CH3 domain, which heterodimerizes with the first CH3 domain, has a residue 3 66, 368 and / or 407 to form the "knob" or creates a "hole" or "knob" complementary to the "hole". The modification to the CH3 domain of introduces a tyrosine (Y) residue at position 366. In one embodiment, the modification to the first CH3 is T366Y. The modification to the first CH3 domain is a tryptophan (W) residue at position 366. In one embodiment, the modification to the first CH3 is T366W. In some embodiments, the heterologous sequence is a first CH3 domain modified at position 366. Modifications to the second CH3 domain (e.g., a nucleotide sequence introduced at position 366) that allow dimerization having a tyrosine (Y) or tryptophan (W), e.g., modified T366Y or T36 6W) has a modification at position 366, a modification at position 368, and a modification at position 407. In one embodiment, the modification at position 366 introduces a serine (S) residue. The modification at position 368 introduces an alanine (A), and the modification at position 407 introduces a valine (Va). (V). In one embodiment, the modifications are T366S, L368A and Y40 In one embodiment, the first CH3 domain of the multispecific molecule comprises a modified T3 a second CH3 domain that contains 66Y and heterodimerizes with the first CH3 domain contains the modifications T366S, L368A and Y407V, or vice versa. In one embodiment, the first CH3 domain of the multispecific molecule comprises the modification T366W and the first The second CH3 domain that heterodimerizes with the CH3 domain is modified T366S, Contains L368A and Y407V or vice versa.

[0390] Further steric or "distortion" (e.g., knobs-in-holes) modifications may be made in accordance with the PCT publication number. International Publication No. 2014 / 145806 (e.g., International Publication No. 2014 / 145 806 pamphlet), Figures 3, 4 and 12, PCT Publication No. WO 2014 / 02314 No. 110601 and PCT Publication No. WO 2016 / 086186 Brochure, International Publication No. 2016 / 086189, International Publication No. 2016 / 086196 and WO 2016 / 182751 Examples of KIH mutants include: , L368D and K3 paired with a second constant chain containing S364K and E357Q modifications. It contains a first constant chain containing a 70S modification.

[0391] Suitable for use in any of the CD3 binding molecules (e.g., MBM) of the present disclosure. Knob-in-hole modified pairs such as those described in WO 1996 / 027011 are also suitable. Fret and Merchant et al., 1998, Nat. Biotechnol. l., 16:677-681.

[0392] In a further embodiment, the CH3 domain is modified to introduce a pair of cysteine ​​residues. Without being bound by theory, it is believed that the disulfide bond formation The introduction of a pair of cysteine ​​residues capable of binding to a heterodimer containing paired CH3 domains allows for the formation of a heterodimer containing ... paired CH3 domain. It is believed to confer stability to the merized CD3 binding molecule (e.g., MBM). In one embodiment, the first CH3 domain contains a cysteine ​​at position 354 and a cysteine ​​at position 355. The second CH3 domain, which heterodimerizes with the H3 domain, has a sequence at position 349. In one embodiment, the first CH3 domain comprises a sigma at position 354. stein (e.g., containing the modification S354C) and tyrosine (Y) at position 366 (e.g., For example, the modified T366Y) and heterodimerizes with the first CH3 domain. The second CH3 domain contains a cysteine ​​at position 349 (e.g., the modification Y349C). ), serine at position 366 (including, for example, the modification T366S), ara at position 368 a valine at position 407 (e.g., containing the modification Y4 In one embodiment, the first CH3 domain comprises a nucleotide sequence at position 354. Cysteine ​​(including, for example, the modified S354C) and tryptophan at position 366 (W) (e.g., containing the modification T366W), which together with the first CH3 domain form a heterologous The second CH3 domain that dimerizes contains a cysteine ​​at position 349 (e.g., modified Y3 49C), serine at position 366 (including, for example, the modification T366S), alanine at position 104 (e.g., containing the modification L368A) and valine at position 407 (e.g., containing the modification For example, containing the modification Y407V.

[0393] A further mechanism that finds use in generating heterodimers is Gunasekara n et al.,2010,J.Biol.Chem.285(25):19637 As described, this is sometimes referred to as "electrostatic steering." In this embodiment, the formation is referred to as a "charge pair." As will be appreciated by those skilled in the art, these can also affect pI and therefore purification, and therefore sometimes referred to as pI variants. However, they are not generated to force heterodimerization. These were not used as purification tools and are therefore classified as "stereovariants." includes, but is not limited to, D221E / P228R / K409R paired with D221R / P228R / K409R. Paired with P228E / L368E and C220R / E224R / P228R / K409R This includes the C220E / P228E / 368E.

[0394] Further variants include the pI variants outlined herein or those described in U.S. Patent Application Publication No. 2012 Other variants, such as the other stereovariants shown in Figure 37 of US Patent Application Publication No. 2001 / 0149876, and optionally They can be selectively and independently combined.

[0395] In one embodiment, the steric variants outlined herein include any pI variant (or Fc variants, FcRn variants, and other variants) in one or both F regions. can be selectively and independently incorporated, and independently or optionally, a CD3 binding moiety Children can be included or excluded.

[0396] A list of suitable distortion variants can be found in Table 5 and will have particular utility in many embodiments. In many embodiments, including but not limited to, S364K / E 357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411T / E360E / Q362E:D401K;L36 8D / K370S:S364K / E357L;andK370S:S364K / E357 The pairs of sets containing Q have specific usages. Regarding nomenclature, "S364K / E357Q The "L368D / K370S" pair is a double mutant set S364K / one carrying E357Q and the other carrying the double mutant set L368D / K370S means.

[0397] [Table 93]

[0398] [Table 94]

[0399] [Table 95]

[0400] [Table 96]

[0401] [Table 97]

[0402] In one embodiment, the CD3 binding molecule comprises a first Fc region and a second Fc region. In one embodiment, the first Fc region contains the following mutations: L368D and K370S. and the second Fc region comprises the following mutations: S364K and E357Q. wherein the first Fc region contains the following mutations: S364K and E357Q, and the second Fc region contains the following mutations: S364K and E357Q. The c region contains the following mutations: L368D and K370S.

[0403] 7.4.1.5.2. Alternative Knobs and Holes: IgG Heterodimerization of the polypeptide chain of a CD3-binding molecule (e.g., MBM) containing paired CH3 domains. Heterodimerization involves one or more modifications in the CH3 domain derived from the IgG1 antibody class. In one embodiment, the modification can be increased by introducing a second CH3 domain. It contains a K409R modification to one CH3 domain paired with an F405L modification in the Further modifications may also or alternatively be made to 366, 368, 370, 399, 405, 4 Preferably, the heterologous sequences of polypeptides containing such modifications are at positions 07 and 409. Dimerization is carried out under reducing conditions, for example, at 25 to 37°C, for example, at 25°C or 37°C for 1 to 10 hours. , for example, 1.5 to 5 hours, for example, 10 to 100 mM 2-MEA (e.g., For example, 25, 50 or 100 mM 2-MEA.

[0404] The amino acid substitutions described herein can be made by modifying the CH3 domain (see, e.g., McPherson, ed., 1991, Dire cted Mutagenesis:a Practical Approach;Ad (See Elman et al., 1983, DNA, 2:183).

[0405] IgG heterodimerization techniques are described, for example, in WO 2008 / 119353. , International Publication No. 2011 / 131746 and International Publication No. 2013 / 06 Further details are provided in Brochure No. 0867.

[0406] In any of the embodiments described in this section, the CH3 domain is selected from the group consisting of 7.4.1. It can be further modified to introduce a pair of cysteine ​​residues as described in Section 5.1. .

[0407] 7.4.1.5.3.pI (isoelectric point) variants Generally, as will be appreciated by those skilled in the art, pI variants fall into two general categories: -: those that increase the pI of a protein (basic change) and those that decrease the pI of a protein As described herein, all combinations of these variants One Fc region must exhibit a pI that is not significantly different from the wild-type or wild-type. Alternatively, each Fc region may be a variant of the other that is more basic or more acidic. , one becomes more basic and one becomes more acidic.

[0408] Exemplary combinations of pI variants are shown in Table 6. As such, these changes are shown relative to IgG1, but all isotypes are Similar to the isotypic hybrids, the heavy chain constant domains can be altered in this manner. When derived from IgG2-4, R133E and R133Q can also be used.

[0409] [Table 98]

[0410] In one embodiment, for example, FIGS. 1B to 1W, 1Y to 1AH, 2B to 2L, and 2 In the N~2V format, the pI variant combination is 208D / 295E / 384D / 41 8E / 421D mutant (N208D / Q295E / N38 for human IgG1) 4D / Q418E / N421D) and one Fc region (negative Fab side) containing a positively charged a second Fc region (positive Fc region) containing an scFv linker, e.g., L36 (described in Section 7.4.3); However, as will be appreciated by those skilled in the art, the first Fc The region contains the CH1 domain including position 208. Therefore, it does not contain the CH1 domain. Constructs (e.g., in the format shown in Figure 2K, e.g., CH1 as one of the domains) In the case of MBM without domains, the negative pI mutant Fc set is 295E / 384D / 418E / 421D variant (Q295E / N for human IgG1) 384D / Q418E / N421D).

[0411] In one embodiment, the first Fc region has a set of substitutions from Table 6 and the second Fc region has a set of substitutions from Table 6. The c region is joined to a charged linker (e.g., selected from those described in Section 7.4.3). To be continued.

[0412] In one embodiment, the CD3 binding molecule of the present disclosure comprises a first Fc region and a second Fc region. In one embodiment, the first Fc region comprises the following mutations: N208D, Q 295E, N384D, Q418E, and N421D. The Fc region of contains the following mutations: N208D, Q295E, N384D, Q418E and Includes N421D.

[0413] 7.4.1.5.4. Isotopic Variants Additionally, many embodiments of the present disclosure provide for the conversion of one IgG isotype to another. , which depends on the "import" of pI amino acids at specific positions and is therefore undesirable. Reduce or eliminate the possibility of introducing immunogenicity into the variants. Many of these are listed in U.S. Pat. This is shown in Figure 21 of Publication No. 2014 / 0370013. is a common isotype of therapeutic antibody for a variety of reasons, including enhanced effector function. However, the heavy chain constant region of IgG1 has a higher pI than that of IgG2. (8.10 vs. 7.31). Introducing IgG2 residues into the IgG1 backbone at specific positions This reduces (or increases) the pI of the resulting Fc region, resulting in a longer serum half-life. For example, IgG1 has glycine at position 137 (pI 5.97), and IgG2 has It has glutamic acid (pI 3.22); importing glutamic acid results in This affects the pI of the resulting protein. As explained below, this has a significant effect on the pI of the mutant antibody. Generally, many amino acid substitutions are required to affect the As will be seen, it should be noted that even changes in the IgG2 molecule can extend the serum half-life. sea ​​bream.

[0414] In other embodiments, as further described below, the resulting protein To reduce the overall charge state (e.g., replacing higher pI amino acids with lower pI ones), amino acid modifications) or to allow for structural adjustments for stability. , non-isotypic amino acid changes are made.

[0415] Furthermore, both the heavy and light chain constant domains of the CD3 binding molecule, which comprises two half antibodies, are expressed as pI By modifying the antibody, significant changes can be seen in each half antibody. If the difference is at least 0.5, ion exchange chromatography or isoelectric focusing or Separation by other methods sensitive to the isoelectric point becomes possible.

[0416] 7.4.1.5.5. Calculation of pI The pI of a half antibody comprising an Fc region and an ABM or ABM chain is determined by the pI of the variant heavy chain constant domain. and the pI of the whole half antibody containing the variant heavy chain constant domain and the ABM or ABM chain. Thus, in one embodiment, the pI change is as described in U.S. Patent Application Publication No. 2014 Using the chart in Figure 19 of the / 0370013 specification, As described herein, modified half antibodies are generally calculated based on the half antibody Alternatively, the pI of each half antibody can be compared.

[0417] 7.4.1.5.6. pI Variants That Also Confer Better FcRn In Vivo Binding If the pI variant reduces the pI of the Fc region, it is believed to improve serum retention in vivo. This may have the further advantage of

[0418] pI mutations are responsible for Fc sequestration upon binding to FcRn at pH 6 in endosomes. The Fc region is thought to confer a longer half-life to antigen-binding molecules in vivo ( Ghetie and Ward,1997,Immunol Today.18(12 ):592-598). The endosomal compartment then recycles Fc to the cell surface. When the compartment opens to the extracellular space, the pH becomes approximately 7.4, and the blood In mice, Dall' Acqua et al. FcRn binding increased at pH 7.4 and H6, but the serum concentration of the FcRn variants was actually decreased. , which has been shown to have the same half-life as wild-type Fc (Dall' Acqua et al. , 2002, J. Immunol. 169:5171-5180). F at pH 7.4 The increased affinity of Fc for cRn is thought to prevent the release of Fc into the blood. Therefore, Fc mutations that increase the half-life of Fc in vivo would ideally be more efficient. Increased FcRn binding at lower pH while still allowing Fc release at pH The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Therefore, it is surprising that His residues are found at key positions in the Fc / FcRn complex. It's not something that should be done.

[0419] Antibodies with variable regions that have lower isoelectric points may also have longer serum half-lives. It has been suggested (Igawa et al., 2010, PEDS.23(5):385 However, this mechanism is not yet fully understood. As described herein, antibodies with reduced pI and extended half-lives Constant region variants have been proposed to develop more modular CD3-binding molecules to improve their pharmacokinetic properties. This will provide a tailored approach.

[0420] 7.4.1.5.7.Polar crosslinking Heterodimers of polypeptide chains of CD3-binding molecules (e.g., MBM) containing an Fc domain The solubility can be increased by introducing modifications based on the principle of "polar cross-linking" The principle of "cross-linking" is to link residues at the binding interface of two polypeptide chains in a heterodimeric form. While interacting with residues with similar (or complementary) physical properties, the homodimeric form In particular, these modifications allow the heterodimer to interact with residues of different physical properties. In the formation of ATP, polar residues interact with polar residues, while hydrophobic residues interact with hydrophobic residues. In contrast, in homodimer formation, the residues are designed to be sparsely populated with polar residues. Modified to interact with aqueous residues. Favorable interactions in heterodimeric form and unfavorable interactions in the homodimeric form, which in turn contribute to the formation of the Fc region in the homodimer. The resulting nucleotide sequence is such that it has a higher propensity to form heterodimers than to form dimers.

[0421] In an exemplary embodiment, the modifications are at residues 364, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415 It is generated at one or more of positions 99, 405, 409 and 411.

[0422] In one embodiment, S364L, T366V, L368Q, N399K, F405 one or more modifications selected from S, K409F and R411K in the two CH3 domains One or more modifications selected from Y407F, K409Q and T411N are introduced into the Decorations can be introduced into the second CH3 domain.

[0423] In another embodiment, S364L, T366V, L368Q, D399K, F405 one or more modifications selected from the group consisting of S, K409F and T411K in one CH3 domain, while one selected from Y407F, K409Q and T411D One or more modifications are introduced into the second CH3 domain.

[0424] In one exemplary embodiment, a substitution of the threonine at position 366 of one CH3 domain The original residue is replaced with a valine, while the tyrosine at position 407 in the other CH3 domain The original residue is replaced with phenylalanine.

[0425] In another exemplary embodiment, the residue of serine at position 364 of one CH3 domain is The residue at position 368 of the same CH3 domain is replaced by leucine, while the residue at position 368 of the same CH3 domain is replaced by leucine. The original residue is replaced with glutamine.

[0426] In yet another exemplary embodiment, a phenylalanine amino acid sequence at position 405 of one CH3 domain is The original residue of α-alanine was replaced by serine, and the lysine at position 409 of this CH3 domain The original residue of α-glucan is replaced by phenylalanine, while the residue at position 409 of the CH3 domain is The original lysine residue in is replaced with glutamine.

[0427] In yet another exemplary embodiment, an aspartate amino acid at position 399 of one CH3 domain is The original residue of paragic acid was replaced by lysine, and the trehalose at position 411 of the same CH3 domain The original residue of onine is replaced by lysine, while the other residue at position 411 of the CH3 domain The original threonine residue is replaced with aspartic acid.

[0428] The amino acid substitutions described herein can be made by modifying the CH3 domain (see, e.g., McPherson, ed., 1991, Dire cted Mutagenesis:a Practical Approach;Ad (See Elman et al., 1983, DNA, 2:183). Polar crosslinks The method is described, for example, in WO 2006 / 106905 and WO 200 9 / 089004 Brochure and K. Gunasekaran, et al. (20 10)The Journal of Biological Chemistry,2 85:19637-19646.

[0429] Further polar crosslinking modifications are described, for example, in PCT Publication No. WO 2014 / 145806 No. 2014 / 145806 (e.g., Figure 6 in WO 2014 / 145806), PCT Publication Number International Publication No. 2014 / 110601 Pamphlet and PCT Publication Number Country International Publication No. 2016 / 086186 Brochure, International Publication No. 2016 / 086189 Brochure, International Publication No. 2016 / 086196 Brochure and International Publication No. 201 No. 6 / 182751, the contents of which are incorporated herein in their entirety. Examples of polar bridge mutants are N208D, Q295E, N384D, Q41 It contains a constant chain containing 8E and N421D modifications.

[0430] In any of the embodiments described herein, the CH3 domain is selected from the group consisting of 7.4.1 It may be further modified to introduce a pair of cysteine ​​residues as described in Section 5.1. do.

[0431] Further techniques for promoting heterodimerization are described, for example, in WO 2016 / 10 Pamphlet No. 5450, International Publication No. 2016 / 086186, International Publication Pamphlet No. 2016 / 086189, Pamphlet No. WO 2016 / 086196 Lett, International Publication No. 2016 / 141378 and International Publication No. 2014 / 145806 brochure and International Publication No. 2014 / 110601 brochure Any of the above techniques can be used in combination with a CD3 binding molecule (e.g., , MBM).

[0432] 7.4.1.5.8. Combining Heterodimerization Variants with Other Fc Variants As will be appreciated by those skilled in the art, the heterodimerization mutants described (skewed and / or All of the Fc domains (including pI variants) are intended to be fully functional as long as the Fc region of the Fc domain retains the ability to dimerize. and can be combined in any way, optionally and independently. All of these variants can be combined in any heterodimerization format.

[0433] In the case of pI variants, embodiments that find particular use are shown in Table 6, but may also be used to facilitate purification. The fundamental principle is to change the pI difference between the two Fc regions of an Fc heterodimer in order to Therefore, other combinations can be generated.

[0434] Furthermore, heterodimerization mutants, distortions and pIs are all generally referred to herein as As outlined, independently and optionally, Fc ablation variants, Fc mutations The antibody is combined with an FcRn variant.

[0435] In certain embodiments, certain of the distortions and pI variants that find use in the present disclosure The combination is T366S / L368A / Y407V:T366W (optionally crosslinked disulfide). Includes fid, T366S / L368A / Y407V / Y349C:T366W / S35 4C), one Fc region contains Q295E / N384D / Q418E / N481D The other Fc region is connected to a positively charged scFv linker (if the format contains an scFv domain). As will be appreciated by those skilled in the art, "knob-in-hole" mutants include those with a pI of Since it does not change the Fc region, it can be used with either one of the Fc regions of the Fc heterodimer. .

[0436] In certain embodiments, the first and second Fc regions that find use in the present disclosure comprise amino and / or The second Fc region contains an ablation mutant with substitutions 233P / L234V / L235A / G 236del / S267K, and the first and / or second Fc region comprises the pI variant substitution N 208D / Q295E / N384D / Q418E / N421D(pl_(-)_isos teric_A).

[0437] Hinge Area CD3 binding molecules (e.g., MBMs) can, for example, link an antigen binding module to an Fc region. The hinge region may also include a hinge region that is a natural or modified hinge region. The region is typically found at the N-terminus of the Fc region.

[0438] A native hinge region is the hinge region that can normally be found between the Fab and Fc domains in a natural antibody. A modified hinge region is any hinge region that differs in length and / or composition from the native hinge region. Such hinges are found in humans, mice, rats, rabbits, sharks, pigs, and hamsters. The hinge region may include hinge regions from other species, such as rabbit, camel, llama, or goat hinge regions. The modified hinge region is derived from an antibody of a different class or subclass than that of the heavy chain Fc region. Alternatively, the modified hinge region may comprise a portion or repeat of the natural hinge. Each unit in the repeat may comprise a repeat unit derived from a natural hinge region. In this case, the native hinge region may contain one or more cysteine ​​or other residues substituted with serine or alanine, etc. by converting a neutral residue to a cysteine ​​residue, or by converting a suitably placed residue to a cysteine ​​residue. By such means, the number of cysteine ​​residues in the hinge region can be modified. This approach is described in U.S. Patent No. 5,677,444 by Bodmer et al. 25. By varying the number of cysteine ​​residues in the hinge region, For example, to facilitate assembly of the light and heavy chains or to increase the stability of the CD3 binding molecule. Other modified hinge regions can be entirely synthetic and can be of any length. They can be designed to have desired properties, such as cysteine ​​composition and flexibility.

[0439] Some modified hinge regions are described, for example, in U.S. Pat. No. 5,677,425; Publication No. 9915549, International Publication No. 2005003170 , International Publication No. 2005003169 Brochure, International Publication No. 2005003170 Brochure, International Publication No. 9825971 Brochure and International Publication No. 2005003 This has already been mentioned in pamphlet No. 171.

[0440] Examples of suitable hinge sequences are shown in Table 7.

[0441] [Table 99]

[0442] [Table 100]

[0443] In one embodiment, the heavy chain Fc region has an intact hinge region at its N-terminus. .

[0444] In one embodiment, the heavy chain Fc region and hinge region are derived from IgG4, and the hinge region contains the modified sequence CPPC (SEQ ID NO: 9). The core hinge region of human IgG4 contains the sequence C containing the sequence CPSC (SEQ ID NO: 10) compared to IgG1 containing PPC (SEQ ID NO: 9). The serine residues present in the IgG4 sequence also allow for increased flexibility in this region. Thus, part of the molecule is in the IgG molecule to form interchain disulfides. Disulfide bonds within the same protein chain (intrachain disulfides) rather than cross-linking to other heavy chains in the chain. (Angel et al., 1993, Mol lmmunol 30(1):105-108). The serine residue was changed to proline, resulting in the same chain structure as IgG1. Obtaining the IgG4 hinge sequence allows for the complete formation of interchain disulfides in the IgG4 hinge region. This modified isotype results in a reduced heterogeneity in the purified product. It is called IgG4P.

[0445] ABM Linker In certain embodiments, the present disclosure provides a CD3 binding molecule (e.g., For example, a MBM), wherein two or more components of the ABM (e.g., the VH of an scFv) are and VL), two or more ABM or ABM and non-ABM domains (e.g., Fc region, etc. The dimerization domains (dimerization domains) are linked to each other by a peptide linker. For example, as described in Section 7.13.2, the drug may be coupled to a CD3 binding molecule (e.g., In contrast to the ADC linkers used to connect the AB It is called the "M linker."

[0446] Peptide linkers can range from 2 amino acids to 60 or more amino acids, and certain In embodiments, the peptide linker is between 3 amino acids and 50 amino acids, between 4 and 30 amino acids, Amino acids, 5 to 25 amino acids, 10 to 25 amino acids, or 12 to 20 amino acids In certain embodiments, the peptide linker is in the range of 2 amino acids, 3 amino acids, Amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids Amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids Amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids , 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids Acid, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 4 4 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 ​​amino acids amino acids, 49 amino acids or 50 amino acids in length.

[0447] Charged and / or flexible linkers are particularly preferred.

[0448] Examples of flexible ABM linkers that can be used in CD3 binding molecules (e.g., MBMs) include: , Chen et al.,2013,Adv Drug Deliv Rev.65( 10):1357-1369 and Klein et al., 2014, Protein Engineering,Design&Selection 27(10):325 A particularly useful flexible linker is (GGG (GS)n (SEQ ID NO: 24) (also referred to as (G4S)n (SEQ ID NO: 24)). In an embodiment, n is any number from 1 to 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 or any range bounded by any two of the above numbers, e.g. 1-5, 2-5, 3-6, 2-4, 1-4, etc. and others.

[0449] In addition to ABM linkers suitable for use in the CD3 binding molecules (e.g., MBMs) of the present disclosure, An example is shown in Table 8 below.

[0450] [Table 101]

[0451] [Table 102]

[0452] [Table 103]

[0453] [Table 104]

[0454] [Table 105]

[0455] In various embodiments, the present disclosure provides CD3 binding molecules (e.g., CD3A-CD3B ... Each of the ABM linkers is optionally selected from Table 8 above. The length of the selected amino acids is 2 to 60 amino acids, preferably 4 to 30 amino acids, 5 to 60 amino acids. 25 amino acids, 10 to 25 amino acids, or 12 to 20 amino acids in length. In certain embodiments, the CD3 binding molecule (e.g., MBM) is a 2, 3, 4 The ABM linker comprises one, five, or six ABM linkers. The ABM linker is a CD3 binding molecule (e.g., , MBM) on one, two, three, four or even more polypeptide chains. do.

[0456] 7.5. Bispecific binding molecular morphology Exemplary BBM configurations are shown in Figure 1. Figure 1A illustrates the BBM configurations shown in Figures 1B-1AH. The components of the antibody are shown below: scFv, Fab, scFab, and non-immunoglobulin-based ABM. and Fc domains are described in Sections 7.3 and 7.4, respectively, for these components. The components of the BBM configuration shown in Figure 1 can have the properties described in Sections 7.3 and 7. By any of the means described in Section 4 (e.g., direct conjugation, ABM linker, disulfide (e.g., by Fc domains modified with Fc domain binding or knob-in-hole interactions) The orientation and association of the various components shown in FIG. 1 is exemplary. as will be appreciated by those skilled in the art, other orientations and associations may be suitable (e.g., (e.g., as described in Sections 7.3 and 7.4).

[0457] The BBM is not limited to the configuration shown in Figure 1. Other configurations that may be used are known to those skilled in the art. For example, International Publication No. 2014 / 145806; International Publication No. 201 7 / 124002 Brochure; Liu et al., 2017, Front Im munol.8:38;Brinkmann&Kontermann,2017,mAb s 9:2,182-212; U.S. Patent Application Publication No. 2016 / 0355600; Klein et al., 2016, MAbs 8(6):1010-20; and US See Patent Application Publication No. 2017 / 0145116.

[0458] 7.5.1. Exemplary Bivalent BBM BBMs can be bivalent, i.e., they have two antigen-binding domains, and One or two of them bind to CD3 (ABM1), and one of them binds to a second target antigen (A BM2), for example, binds to CD2 or TAA.

[0459] Exemplary bivalent BBM forms are shown in Figures 1B-1F.

[0460] As shown in Figures 1B-1D, a BBM can contain two half antibodies, one of which is an AB. One half antibody contains one ABM and the other contains one ABM, and the two half antibodies are paired via the Fc domain. are.

[0461] In the embodiment of FIG. 1B, the first (or left) half antibody comprises a Fab and an Fc region. The second (or right) half antibody comprises a Fab and an Fc region. They associate via the Fc region to form an Fc domain.

[0462] In the embodiment of FIG. 1C, the first (or left) half antibody comprises a Fab and an Fc region. The second (or right) half antibody comprises an scFv and an Fc region. , are associated via the Fc region to form an Fc domain.

[0463] In the embodiment of FIG. 1D, the first (or left) half antibody comprises an scFv and an Fc region. The first and second half antibodies comprise an scFv and an Fc region. are associated via the Fc region to form an Fc domain.

[0464] As shown in Figures 1E-1F, the bivalent BBM binds to one Fc region of the Fc domain. It may contain two ABMs combined.

[0465] In the embodiment of FIG. 1E, the BBM comprises Fab, scFv, and Fc domains, Here, the scFv is located between the Fab and Fc domains.

[0466] In the embodiment of FIG. 1F, the BBM (in the "one-arm scFv-mAb" format) b, scFv and Fc domains, where Fab is between the scFv and Fc domains Located in.

[0467] In the embodiment shown in Figures 1B to 1F, each of X and Y is ABM1 or ABM2. and wherein the BBM includes one ABM1 and one ABM2. Accordingly, the present disclosure provides a bivalent BBM as shown in any one of Figures 1B-1F, where X is ABM1 and Y is ABM2 (this form of ABM is conveniently referred to as "B 1"). Thus, the present disclosure provides a method for manufacturing a semiconductor device according to any one of the embodiments shown in FIGS. 1B-1F. Also provided are bivalent BBMs such that X is ABM2 and Y is ABM1 (ABM This form of is conveniently designated "B2").

[0468] 7.5.2. Exemplary Trivalent BBM BBMs can be trivalent, i.e., they have three antigen-binding domains, and One or two of these bind to CD3 (ABM1), and one or two of these bind to a second target. Binds to an antigen (ABM2), such as CD2 or TAA.

[0469] Exemplary trivalent BBM configurations are shown in Figures 1G-1Z.

[0470] As shown in Figures 1G-1N, 1Q-1W, and 1Y-1Z, the BBM consists of two half antibodies. one containing two ABMs and the other containing one ABM, and the two half antibodies may contain Fc They are paired via domains.

[0471] In the embodiment of FIG. 1G, the first (or left) half antibody comprises a Fab and an Fc region. The second (or right) half antibody comprises the scFv, Fab and Fc regions. The half antibodies are associated via their Fc regions to form an Fc domain.

[0472] In the embodiment of Figure 1H, the first (or left) half antibody comprises a Fab and an Fc region. The second (or right) half antibody comprises the Fab, scFv and Fc regions. The half antibodies are associated via their Fc regions to form an Fc domain.

[0473] In the embodiment of FIG. 1I, the first (or left) half antibody comprises an scFv and an Fc region. The first and second halves comprise two Fab and Fc regions. Antibodies associate via their Fc regions to form Fc domains.

[0474] In the embodiment of Figure 1J, the first (or left) half antibody comprises two Fav and Fc regions. and the second (or right) half antibody comprises the Fab and Fc regions. The antibodies associate via the Fc region to form an Fc domain.

[0475] In the embodiment of FIG. 1K, the first (or left) half antibody comprises an scFv and an Fc region. The first and second half antibodies comprise two scFvs and an Fc region. The half antibodies are associated via their Fc regions to form an Fc domain.

[0476] In the embodiment of FIG. 1L, the first (or left) half antibody comprises an scFv and an Fc region. The first and second (or right) half antibodies comprise scFv, Fab and Fc regions. The half antibodies are associated via their Fc regions to form an Fc domain.

[0477] In the embodiment of FIG. 1M, the first (or left) half antibody comprises an scFv and an Fc region. The first and second half antibodies comprise the Fab, scFv, and Fc regions. The half antibodies are associated via their Fc regions to form an Fc domain.

[0478] In the embodiment of FIG. 1N, the first (or left) half antibody has a bispecific antibody-type binding domain. The first (or right) half antibody comprises an Fab and an Fc region, and the second (or right) half antibody comprises an Fab and an Fc region. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0479] In the embodiment of Figure 1Q, the first (or left) half antibody comprises a Fab and an Fc region. The second (or right) half antibody comprises a Fab, an Fc region, and an scFv. The half antibodies are associated via their Fc regions to form an Fc domain.

[0480] In the embodiment of FIG. 1R, the first (or left) half antibody comprises an scFv and an Fc region. The first and second (or right) half antibodies comprise a Fab, an Fc region, and an scFv. The half antibodies are associated via their Fc regions to form an Fc domain.

[0481] In the embodiment of FIG. 1S, the first (or left) half antibody comprises an scFv and an Fc region. The second (or right) half antibody comprises an scFv, an Fc region and a second scFv. and the second half antibody are associated via their Fc regions to form an Fc domain.

[0482] In the embodiment of FIG. 1T, the first (or left) half antibody comprises an scFv, an Fc region, and an F ab, and the second (or right) half antibody comprises the Fab and Fc regions. The half antibodies are associated via their Fc regions to form an Fc domain.

[0483] In the embodiment of FIG. 1U, the first (or left) half antibody comprises two Fab and Fc regions. and the second (or right) half antibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0484] In the embodiment of Figure 1V, the first (or left) half antibody comprises Fab, scFv, and Fc The second (or right) half antibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second half antibodies are associated via the Fc region to form an Fc domain. do.

[0485] In the embodiment of FIG. 1W, the first (or left) half antibody comprises a Fab and an Fc region. The second (or right) half antibody comprises an scFv, a non-immunoglobulin-based ABM, and an Fc region. The first and second half antibodies are associated via the Fc region to form an Fc domain. do.

[0486] In the embodiment of FIG. 1Y, the first (or left) half antibody comprises an scFv and an Fc region. The first and second half antibodies comprise the Fab, scFv, and Fc regions. The half antibodies are associated via their Fc regions to form an Fc domain.

[0487] In the embodiment of FIG. 1Z, the first (or left) half antibody comprises a Fab, an Fc region, and a sc The first and second half antibodies comprise a Fab region, and the second (or right) half antibody comprises a Fab and an Fc region. The half antibodies are associated via their Fc regions to form an Fc domain.

[0488] Alternatively, as shown in Figures 1O and 1P, a trivalent BBM can comprise two half antibodies: It contains one complete ABM (Fab in Figures 1O and 1P) and part of another ABM (one of which is V As soon as the two half antibodies are paired via the Fc domain, VH and VL then associate to form a complete antigen-binding Fv domain.

[0489] As shown in Figure 1X, the BBM can be single-stranded. It comprises three scFv domains linked via

[0490] In the embodiment shown in Figures 1G to 1Z, each of X, Y and A is ABM1 or AB M2, provided that BBM is at least ABM1 and at least one A Thus, a trivalent MBM contains one or two ABM1 and one or two ABM2. In one embodiment, the trivalent BBM comprises two ABM1s and one ABM2. In another embodiment, the trivalent BBM comprises one ABM1 and two ABM2. Includes:

[0491] Therefore, in the present disclosure, a trivalent BBM as shown in any one of Figures 1G-1Z is wherein X is ABM1, Y is ABM1, and A is ABM2 (AB This form of M is conveniently designated "T1").

[0492] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, Here, X is ABM1, Y is ABM2, and A is ABM1 (this morphology is conveniently designated as "T2").

[0493] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, Here, X is ABM2, Y is ABM1, and A is ABM1 (this Form is conveniently designated as "T3").

[0494] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, Here, X is ABM1, Y is ABM2, and A is ABM2 (this Form is conveniently designated as "T4").

[0495] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, Here, X is ABM2, Y is ABM1, and A is ABM2 (this Form is conveniently designated as "T5").

[0496] The present disclosure further provides a trivalent BBM as shown in any one of Figures 1G-1Z, Here, X is ABM2, Y is ABM2, and A is ABM1 (this form is conveniently designated as "T6").

[0497] 7.5.3. Exemplary 4-valent BBM BBMs can be tetravalent, i.e., they have four antigen-binding domains, and One, two, or three of these bind to CD2 (ABM1), and one, two, or three of these bind to binds to a second target antigen (ABM2), e.g., CD2 or a TAA.

[0498] Exemplary tetravalent BBM configurations are shown in Figures 1AA-1AH.

[0499] As shown in Figures 1AA-1AH, a tetravalent BBM can include two half antibodies, each contains two complete ABMs, and the two half antibodies are paired via the Fc domain.

[0500] In the embodiment of FIG. 1AA, the first (or left) half antibody comprises a Fab, an Fc region, and a s The first (or right) half antibody comprises a Fab, an Fc region, and an scFv. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0501] In the embodiment of FIG. 1AB, the first (or left) half antibody comprises Fab, scFv, and F The first (or right) half antibody comprises the Fab, scFv and Fc regions, and the second (or right) half antibody comprises the Fab, scFv and Fc regions. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0502] In the embodiment of FIG. 1AC, the first (or left) half antibody comprises scFv, Fab, and F The first (or right) half antibody comprises the scFv, Fab and Fc regions. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0503] In the embodiment of FIG. 1AD, the first (or left) half antibody comprises a Fab, an Fc region, and and the second (or right) half antibody comprises a Fab, an Fc region, and a second Fab. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0504] In the embodiment of FIG. 1AE, the first (or left) half antibody is an scFv, the second scF and Fc regions, and the second (or right) half antibody comprises a scFv, a second scFv, and an F The first and second half antibodies are associated via the Fc region to form an Fc domain. Complete.

[0505] In the embodiment of FIG. 1AF, the first (or left) half antibody comprises Fab, scFv, and F The first (or right) half antibody comprises the Fab, scFv and Fc regions, and the second (or right) half antibody comprises the Fab, scFv and Fc regions. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0506] In the embodiment of FIG. 1AG, the first (or left) half antibody comprises a Fab, an Fc region, and a s The first (or right) half antibody comprises a cFv, and the second (or right) half antibody comprises an scFv, an Fc region, and an Fab. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0507] In the embodiment of FIG. 1AH, the first (or left) half antibody comprises an scFv, an Fc region, and The first (or right) half antibody comprises an scFv, an Fc region, and an Fab. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0508] In the embodiments shown in FIGS. 1AA to 1AH, each of X, Y, A, and B is not necessarily ABM1 or ABM2, not necessarily in that order, but BBM may be at least A tetravalent ABM comprises at least one ABM1 and at least one ABM2. In some embodiments, the ABM1 gene comprises one, two, or three ABM1 genes and one, two, or three ABM2 genes. In another embodiment, the tetravalent BBM comprises three ABM1s and one ABM2. In yet another embodiment, the tetravalent BBM comprises two ABM1s and two ABM2s. Thus, a tetravalent BBM contains one ABM1 and three ABM2.

[0509] Thus, in the present disclosure, a tetravalent BB M, where X is ABM1 and each of Y, A and B is ABM2 ( This form of ABM is conveniently designated as "Tv 1").

[0510] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where Y is ABM1, and each of X, A, and B is ABM2 (ABM This form is conveniently designated "Tv 2").

[0511] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where A is ABM1, and each of X, Y, and B is ABM2 (ABM This form is conveniently designated "Tv 3").

[0512] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where B is ABM1, and X, Y, and A are each ABM2 (ABM This form is conveniently designated "Tv 4").

[0513] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where X and Y are both ABM1, and A and B are both ABM2 ( This form of ABM is conveniently designated as "Tv 5").

[0514] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where X and A are both ABM1, and Y and B are both ABM2 ( This form of ABM is conveniently designated as "Tv 6").

[0515] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where X and B are both ABM1, and Y and A are both ABM2 ( This form of ABM is conveniently designated as "Tv 7").

[0516] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where Y and A are both ABM1, and X and B are both ABM2 ( This form of ABM is conveniently designated as "Tv 8").

[0517] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where Y and B are both ABM1, and X and A are both ABM2 ( This form of ABM is conveniently designated as "Tv 9").

[0518] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where A and B are both ABM1, and X and Y are both ABM2 ( This form of ABM is conveniently designated as "Tv 10").

[0519] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where X, Y, and A are ABM1 and B is ABM2 (ABM This form is conveniently designated "Tv 11").

[0520] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where X, Y, and B are ABM1 and A is ABM2 (ABM This form is conveniently designated "Tv 12").

[0521] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where X, A, and B are ABM1 and Y is ABM2 (ABM This form is conveniently designated "Tv 13").

[0522] The present disclosure further provides a tetravalent BBM as shown in any one of Figures 1AA-1AH. where Y, A, and B are each ABM1, and X is ABM2 (ABM This form is conveniently designated "Tv 14").

[0523] 7.6. Trispecific binding molecular morphology Exemplary TBM configurations are shown in Figure 2. Figure 2A shows the TBM configurations shown in Figures 2B-2V. scFv, Fab, non-immunoglobulin-based ABM and Fc are Each may have the properties described for these components in Sections 7.3 and 7.4. The components of the TBM configuration shown in Figure 2 are the means described in Sections 7.3 and 7.4. By any of the following (e.g., direct conjugation, ABM linker, disulfide bond, knob-in These can be associated with each other (e.g., by Fc domains modified with hole interactions). The orientation and association of the various components shown is exemplary only; As will be appreciated, other orientations and associations may be suitable (e.g., see Sections 7.3 and 7.4). as described in Sect.

[0524] The TBM is not limited to the configuration shown in Figure 2. Other configurations that can be used are well known in the art. For example, International Publication No. 2014 / 145806; International Publication No. No. 2017 / 124002 Brochure; Liu et al., 2017, Fron t Immunol.8:38;Brinkmann&Kontermann,2017 , mAbs 9:2,182-212; U.S. Patent Application Publication No. 2016 / 0355600 Specification; Klein et al., 2016, MAbs 8(6):1010-20; and U.S. Patent Application Publication No. 2017 / 0145116.

[0525] 7.6.1. Exemplary Trivalent TBM TBMs can be trivalent, i.e., they have three antigen-binding domains, and One of them binds to CD3, one of them binds to TAA, and one of them binds to CD2. or binds to a second TAA.

[0526] Exemplary trivalent TBM forms are shown in Figures 2B-2P.

[0527] As shown in Figures 2B-2K and 2N-2P, the TBM may contain two half antibodies. One half antibody contains two ABMs and the other contains one ABM. The two half antibodies share an Fc domain. are paired via

[0528] In the embodiment of FIG. 2B, the first (or left) half antibody comprises an scFv and an Fc region. The first and second half antibodies comprise the Fab, scFv, and Fc regions. The half antibodies are associated via their Fc regions to form an Fc domain.

[0529] In the embodiment of FIG. 2C, the first (or left) half antibody comprises two Fab and Fc regions. and the second (or right) half antibody comprises the Fab and Fc regions. The antibodies associate via the Fc region to form an Fc domain.

[0530] In the embodiment of FIG. 2D, the first (or left) half antibody comprises Fab, scFv, and Fc The first and second half antibodies comprise a Fab and Fc region, and the second (or right) half antibody comprises a Fab and Fc region. The half antibodies are associated via their Fc regions to form an Fc domain.

[0531] In the embodiment of FIG. 2E, the first (or left) half antibody comprises an scFv and an Fc region. The first and second halves comprise two Fab and Fc regions. Antibodies associate via their Fc regions to form Fc domains.

[0532] In the embodiment of FIG. 2F, the first (or left) half antibody comprises an scFv, an Fc region, and an F ab, and the second (or right) half antibody comprises the Fab and Fc regions. The half antibodies are associated via their Fc regions to form an Fc domain.

[0533] In the embodiment of Figure 2G, the first (or left) half antibody comprises an scFv and an Fc region. The first and second (or right) half antibodies comprise a Fab, an Fc region, and an scFV. The half antibodies are associated via their Fc regions to form an Fc domain.

[0534] In the embodiment of Figure 2H, the first (or left) half antibody comprises two Fab and Fc regions. and the second (or right) half antibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0535] In the embodiment of Figure 2I, the first (or left) half antibody comprises Fab, scFv, and Fc The second (or right) half antibody comprises a non-immunoglobulin-based ABM and an Fc region. The first and second half antibodies are associated via the Fc region to form an Fc domain. do.

[0536] In the embodiment of Figure 2J, the first (or left) half antibody comprises a Fab and an Fc region. The second (or right) half antibody comprises an scFv, a non-immunoglobulin-based ABM, and an Fc region. The first and second half antibodies are associated via the Fc region to form an Fc domain. do.

[0537] In the embodiment of Figure 2K, the first (or left) half antibody comprises an scFv and an Fc region. The second (or right) half antibody comprises an scFv, an Fc region and a second scFv. and the second half antibody are associated via their Fc regions to form an Fc domain.

[0538] In the embodiment of Figure 2N, the first (or left) half antibody comprises a Fab, an Fc region, and a sc The first and second half antibodies comprise an Fv region, and the second (or right) half antibody comprises an Fab and an Fc region. The half antibodies are associated via their Fc regions to form an Fc domain.

[0539] In the embodiment of Figure 2O, the first (or left) half antibody comprises a Fab, an Fc region, and a sc The first and second half antibodies comprise a Fab region, and the second (or right) half antibody comprises a Fab and an Fc region. The half antibodies are associated via their Fc regions to form an Fc domain.

[0540] In the embodiment of Figure 2P, the first (or left) half antibody is a Fab, a non-immunoglobulin The second (or right) half antibody comprises a base ABM and an Fc region, and the second (or right) half antibody comprises an scFv and an Fc region. The first and second half antibodies are associated via the Fc region to form an Fc domain. do.

[0541] Alternatively, as shown in Figure 2L, a trivalent TBM can contain two half antibodies and one full antibody. It contains a complete ABM and a portion of another ABM (one is VH, the other is VL). As soon as the half antibodies are paired via the Fc domain, the VH and VL associate to form a complete Forms the antigen-binding Fv domain.

[0542] As shown in Figure 2M, the TBM can be single-stranded. It comprises three scFv domains linked via

[0543] In each of the configurations shown in Figures 2B-2P, the domains designated as X, Y, and Z Each of the subunits may be, but not necessarily in that order, ABM1, ABM2, or AB In other words, X can be ABM1, ABM2 or ABM3, and Y can be ABM1. , ABM2 or ABM3, and Z can be ABM1, ABM2 or ABM3, However, the TBM includes one ABM1, one ABM2 and one ABM3.

[0544] Therefore, in the present disclosure, a trivalent TB as shown in any one of Figures 2B-2P M, where X is ABM1, Y is ABM3, and Z is ABM2 ( This form of ABM is conveniently designated "T1").

[0545] The present disclosure also provides a trivalent TBM as shown in any one of Figures 2B-2P, wherein , X is ABM1, Y is ABM2, and Z is ABM3 (this form of ABM is , denoted as "T2" for convenience).

[0546] The present disclosure further provides a trivalent TBM as shown in any one of Figures 2B-2P, Here, X is ABM3, Y is ABM1, and Z is ABM2 (this Form is conveniently designated as "T3").

[0547] The present disclosure also provides a trivalent TBM as shown in any one of Figures 2B-2P. where X is ABM3, Y is ABM2, and Z is ABM1 (ABM This form is conveniently designated "T4").

[0548] The present disclosure also provides a trivalent TBM as shown in any one of Figures 2B-2P. where X is ABM2, Y is ABM1, and Z is ABM3 (ABM This form is conveniently designated "T5").

[0549] The present disclosure also provides a trivalent TBM as shown in any one of Figures 2B-2P. where X is ABM2, Y is ABM3, and Z is ABM1 (ABM This form is conveniently designated "T6").

[0550] 7.6.2. Exemplary Tetravalent TBM TBMs can be tetravalent, i.e., they have four antigen-binding domains, One or two of them bind to CD3, one or two of them bind to TAA, and One or two of them bind to CD2 or a second TAA.

[0551] Exemplary tetravalent TBM forms are shown in Figures 2Q-2S.

[0552] As shown in Figures 2Q-2S, a tetravalent TBM can contain two half antibodies, each containing 2 It contains one complete ABM and two half antibodies paired via their Fc domains.

[0553] In the embodiment of FIG. 2Q, the first (or left) half antibody comprises a Fab, an Fc region, and a second the first (or right) half antibody comprises a Fab, an Fc region, and a second Fab. The first and second half antibodies are associated via their Fc regions to form an Fc domain.

[0554] In the embodiment of Figure 2Q, the first (or left) half antibody comprises a Fab, an Fc region, and a sc The first (or right) half antibody comprises an Fab, an Fc region, and an scFv. and the second half antibody are associated via their Fc regions to form an Fc domain.

[0555] In the embodiment of FIG. 2S, the first (or left) half antibody comprises a Fab, an Fc region, and a sc The first (or right) half antibody comprises an Fv, and the second (or right) half antibody comprises an scFv, an Fc region, and an Fab. and the second half antibody are associated via their Fc regions to form an Fc domain.

[0556] In the embodiments shown in FIGS. 2Q to 2S, each of X, Y, Z, and A does not necessarily mean that Represents, but not necessarily in order, ABM1, ABM2, or ABM3, except that TBM , at least one ABM1, at least one ABM2 and at least one ABM3 Thus, a tetravalent ABM comprises two antibodies against CD3, CD2, or one of the second TAAs. In some cases, the tetravalent TBM has two CD3 ABMs.

[0557] Thus, the present disclosure provides a tetravalent TBM as shown in any one of Figures 2Q-2S. wherein X, Y, Z, and A are CD3, CD2, or a second CD3, CD2, or a second CD2 ... ABM for TAA.

[0558] [Table 106]

[0559] 7.6.3. Exemplary Pentavalent TBM TBMs can be pentavalent, i.e., they have five antigen-binding domains, One, two, or three of them bind to CD3, and one, two, or three of them bind to TAA. bind, one, two or three of which bind to CD2 or a second TAA.

[0560] An exemplary pentavalent TBM form is shown in Figure 2T.

[0561] As shown in Figure 2T, a pentavalent TBM can contain two half antibodies, one containing two full antibodies. The two half antibodies are connected via the Fc domain, one containing an ABM and the other containing one complete ABM. are paired.

[0562] In the embodiment of Figure 2T, the first (or left) half antibody comprises Fab, scFv and Fc The first (or right) half antibody comprises a Fab, an Fc region, and an scFv. and the second half antibody are associated via their Fc regions to form an Fc domain.

[0563] In the embodiment shown in FIG. 2T, X, Y, Z, A, and B are not necessarily in that order. represents, but is not necessarily in order of ABM1, ABM2, or ABM3, provided that TBM It contains at least one ABM1, one ABM2 and one ABM3. A titered TBM consists of two antibodies against CD3, a TAA, and two antibodies against CD2 or a second TAA. ABM or 3 A's for one of CD3, TAA and CD2 or a second TAA In some cases, the pentavalent TBM has two or three CD3 ABMs. In one embodiment, the pentavalent TBM comprises three ABM1s, one ABM2, and one ABM3. Has BM3.

[0564] Thus, the present disclosure provides a pentavalent TBM as shown in FIG. 2T, where X, Y , Z, A and B are CD3, TAA and CD2 or a second TA as shown in Table 10. This is ABM for A.

[0565] [Table 107]

[0566] [Table 108]

[0567] [Table 109]

[0568] 7.6.4. Exemplary Hexavalent TBM TBMs can be hexavalent, i.e., they have six antigen-binding domains, One, two, three or four of which bind to CD3, and one, two, three or four of which One binds to a TAA, and one, two, three, or four of them bind to CD2 or a second TAA. Combine.

[0569] Exemplary hexavalent TBM forms are shown in Figures 2U-2V.

[0570] As shown in Figures 2U-2V, a pentavalent TBM can contain two half antibodies, one of which binds two The two half antibodies, one containing a complete ABM and the other containing one complete ABM, have the Fc domain are paired via

[0571] In the embodiment of Figure 2U, the first (or left) half antibody comprises a Fab, a second Fab, a F The second (or right) half antibody comprises a Fab, a second Fab, an Fc region, and an scFv. The first and second half antibodies are associated via the Fc region to form an Fc domain. Form the main.

[0572] In the embodiment of Figure 2V, the first (or left) half antibody comprises a first Fv, a second Fv, The third half antibody comprises a first Fv and a second Fc region, and the second (or right) half antibody comprises a first Fv, a second Fv, and a third Fc region. The first and second half antibodies are associated via the Fc region. The Fc domain is formed by

[0573] In the embodiments shown in FIGS. 2U to 2V, each of X, Y, Z, A, B, and C must be represents ABM1, ABM2, or ABM3, but not necessarily in that order, provided that: A TBM comprises at least one ABM1, one ABM2 and one ABM3. Thus, a hexavalent TBM can be used to identify: (i) a CD3 TAA and a CD2 or second TAA, respectively; (ii) two ABMs for CD3, TAA and one for CD2 or a second TAA or (iii) CD3, TAA and CD2 or a second TA For example, a hexavalent ABM may contain four ABMs against one of the CD3 ABM for the TAA, two ABMs for the TAA, and one ABM for CD2 or a second TAA As another example, a hexavalent ABM may include three ABMs for CD3, one ABM for TAAs, and one ABM for TAAs. In some cases, the antibody may contain one ABM directed against a TAA and two ABMs directed against CD2 or a second TAA. In some embodiments, the hexavalent TBM has two, three, or four CD3 ABMs. In another embodiment, the hexavalent TBM has three CD3 ABMs. The BM has four CD3 ABMs.

[0574] Therefore, in the present disclosure, a hexavalent TB as shown in any one of Figures 2U to 2V M, where X, Y, Z, A, B, and C are selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD36, CD26, CD27, CD28, CD37, ABM to a TAA and CD2 or a second TAA.

[0575] [Table 110]

[0576] Table 111

[0577] Table 112

[0578] Table 113

[0579] Table 114

[0580] Table 115

[0581] Table 116

[0582] Table 117

[0583] Table 118

[0584] Table 119

[0585] Table 120

[0586] Table 121

[0587] [Table 122]

[0588] 7.7. Exemplary Multispecific Binding Molecules Exemplary MBM forms are shown in Figures 3A-3E. scFv, Fab, non-immunoglobulin The base ABM and Fc are described in Sections 7.2-7.4, respectively, for these components. The components of the MBM configuration may have the properties described in Sections 7.3 and 7.4. by any of the means known in the art (e.g., direct conjugation, ABM linker, disulfide bond, etc.) , by Fc domains modified with knob-in-hole interactions) The orientation and association of the various components shown in Figures 3A-3E are illustrative only. as will be appreciated by those skilled in the art, other orientations and associations may be suitable (e.g., , as described in Sections 7.3 and 7.4).

[0589] The MBM is not limited to the configuration shown in Figures 3A-3E. Other configurations that can be used include: Such methods are known to those skilled in the art, for example, WO 2014 / 145806; International Publication No. 2017 / 124002; Liu et al., 2017, Front Immunol.8:38;Brinkmann&Kontermann, 2017, mAbs 9:2, 182-212; U.S. Patent Application Publication No. 2016 / 0355 Specification No. 600; Klein et al., 2016, MAbs 8(6):1010 -20; and U.S. Patent Application Publication No. 2017 / 0145116.

[0590] 7.7.1. Exemplary MBM MBMs can be bispecific, e.g., they have two antigen-binding domains, One antigen-binding domain binds to CD3 and one antigen-binding domain binds to a TAA do.

[0591] 7.7.2. Exemplary Trivalent MBMs The MBMs can be trivalent, e.g., they have three antigen binding domains, where 3 At least one of the three antigen-binding domains binds to CD3, and 0 of the three antigen-binding domains ~One binds to CD2, and at least one of the three antigen-binding domains binds to a TAA .

[0592] 7.7.3. Exemplary Tetravalent MBM The MBMs can be tetravalent, e.g., they have four antigen binding domains, where 4 At least one of the four antigen-binding domains binds to CD3, and 0 of the four antigen-binding domains ~Two bind to CD2, and at least one of the four antigen-binding domains binds to a TAA .

[0593] 7.8.TCR ABM The MBM contains an ABM that specifically binds to components of the TCR complex. The highly variable alpha (α) is normally expressed as part of a complex with the non-mutated CD3 chain molecule. A disulfide-linked membrane-anchored heterodimeric protein consisting of a β- and a β-chain. T cells that express this receptor are called α:β (or αβ) T cells. However, a small number of T cells express a different receptor, with variable gamma (γ) and delta (δ) chains. These cells are called γδ T cells.

[0594] In a preferred embodiment, the MBM is CD3, e.g., a CD4 antibody found in Table 1 or Table 19. It contains an ABM that specifically binds to the D3 antigen-binding domain.

[0595] 7.8.1.CD3 ABM The MBM may contain an ABM that specifically binds to CD3. The term "CD3" refers to The three differentiated coreceptors (or coreceptor complexes or polynucleotides of the coreceptor complex) of the T cell receptor The CD3 protein may also include variants. The CD3 protein may also include post-translational modifications of the CD3 amino acid sequence. Post-translational modifications include, but are not limited to, N-linked and O-linked glycosylation. can be done.

[0596] In one embodiment, the MBM is an anti-CD3 antibody or an antigen-binding domain thereof, AB Exemplary anti-CD3 VH, VL, and scFVs that can be used in MBM. The sequences are shown in Tables 1 and 19.

[0597] In one embodiment, the CD3 ABM comprises the CDR sequences of NOV292. In some embodiments, the CD3 ABM comprises the CDR sequences of NOV123. In one embodiment, the CD3 ABM comprises the CDR sequences of NOV453. The CD3 ABM comprises the CDR sequences of NOV229. The ABM comprises the CDR sequences of NOV110. In one embodiment, the CD3 ABM In one embodiment, the CD3 ABM comprises the CDR sequences of NOV832. In one embodiment, the CD3 ABM comprises the CDR sequences of NOV580. In one embodiment, the CD3 ABM comprises the CDR sequences of NOV567. In one embodiment, the CD3 ABM comprises the CDR sequences of NOV221. .

[0598] MBM can be prepared by cloning the complete heavy and light chain variable region of either the CD3 sequence found in Table 1 or Table 19. In one embodiment, the MBM may comprise the VH and VL sequences of NOV292. In one embodiment, the MBM comprises a CD3 ABM comprising the VH of NOV123 and In one embodiment, the MBM comprises a CD3 ABM comprising a VL sequence. In one embodiment, the MBM comprises a CD3 ABM comprising the VH and VL sequences of N In one embodiment, the antibody comprises a CD3 ABM comprising the VH and VL sequences of OV229. The BM comprises a CD3 ABM comprising the VH and VL sequences of NOV110. In this case, the MBM comprises a CD3 ABM comprising the VH and VL sequences of NOV832. In an embodiment, the MBM comprises a CD3 ABM comprising the VH and VL sequences of NOV589. In one embodiment, the MBM comprises a CD3 antibody comprising the VH and VL sequences of NOV580. In one embodiment, the MBM comprises the VH and VL sequences of NOV567. In one embodiment, the MBM comprises a CD3 ABM comprising the VH of NOV221 and It contains the CD3 ABM, which includes the VL sequence.

[0599] TCR-α / β ABM MBM specifically binds to the TCR-α chain, the TCR-β chain, or the TCR-αβ dimer. BM. Exemplary anti-TCR-α / β antibodies are known in the art ( See, for example, U.S. Patent Application Publication No. 2012 / 0034221; Borst et al. l.,1990,Hum Immunol.29(3):175-88(Antibody BMA03 1) which describes the VH, VL and Kabat The CDR sequences are shown in Table 12.

[0600] [Table 123]

[0601] In one embodiment, the TCR ABM may comprise the CDR sequences of antibody BMA031. In another embodiment, the TCR ABM comprises the VH and VL sequences of antibody BMA031. It can be seen.

[0602] TCR-γ / δ ABM MBM specifically binds to the TCR-γ chain, TCR-δ chain, or TCR-γδ dimer. BM. Exemplary anti-TCR-γ / δ antibodies are known in the art ( See, for example, U.S. Pat. No. 5,980,892 (ATC under Accession No. HB 9578). See, for example, the references to the δTCS1 gene, which describes the δTCS1 gene produced by the hybridoma deposited by I want to be illuminated).

[0603] 7.9.CD2 ABM Immunoglobulin-based CD2 ABM In one embodiment, the MBM is an anti-CD2 antibody or an antigen-binding domain thereof, AB Exemplary anti-CD2 antibodies are known in the art (see, e.g., U.S. Pat. No. 6,629,299). 6,849,258, China Patent Application Publication No. 102827281A, U.S. Patent Application Publication No. 2003 / 0139579 A1 and U.S. Pat. No. 5,795,5 (See US Pat. No. 72,422,422.) Table 13 lists anti-CD2 antibodies or Exemplary CDR, VH and VL sequences that may be included in the antigen-binding fragment are provided.

[0604] [Table 124]

[0605] [Table 125]

[0606] In one embodiment, the CD2 ABM comprises the CDR sequences of CD2-1 (SEQ ID NO: 87- 92). In one embodiment, the CD2 ABM comprises the heavy and light chain fragments of CD2-1. In one embodiment, the CD2 ABM comprises a variant sequence (SEQ ID NOs: 93-94). In one embodiment, the heavy and light chain variable sequences of ICD2-1 (SEQ ID NOs: 95-96) are included. The CD2 ABM is composed of the heavy and light chain variable sequences of hu2CD2-1 (SEQ ID NOs: 97-99). 8).

[0607] In another embodiment, the CD2 ABM is distributed by Chinese Culture Collection Committee General Mic Hive deposited at the Microbiology Center under accession number CGMCC 6132 The antibody 9D1 produced by the antibody 9D1 gene may include the CDR sequences of the antibody 9D1 produced by the antibody 9D1 gene, which is disclosed in the Chinese Patent Application Publication No. In another embodiment, CD2 ABM was founded by the American Type Culture Company on June 22, 1999. The hybridoma is produced by the hybridoma deposited in the collection under accession number PTA-802. The antibody may comprise the CDR sequences of the antibody LO-CD2b, which is described in U.S. Patent Application Publication No. 2003 / 0149994. In yet another embodiment, CD2 ABM was deposited with the ATCC on April 9, 1993 under accession number 69277 and is U.S. Pat. cloning in recombinant E. coli as described in US Pat. No. 5,795,572 The CD2 SFv-Ig CDR sequences are produced by expression of a cloned construct. obtain.

[0608] In other embodiments, the CD2 ABM may comprise the VH and VL sequences of antibody 9D1. In another embodiment, the CD2 ABM comprises the VH and VL sequences of antibody LO-CD2b. In yet another embodiment, the CD2 ABM may comprise ATCC Accession No. 6927 Produced by expression of a cloned construct in recombinant E. coli harboring The VH and VL sequences of the CD2 SFv-Ig may be included.

[0609] 7.9.2.CD58-based CD2 ABM In certain embodiments, the present disclosure provides an MBM comprising a CD2 ABM that is a ligand. CD2 ABM is a human antigen-binding protein whose natural ligand is CD58, also known as LFA-3. The CD58 / LFA-3 protein binds specifically to CD2 on the surface of various cell types. It is a glycoprotein expressed in the IL-1 receptor (Dustin et al., 1991, Annu. Re v.Immunol.9:27), in both antigen-dependent and antigen-independent manners, APCs and plays a role in mediating T cell interactions (Wallner et al., 1987, J. Exp. Med. 166:923). Thus, in certain embodiments, CD2 A BM is a CD58 portion. As used herein, a CD58 portion refers to CD5 At least 70% sequence identity to the CD2 binding portion of CD58, e.g., CD2 At least 70%, 71%, 72%, 73%, 74%, 75%, 76% of the bonded portion ,77%,78%,79%,80%,81%,82%,83%,84%,85%,86% ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96% , 97%, 98% or 99% identity to the amino acid sequence of human CD58. has the Uniprot identifier P19256. CD5 containing 123 (i.e., the sequence shown as CD58-4 in Table 14 below) It has been established that the 8 fragment is sufficient for binding to CD2. Wang et al. al., 1999, Cell 97:791-803. Thus, in certain embodiments The CD58 portion has at least 70% sequence identity to amino acids 30 to 123 of CD58. identity, e.g., at least 70%, 71% to the amino acid sequence designated CD58-4 ,72%,73%,74%,75%,76%,77%,78%,79%,80%,81% ,82%,83%,84%,85%,86%,87%,88%,89%,90%,91% , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity It contains an amino acid sequence containing

[0610] The interaction between CD58 and CD2 has been mapped by X-ray crystallography and molecular modeling. Residues E25, K29, K30, K32, D33, K34, E37, and D84 and substitution of K87 (numbering refers to the mature polypeptide) reduced binding to CD2. Ikemizu et al., 1999, Proc. Natl. Acad. Sc i.USA 96:4289-94. Thus, in a preferred embodiment, CD58 Parts are E25, K29, K30, K32, D33, K34, E37, D84 and K87 The wild-type residue is retained.

[0611] In contrast, the following substitutions (numbering referring to the full-length polypeptide) No effect on the case: F29S; V37K; V49Q; V86K; T113S; and L121G. Thus, the CD58 moiety can contain one, two, three, four, or five of the aforementioned substitutions. Or it could include all six.

[0612] Exemplary CD58 moieties are shown in Table 14 below.

[0613] [Table 126]

[0614] CD48-based CD2 ABM In certain embodiments, the present disclosure provides an MBM comprising a CD2 ABM that is a CD48 portion. As used herein, a CD48 portion refers to the CD2-binding portion of CD48. at least 70% sequence identity to the CD2-binding portion of CD48, e.g., At most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 7 9%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or The amino acid sequence of human CD48 contains 99% identity to the signal peptide (amino acids 1-26) and GPI anchor (amino acids 221-243), Unip rot identifier P09326(www.uniprot.org / uniprot / P09 326). In certain embodiments, the CD48 portion has the Uniprot identifier P09 At least 70% of the amino acid sequence consisting of amino acids 27 to 220 of 326 Identity (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76% ,77%,78%,79%,80%,81%,82%,83%,84%,85%,86% ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96% Human CD48 contains an amino acid sequence containing I g-like C2 type I domain (amino acids 29-127 of Uniprot identifier P09326) and Ig-like C2 type 2 domain (amino acids 132 to 134 of Uniprot identifier P09326) 212). Thus, in one embodiment, the CD48 portion has a Unipro C2-I for the amino acid sequence consisting of amino acids 29 to 212 of t identifier P09326 for the type domain (amino acids 29-127 of Uniprot identifier P09326) and / or Ig-like C2 type 2 domain (amino acid 132 of Uniprot identifier P09326) ~212) to at least 70% sequence identity (e.g., at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) In one embodiment, the CD48 portion comprises an amino acid sequence comprising the Uniprot identifier The sequence may include one or more naturally occurring variants relative to the sequence of the P09326 gene. For example, the CD48 domain The CD48 portion can include an E102Q substitution. or a CD2-binding portion thereof, e.g., having the Uniprot identifier P09326-2 It may comprise an amino acid sequence corresponding to an isoform or a CD2-binding portion thereof.

[0615] 7.10. Tumor-associated antigen ABM The MBM comprises at least one ABM that specifically binds to a tumor-associated antigen (TAA). For example, the BBM can include ABM2, which specifically binds to a TAA, and the TBM can include ABM2, which specifically binds to a TAAA. ABM2 ("TAA1") that specifically binds to A and A that specifically binds to a different TAA Preferably, the TAA (or TAA1 and TAA2) In this case, each TAA is a human TAA. The antigen may also be present in normal cells. In certain embodiments, the TAA may inhibit the proliferation of tumor cells relative to normal cells. In another embodiment, the TAA is preferentially expressed or upregulated in the cell line Mycobacterium tuberculosis. is a car.

[0616] Any type of tumor and any type of TAA can be targeted by MBM Exemplary types of cancer that can be targeted include acute lymphoblastic leukemia, Leukemia, acute myeloid leukemia, bile duct cancer, B-cell leukemia, B-cell lymphoma, bile duct cancer, bone cancer, brain Cancer, breast cancer, triple-negative breast cancer, cervical cancer, Burkitt's lymphoma, chronic lymphocytic leukemia disease, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, Glioma, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, medullary thyroid cancer, Melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, lung duct cancer, These include kidney cancer, sarcoma, skin cancer, testicular cancer, urothelial carcinoma and other bladder cancers. Thus, one of skill in the art will understand that TAAs are known for virtually every type of cancer. Deaf.

[0617] Exemplary types of B-cell malignancies that can be targeted include Hodgkin's lymphoma, non-Hodgkin's lymphoma, and Examples of NHL include diffuse lymphoma (NHL) and multiple myeloma. Large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL) ) / Small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (Waldenström's macroglobulinemia) phosphatase), hairy cell leukemia, primary central nervous system (CNS) lymphoma, primary mediastinal large cell lymphoma Type B-cell lymphoma, mediastinal gray zone lymphoma (MGZL), splenic marginal zone B-cell lymphoma, MA LT-type extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, and primary effusion lymphoma Examples include tumors.

[0618] Examples of MBMs that can be generated (e.g., targeted by ABM2 and / or ABM3) Typical TAA are ABCF1;ACVR1;ACVR1B;ACVR2;ACVR 2B;ACVRL1;ADORA2A;ADRB3;aggrecan;AGR2;AICDA ;AIF1;AIG1;AKAP1;AKAP2;ALK;AMH;AMHR2;ANG PT1;ANGPT2;ANGPTL3;ANGPTL4;ANPEP;APC;APO C1; AR; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; B AFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (M DR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; B MP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (preptin ); BRCA1; C19orf10 (IL27w); C3; C4A; C5; C5R1; ka dherin 17; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / J AB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MC P-4); CCL15 (MIP-1d); CCL16 (HCC-4); CCL17 (TA RC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1 ); MCAF; CCL20 (MIP-3a); CCL21 (MIP-2); SLC; ex odus-2; CCL22 (MDC / STC-1); CCL23 (MPIF-1); CC L24 (MPIF-2 / eotaxin-2); CCL25 (TECT); CCL26 (e otaxin-3); CCL27 (CTACK / ILC); CCL28; CCL3 (MIP -1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP- 3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; C CNE2; CCR1 (CKR1 / HM145); CCR2 (mcp-1RB / RA); C CR3 (CKR3 / CMKBR3); CCR4; CCR5 (CMKBR5 / ChemR1 3); CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 ( CKR7 / EBI1); CCR8 (CMKBR8 / TER1 / CKR-L1); CCR9 (GPR-9-6);CCRL1(VSHK1);CCRL2(L-CCR);CD16 4;CD19;CD1C;CD20;CD200;CD-22;CD24;CD28;C D3;CD37;CD38;CD3E;CD3G;CD3Z;CD4;CD32b;CD 40;CD40L;CD44;CD45RB;CD52;CD69;CD72;CD74 ;CD79A;CD79B;CD8;CD80;CD81;CD83;CD86;CD9 7;CD179a;CDH1(E-カドヘリン);CDH10;CDH12;CDH13 ;CDH18;CDH19;CDH20;CDH5;CDH7;CDH8;CDH9;C DK2;CDK3;CDK4;CDK5;CDK6;CDK7;CDK9;CDKN1A (p21Wap1 / Cip1);CDKN1B(p27Kip1);CDKN1C;CD KN2A(p16INK4a);CDKN2B;CDKN2C;CDKN3;CEBPB ;CER1;CHGA;CHGB;Kitchenase;CHST10;CKLFSF2;CKL FSF3;CKLFSF4;CKLFSF5;CKLFSF6;CKLFSF7;CKL FSF8;CLDN3;CLDN6;CLDN7(クローディン-7);CLN3;CL U(クラステリン);CMKLR1;CMKOR1(RDC1);CNR1;COL18 A1;COL1A1;COL4A3;COL6A1;CR2;CRP;CSF1(MC SF);CSF2(GM-CSF);CSF3(GCSF);CTLA4;CTNNB1 (b-カテニン);CTSB(カテプシンB);CX3CL1(SCYD1);CX3C R1(V28);CXCL1(GRO1);CXCL10(IP-10);CXCL11 (1-TAC / IP-9);CXCL12(SDF1);CXCL13;CXCL14; CXCL16;CXCL2(GRO2);CXCL3(GRO3);CXCL5(ENA -78 / LIX);CXCL6(GCP-2);CXCL9(MIG);CXCR3(G PR9 / CKR-L2);CXCR4;CXCR6(TYMSTR / STRL33 / Bo nzo);CYB5;CYC1;CYSLTR1;CGRP;C1q;C1r;C1;C 4a;C4b;C2a;C2b;C3a;C3b;DAB2IP;DES;DKFZp4 51J0118;DNCL1;DPP4;E-selectin;E2F1;ECGF1;ED G1;EFNA1;EFNA3;EFNB2;EGF;EGFR;EGFRvIII;E LAC2;ENG;ENO1;ENO2;ENO3;EPHB4;EPO;ERBB2( Her-2);EREG;ERK8;ESR1;ESR2;F3(TF);Factor VII ;Factor IX;Factor V;Factor VIIa;Factor X;Factor XII;Factor XIII; FADD;FasL;FASN;FCER1A;FCER2;Fc gamma receptor;FCG R3A;FCRL5;FGF;FGF1(aFGF);FGF10;FGF11;FGF 12;FGF12B;FGF13;FGF14;FGF16;FGF17;FGF18; FGF19;FGF2(bFGF);FGF20;FGF21;FGF22;FGF23 ;FGF3(int-2);FGF4(HST);FGF5;FGF6(HST-2); FGF7(KGF);FGF8;FGF9;FGFR3;FIGF(VEGFD);FI L1(EPSILON);FIL1(ZETA);FLJ12584;FLJ25530 ;FLRT1 (fibronectin);FLT1;Folate receptor alpha;Folate receptor beta ;FOS;FOSL1(FRA-1);Face GM1;FY(DARC);GABRP (MARKET);GAGEB1;GAGEC1;GALNAC4S-6ST;GATA3 ;GDF5;GFI1;GGT1;GM-CSF;GloboH;GNAS1;GNRH 1;GPNMB;GPR2(CCR10);GPR20;GPR31;GPR44;GP R64;GPR81(FKSG80);GPRC5D;GRCC10(C10);GRP ;GSN(glass);GSTP1;gP(gP)IIb / IIIa;HAV CR1;HAVCR2;HDAC4;HDAC5;HDAC7A;HDAC9;Her2 ;HER3;HGF;HIF1A;HIP1;Hamster and Hematopoietic Progenitors;HLA -A;HLA-DRA;HM74;HMGB1;HMOX1;HMWMAA;HUMCY T2A;ICEBERG;ICOSL;ID2;IFN-a;IFNA1;IFNA2; IFNA4;IFNA5;IFNA6;IFNA7;IFNB1;IFN-γ;IFNW 1;IGBP1;IGF1;IGF1R;IGF2;IGFBP2;IGFBP3;IG FBP6;IL-1;IL-α;IL-1-β;IL10;IL10RA;IL10RB ;IL11;IL11RA;IL-12;IL12A;IL12B;IL12RB1;I L12RB2;IL13;IL13RA1;IL13RA2;IL14;IL15;IL IL15RA;IL16;IL17;IL17B;IL17C;IL17R;IL18;IL 18BP;IL18R1;IL18RAP;IL19;IL1A;IL1B;IL1F1 0;IL1F5;IL1F6;IL1F7;IL1F8;IL1F9;IL1HY1;I L1R1;IL1R2;IL1RAP;IL1RAPL1;IL1RAPL2;IL1R L1;IL1RL2;IL1RN;IL2;IL20;IL20RA;IL21R;IL 22;IL22R;IL22RA2;IL23;IL24;IL25;IL26;IL2 7;IL28A;IL28B;IL29;IL2RA;IL2RB;IL2RG;IL3 ;IL30;IL3RA;IL4;IL4R;IL5;IL5RA;IL6;IL6R; IL6ST(protease130);IL7;IL7R;IL8;IL8RA;IL8R B;IL8RB;IL9;IL9R;ILK;INHA;INHBA;INSL3;IN SL4;IRAQ1;IRAQ2;ITGA1;ITGA2;ITGA3;ITGA6( a6case);ITGAV;ITGB3;ITGB4(b4case);JA G1;JAK1;JAK3;JUN;K6HF;KAI1;KDR;KITLG;KLF 5(GC Box BP);KLF6;KLK10;KLK12;KLK13;KLK1 4;KLK15;KLK3;KLK4;KLK5;KLK6;KLK9;KRT1;KR T19(ツ19);KRT2A;KRTHB6(KR2 Type Type      Type);L -Lingo;LAMAS;LEP(Lingo);Lingo-p75;Lingo-T roy;LRP6;LPS;LTA(TNF-b);LTB;LTB4R(GPR16) ;LTB4R2;LTBR;LY6K;LYPD8;MACMARCKS; mgp;MAP2K7(c-Jun);MDK;MAP2K7(c-Jun);MIB1;MAP2K7; MIF;MIP-2;MKI67(Ki-67);MMP2;MMP9;MS4A1;M SMB;MT3(mucosa-III);MTSS1;MUC1(muco);M YC;MYD88;NCK2;Neurocan;NKG2D;NFKB1;NFKB2;N GF;NGFB(NGF);NGFR;NgR-Lingo;NgR-Nogo66(N ogo);NgR-p75;NgR-Troy;NME1(NM23A);NOX5;N PPB;NR0B1;NR0B2;NR1D1;NR1D2;NR1H2;NR1H3; NR1H4;NRII2;NRII3;NR2C1;NR2C2;NR2E1;NR2E 3;NR2F1;NR2F2;NR2F6;NR3C1;NR3C2;NR4A1;NR 4A2;NR4A3;NR5A1;NR5A2;NR6A1;NRP1;NRP2;NT 5E;NTN4;NY-BR-1;o-acetyl-GD2;ODZ1;OPRD1;OR 51E2;P2RX7;PANX3;PAP;PART1;PATE;PAWR;PCA 3;PCNA;PDGFA;PDGFB;PECAM1;PF4(CXCL4);PGE 2;PGF;PGR;Phosphacan;PIAS2;PIK3CG;PLAC1;Plasmi gen activator; PLAU(uPA); PLG; PLXDC1; polysialic acid; PP BP(CXCL7);PPID;PR1;PRKCQ;PRKD1;PRL;PROC; Protein C; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 ( COX-2);PTN;RAC2(p21Rac2);RAGE;RARB;RGS1; RGS13;RGS3;RNF110(ZNF144);ROBO2;SIO0A2;S CGB1D2 (lipophilin B); SCGB2A1 (mammaglobin 2); SCGB2A 2 (mammaglobin 1); SCYE1 (endothelial monocyte-activating cytokine); SDF2; SE RPIN1;SERPIN3;SERPINB5(snail);SERPIN1( PAI-1);SERPINF1;SHBG;SLA2;SLC2A2;SLC33A1 ;SLC34A2;SLC39A6;SLC43A1;SLIT2;SLITRK6;S PP1;SPRR1B(Spr1);ST6GAL1;STAB1;STAT6;STE AP;STEAP2;THE P;TACSTD2;TB4R2;TBX21;TC P10;TDGF1;TEK;TEM1 / CD248;TEM7R;TGFA;TGFB 1;TGFB111;TGFB2;TGFB3;TGFBI;TGFBR1;TGFBR 2;TGFBR3;TH1L;THBS1(THB-1);THBS2;T HBS4;THPO;TIE(Tie-1);TIMP3;protease;TLR10;TL R2;TLR3;TLR4;TLR5;TLR6;TLR7;TLR8;TLR9;TN F;TNF-α;TNFAIP2(B94);TNFAIP3;TNFRSF11A;T NFRSF1A;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF 6 (Fas);TNFRSF7;TNFRSF8;TNFRSF9;TNFSF10(TR AIL);TNFSF11(TRANC);TNFSF12(APO3L);TNFS F13(April);TNFSF13B;TNFSF14(WHEM-L);TNFS F15(VEGI);TNFSF18;TNFSF4(OX40β);TNFSF 5(CD40 factor);TNFSF6(FasL);TNFSF7(CD27 factor). );TNFSF8(CD30 factor);TNFSF9(4-1BB factor);TOL LIP; Toll-like receptor; TOP2A (topoisomerase 2A); TP53; TPM1 ;TPM2;TRADD;TRAF1;TRAF2;TRAF3;TRAF4;TRAF 5;TRAF6;TREM1;TREM2;TRPC6;TSHR;TSLP;TWEA K; thrombomodulin; thrombin; UPK2; VEGF; VEGFB; VEGFC; Versican; VHLC5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM -1b); XCR1 (GPRS / CCXCR1); YY1; and ZFPM2. .

[0619] In one embodiment, the TAA targeted by the MBM is ADRB3. In some embodiments, the TAA targeted by the MBM is AKAP-4. In an embodiment, the TAA targeted by MBM is ALK. In one embodiment, the TAA targeted by MBM is the androgen receptor. In one embodiment, the TAA targeted by the MBM is B7H3. In one embodiment, the TAA targeted by MBM is BCMA. In one embodiment, the TAA targeted by MBM is BORIS. The TAA targeted by the BM is BST2. The TAA targeted by this is cadherin 17. In one embodiment, The TAA targeted thereby is CAIX. In one embodiment, by MBM The TAA targeted is CD171. In one embodiment, the TAA targeted by MBM is CD171. In one embodiment, the TAA targeted by MBM is CD179a. In one embodiment, the TAA targeted by MBM is CD19. The TAA is CD20. In one embodiment, the TAA targeted by MBM In one embodiment, the TAA targeted by the MBM is C In one embodiment, the TAA targeted by the MBM is CD30 In one embodiment, the TAA targeted by the MBM is CD300LF. In one embodiment, the TAA targeted by the MBM is CD32b. In one embodiment, the TAA targeted by MBM is CD33. In some embodiments, the TAA targeted by the MBM is CD38. In some embodiments, the TAA targeted by the MBM is CD44v6. In one embodiment, the TAA targeted by the MBM is CD72. In one embodiment, the TAA targeted by MBM is CD79a. In one embodiment, the TAA targeted by MBM is CD79b. The TAA targeted by the BM is CD97. In one embodiment, the TAA targeted by MBM is CEA. In one embodiment, the TAA targeted by MBM is CLDN6. In one embodiment, the TAA targeted by MBM is CLEC12A. In one embodiment, the TAA targeted by MBM is CLL-1. In one embodiment, the TAA targeted by MBM is CS-1. A is CXORF61. In one embodiment, the TA targeted by MBM A is cyclin B1. In one embodiment, the TA targeted by MBM A is CYP1B1. In one embodiment, the TAA targeted by MBM In one embodiment, the TAA targeted by the MBM is E In one embodiment, the TAA targeted by the MBM is: In one embodiment, the TAA targeted by the MBM is EPC In one embodiment, the TAA targeted by the MBM is EphA2 In one embodiment, the TAA targeted by the MBM is EphB2. In one embodiment, the TAA targeted by the MBM is ERBB2. In one embodiment, the TAA targeted by the MBM is ERG (TMPRSS2 In one embodiment, the TA targeted by the MBM is a fusion gene. A is ETV6-AML. In one embodiment, T targeted by MBM The AA is a FAP. In one embodiment, the TAA targeted by the MBM is: In one embodiment, the TAA targeted by the MBM is FCR. In one embodiment, the TAA targeted by MBM is FLT3. In one embodiment, the TAA targeted by MBM is FLT3. In one embodiment, the TAA targeted by the MBM is the folate receptor alpha. In one embodiment, the TAA targeted by the MBM is the folate receptor beta. In one embodiment, the TAA targeted by the MBM is Fos-related antigen 1. In one embodiment, the TAA targeted by the MBM is Fucosyl-GM1 In one embodiment, the TAA targeted by MBM is GD2. In one embodiment, the TAA targeted by the MBM is GD2. In one embodiment, the TAA targeted by the MBM is GD3. In one embodiment, the TAA targeted by the MBM is GloboH. In one embodiment, the TAA targeted by MBM is GM3. The TAA targeted by MB is gp100Tn. In one embodiment, In one embodiment, the TAA targeted by MBM is GPC3. In one embodiment, the TAA targeted by MBM is GPNMB. The TAA targeted is GPR20. In one embodiment, the TAA targeted by the MBM In one embodiment, the TAA targeted by MBM is GPRC5D. In one embodiment, the TAA targeted by MBM is GPR64. In one embodiment, the TAA targeted by MBM is HAVCR1. The TAA is HER3. In one embodiment, the TAA targeted by MBM is a HMWMAA. In one embodiment, the TAA targeted by the MBM is In one embodiment, the TAA targeted by the MBM is I In one embodiment, the TAA targeted by MBM is: In one embodiment, the TAA targeted by MBM is IL-1. In one embodiment, the TAA targeted by the MBM is IL-11Ra. In one embodiment, the TAA targeted by the MBM is K In one embodiment, the TAA targeted by the MBM is LAIR1. In one embodiment, the TAA targeted by MBM is LCK. In one embodiment, the TAA targeted by the MBM is LewisY. In some embodiments, the TAA targeted by the MBM is LILRA2. In an embodiment, the TAA targeted by the MBM is LMP2. In one embodiment, the TAA targeted by the MBM is LRP6. In one embodiment, the TAA targeted by MBM is LY6K. The TAA targeted by MBM is LY75. In one embodiment, MBM In one embodiment, the TAA targeted by MBM is LYPD8. In one embodiment, the TAA targeted by the MBM is MAD-CT-1. The TAA targeted thereby is MAD-CT-2. In one embodiment, MBM In one embodiment, the TAA targeted by MBM is mesothelin. In one embodiment, the TAA targeted by MBM is ML-IAP. In one embodiment, the TAA targeted by MBM is MUC1. In one embodiment, the TAA targeted by MBM is MYCN. In one embodiment, the TAA targeted by MBM is NA17. A is NCAM. In one embodiment, the TAA targeted by MBM is In one embodiment, the TAA targeted by MBM is NY In one embodiment, the TAA targeted by the MBM is o-BR-1. In one embodiment, the TAA targeted by the MBM is acetyl-GD2. In one embodiment, the TAA targeted by MBM is: In one embodiment, the TAA targeted by MBM is: In one embodiment, the TAA targeted by the MBM is P In one embodiment, the TAA targeted by MBM is PAX 3. In one embodiment, the TAA targeted by MBM is PAX5. In one embodiment, the TAA targeted by the MBM is PDGFR-beta. In one embodiment, the TAA targeted by the MBM is PLAC1. In one embodiment, the TAA targeted by the MBM is polysialic acid. In one embodiment, the TAA targeted by the MBM is PRSS21. In one embodiment, the TAA targeted by the MBM is PSCA. In embodiments, the TAA targeted by the MBM is RhoC. In one embodiment, the TAA targeted by MBM is ROR1. The TAA targeted by MBM is the sarcoma translocation breakpoint protein In one embodiment, the TAA targeted by MBM is SART3. In some embodiments, the TAA targeted by the MBM is SLC34A2. In some embodiments, the TAA targeted by the MBM is SLC39A6. In some embodiments, the TAA targeted by the MBM is sLe. In some embodiments, the TAA targeted by MBM is SLITRK6. In one embodiment, the TAA targeted by MBM is sperm protein 17. In an embodiment, the TAA targeted by the MBM is SSEA-4. In some embodiments, the TAA targeted by the MBM is SSX2. In one embodiment, the TAA targeted by MBM is TAAG72. In one embodiment, the TAA targeted by MBM is TAARP. In one embodiment, the TAA targeted by MBM is TACSTD2. In one embodiment, the TAA targeted by MBM is TEM1 / CD248. In one embodiment, the TAA targeted by the MBM is TEM7R. In one embodiment, the TAA targeted by MBM is TGS5. The TAA targeted by the BM is Tie 2. In one embodiment, the MBM In one embodiment, the TAA targeted by the MBM is TnAg. In one embodiment, the TAA targeted by MBM is TSHR. In one embodiment, the TAA targeted by the MBM is tyrosinase. In one embodiment, the TAA targeted by MBM is UPK2. In one embodiment, the TAA targeted by MBM is VEGFR2. The TAA is WT1. In one embodiment, the TAA targeted by the MBM is , XAGE1.

[0620] In one embodiment, the TAA targeted by MBM is BCMA, CD19, CD20, CD22, CD123, CD33, CLL1, CD138 (syndecan-1, (also known as SDC1), CS1, CD38, CD133, FLT3, CD52 , TNFRSF13C (BAFFR: also known as B cell-activating factor receptor, T NF receptor superfamily member 13C), TNFRSF13B (TACI: transmembrane receptor TNF receptor superfamily, also known as TNF receptor activator and CAML interactor Myri member 13B), CXCR4 (CXC motif chemokine receptor 4), PD -L1 (programmed cell death ligand 1), LY9 (also known as CD229), lymphocyte antigen 9), CD200, FCGR2B (also known as CD32b, IgG receptor IIb Fc fragment), CD21, CD23, CD24, CD40L, CD 72, CD79a and CD79b.

[0621] In one embodiment, the TAA targeted by the MBM is CD19. In an embodiment, the TAA targeted by the MBM is BCMA. In one embodiment, the TAA targeted by the MBM is CD20. In one embodiment, the TAA targeted by MBM is CD22. The TAA targeted by MBM is CD123. In one embodiment, the MB In one embodiment, the TAA targeted by MBM is CD33. In one embodiment, the TAA targeted by MBM is CLL1. In one embodiment, the TAA targeted by MBM is CD138. In one embodiment, the TAA targeted by MBM is CS1. The AA is CD38. In one embodiment, the TAA targeted by the MBM is , CD133. In one embodiment, the TAA targeted by MBM is F In one embodiment, the TAA targeted by MBM is CD52 In one embodiment, the TAA targeted by the MBM is TNFRSF1 3C. In one embodiment, the TAA targeted by the MBM is TNFRS. In one embodiment, the TAA targeted by the MBM is C-X-C In one embodiment, the TAA targeted by the MBM is PD-L1 R4. In one embodiment, the TAA targeted by the MBM is LY9. In one embodiment, the TAA targeted by the MBM is CD200. In an embodiment, the TAA targeted by the MBM is CD21. In one embodiment, the TAA targeted by the MBM is CD23. In one embodiment, the TAA targeted by MBM is CD24. The TAA targeted by MBM is CD40L. In one embodiment, the TAA targeted by MBM is CD72. In one embodiment, the TAA targeted by MBM is CD79a. The TAA targeted is CD79b.

[0622] In one embodiment, the MBM comprises two TAA selected from the TAAs described in this section. Targets AA (TAA1 and TAA2).

[0623] In one embodiment, TAA1 is CD19 and TAA2 is CD20 ( In one embodiment, TAA1 is CD19 and TAA2 is CD19. is CD22 (or vice versa). 19 and TAA2 is CD123 (or vice versa). In this case, TAA1 is CD19 and TAA2 is BCMA (or vice versa). In one embodiment, TAA1 is CD19 and TAA2 is CD33. (or vice versa). In one embodiment, TAA1 is CD19 and TAA 2 is CLL1 (or vice versa). D19 and TAA2 is CD138 (or vice versa). wherein TAA1 is CD19 and TAA2 is CS1 (or vice versa). In one embodiment, TAA1 is CD19 and TAA2 is CD38. (or vice versa). In one embodiment, TAA1 is CD19 and TAA 2 is CD133 (or vice versa). In one embodiment, the TAA2 is CD19 and the TAA2 is FLT3 (or vice versa). In this case, TAA1 is CD19 and TAA2 is CD52 (or vice versa). In one embodiment, TAA1 is CD19 and TAA2 is TNFRSF In one embodiment, TAA1 is CD19. and TAA2 is TNFRSF13B (or vice versa). In this case, TAA1 is CD19 and TAA2 is CXCR4 (or vice versa). In one embodiment, TAA1 is CD19 and TAA2 is PD-L1 In one embodiment, TAA1 is CD19 (or vice versa). TAA2 is LY9 (or vice versa). In one embodiment, TAA1 is , CD19 and TAA2 is CD200 (or vice versa). In some embodiments, TAA1 is CD19 and TAA2 is FCGR2B (or vice versa). In one embodiment, TAA1 is CD19 and TAA2 is CD 21 (or vice versa). In one embodiment, TAA1 is CD19. and TAA2 is CD23 (or vice versa). A1 is CD19 and TAA2 is CD24 (or vice versa). In an embodiment, TAA1 is CD19 and TAA2 is CD40L (or vice versa). In one embodiment, TAA1 is CD19 and TAA2 is C In one embodiment, TAA1 is CD19. and TAA2 is CD79a (or vice versa). TAA1 is CD19 and TAA2 is CD79b (or vice versa). In one embodiment, TAA1 is CD20 and TAA2 is CD22 (or In one embodiment, TAA1 is CD20 and TAA2 is , CD123 (or vice versa). 20 and TAA2 is BCMA (or vice versa). where TAA1 is CD20 and TAA2 is CD33 (or vice versa). In one embodiment, TAA1 is CD20 and TAA2 is CLL1 ( In one embodiment, TAA1 is CD20 and TAA2 is CD20. is CD138 (or vice versa). D20 and TAA2 is CS1 (or vice versa). where TAA1 is CD20 and TAA2 is CD38 (or vice versa). In one embodiment, TAA1 is CD20 and TAA2 is CD133. (or vice versa). In one embodiment, TAA1 is CD20 and TAA 2 is FLT3 (or vice versa). D20 and TAA2 is CD52 (or vice versa). In this case, TAA1 is CD20 and TAA2 is TNFRSF13C (or vice versa). In one embodiment, TAA1 is CD20 and TAA2 is TN In one embodiment, TAA1 is C D20 and TAA2 is CXCR4 (or vice versa). In this case, TAA1 is CD20 and TAA2 is PD-L1 (or vice versa). In one embodiment, TAA1 is CD20 and TAA2 is LY9. In one embodiment, TAA1 is CD20 and TA A2 is CD200 (or vice versa). In one embodiment, TAA1 is , CD20 and TAA2 is FCGR2B (or vice versa). In some embodiments, TAA1 is CD20 and TAA2 is CD21 (or vice versa). In one embodiment, TAA1 is CD20 and TAA2 is CD2 3 (or vice versa). In one embodiment, TAA1 is CD20 , TAA2 is CD24 (or vice versa). TAA1 is CD20 and TAA2 is CD40L (or vice versa). In an embodiment, TAA1 is CD20 and TAA2 is CD72 (or vice versa). In one embodiment, TAA1 is CD20 and TAA2 is CD 79a (or vice versa). In one embodiment, TAA1 is CD20 and TAA2 is CD79b (or vice versa). TAA1 is CD22 and TAA2 is CD123 (or vice versa). In one embodiment, TAA1 is CD22 and TAA2 is BCMA (or In one embodiment, TAA1 is CD22 and TAA2 is , CD33 (or vice versa). In one embodiment, TAA1 is CD2 2 and TAA2 is CLL1 (or vice versa). , TAA1 is CD22 and TAA2 is CD138 (or vice versa) In one embodiment, TAA1 is CD22 and TAA2 is CS1 (or In one embodiment, TAA1 is CD22 and TAA2 is , CD38 (or vice versa). In one embodiment, TAA1 is CD2 2 and TAA2 is CD133 (or vice versa). where TAA1 is CD22 and TAA2 is FLT3 (or vice versa). In one embodiment, TAA1 is CD22 and TAA2 is CD52 ( In one embodiment, TAA1 is CD22 and TAA2 is CD23. is TNFRSF13C (or vice versa). 1 is CD22 and TAA2 is TNFRSF13B (or vice versa) In one embodiment, TAA1 is CD22 and TAA2 is CXCR4. (or vice versa). In one embodiment, TAA1 is CD22 and TAA 2 is PD-L1 (or vice versa). CD22 and TAA2 is LY9 (or vice versa). In this case, TAA1 is CD22 and TAA2 is CD200 (or vice versa). In one embodiment, TAA1 is CD22 and TAA2 is FCGR2B In one embodiment, TAA1 is CD22 (or vice versa). TAA2 is CD21 (or vice versa). is CD22 and TAA2 is CD23 (or vice versa). In some embodiments, TAA1 is CD22 and TAA2 is CD24 (or vice versa). In one embodiment, TAA1 is CD22 and TAA2 is CD40 In one embodiment, TAA1 is CD22. , TAA2 is CD72 (or vice versa). TAA1 is CD22 and TAA2 is CD79a (or vice versa). In an embodiment, TAA1 is CD22 and TAA2 is CD79b (or vice versa). In one embodiment, TAA1 is CD123 and TAA2 is In one embodiment, TAA1 is CD12 3 and TAA2 is CD33 (or vice versa). , TAA1 is CD123 and TAA2 is CLL1 (or vice versa) In one embodiment, TAA1 is CD123 and TAA2 is CD138. In one embodiment, TAA1 is CD123 and T AA2 is CS1 (or vice versa). In one embodiment, TAA1 is CD123 and TAA2 is CD38 (or vice versa). In this example, TAA1 is CD123 and TAA2 is CD133 (or vice versa). In one embodiment, TAA1 is CD123 and TAA2 is FL In one embodiment, TAA1 is CD123. and TAA2 is CD52 (or vice versa). AA1 is CD123 and TAA2 is TNFRSF13C (or vice versa). In one embodiment, TAA1 is CD123 and TAA2 is TNFR SF13B (or vice versa). In one embodiment, TAA1 is CD1 23 and TAA2 is CXCR4 (or vice versa). In this case, TAA1 is CD123 and TAA2 is PD-L1 (or vice versa). In one embodiment, TAA1 is CD123 and TAA2 is LY9. In one embodiment, TAA1 is CD123 (or vice versa). TAA2 is CD200 (or vice versa). 1 is CD123 and TAA2 is FCGR2B (or vice versa). In some embodiments, TAA1 is CD123 and TAA2 is CD21 (or In one embodiment, TAA1 is CD123 and TAA2 is CD123. is CD23 (or vice versa). 123 and TAA2 is CD24 (or vice versa). In this case, TAA1 is CD123 and TAA2 is CD40L (or vice versa). In one embodiment, TAA1 is CD123 and TAA2 is CD72 In one embodiment, TAA1 is CD123 (or vice versa). , TAA2 is CD79a (or vice versa). A1 is CD123 and TAA2 is CD79b (or vice versa). In some embodiments, TAA1 is BCMA and TAA2 is CD33 (or In one embodiment, TAA1 is BCMA and TAA2 is In one embodiment, TAA1 is BCMA. in and TAA2 is CD138 (or vice versa). TAA1 is BCMA and TAA2 is CS1 (or vice versa). In an embodiment, TAA1 is BCMA and TAA2 is CD38 (or vice versa). In one embodiment, TAA1 is BCMA and TAA2 is C In one embodiment, TAA1 is BCMA D133 (or vice versa). and TAA2 is FLT3 (or vice versa). TAA1 is BCMA and TAA2 is CD52 (or vice versa). In some embodiments, TAA1 is BCMA and TAA2 is TNFRSF13C. In one embodiment, TAA1 is BCMA and T AA2 is TNFRSF13B (or vice versa). TAA1 is BCMA and TAA2 is CXCR4 (or vice versa). In one embodiment, TAA1 is BCMA and TAA2 is PD-L1 ( In one embodiment, TAA1 is BCMA and TAA2 is is LY9 (or vice versa). In one embodiment, TAA1 is BCM A and TAA2 is CD200 (or vice versa). where TAA1 is BCMA and TAA2 is FCGR2B (or vice versa). In one embodiment, TAA1 is BCMA and TAA2 is CD21. In one embodiment, TAA1 is BCMA and TA A2 is CD23 (or vice versa). In one embodiment, the TAA2 is BCMA and the TAA2 is CD24 (or vice versa). In this case, TAA1 is BCMA and TAA2 is CD40L (or vice versa). In one embodiment, TAA1 is BCMA and TAA2 is CD72. In one embodiment, TAA1 is BCMA and T AA2 is CD79a (or vice versa). is BCMA and TAA2 is CD79b (or vice versa). In some embodiments, TAA1 is CD33 and TAA2 is CLL1 (or vice versa). In one embodiment, TAA1 is CD33 and TAA2 is CD1 In one embodiment, TAA1 is CD33. and TAA2 is CS1 (or vice versa). TAA1 is CD33 and TAA2 is CD38 (or vice versa). In some embodiments, TAA1 is CD33 and TAA2 is CD133 (or vice versa). In one embodiment, TAA1 is CD33 and TAA2 is FL In one embodiment, TAA1 is CD33. and TAA2 is CD52 (or vice versa). A1 is CD33 and TAA2 is TNFRSF13C (or vice versa) In one embodiment, TAA1 is CD33 and TAA2 is TNFRSF1 In one embodiment, TAA1 is CD33. and TAA2 is CXCR4 (or vice versa). AA1 is CD33 and TAA2 is PD-L1 (or vice versa). In some embodiments, TAA1 is CD33 and TAA2 is LY9 (or vice versa). In one embodiment, TAA1 is CD33 and TAA2 is C In one embodiment, TAA1 is CD33. and TAA2 is FCGR2B (or vice versa). where TAA1 is CD33 and TAA2 is CD21 (or vice versa). In one embodiment, TAA1 is CD33 and TAA2 is CD23 ( In one embodiment, TAA1 is CD33 and TAA2 is CD33. is CD24 (or vice versa). 33 and TAA2 is CD40L (or vice versa). In this case, TAA1 is CD33 and TAA2 is CD72 (or vice versa). In one embodiment, TAA1 is CD33 and TAA2 is CD79a. In one embodiment, TAA1 is CD33 and TA A2 is CD79b (or vice versa). In one embodiment, TAA1 is , CLL1 and TAA2 is CD138 (or vice versa). In some embodiments, TAA1 is CLL1 and TAA2 is CS1 (or vice versa). In one embodiment, TAA1 is CLL1 and TAA2 is CD38. In one embodiment, TAA1 is CLL1 and T AA2 is CD133 (or vice versa). is CLL1 and TAA2 is FLT3 (or vice versa). In some embodiments, TAA1 is CLL1 and TAA2 is CD52 (or vice versa). In one embodiment, TAA1 is CLL1 and TAA2 is TNFR In one embodiment, TAA1 is SF13C (or vice versa). 1 and TAA2 is TNFRSF13B (or vice versa). In this embodiment, TAA1 is CLL1 and TAA2 is CXCR4 (or vice versa). In one embodiment, TAA1 is CLL1 and TAA2 is PD-L1. In one embodiment, TAA1 is CLL1 (or vice versa). and TAA2 is LY9 (or vice versa). TAA1 is CLL1 and TAA2 is CD200 (or vice versa). In an embodiment, TAA1 is CLL1 and TAA2 is FCGR2B (or In one embodiment, TAA1 is CLL1 and TAA2 is In one embodiment, TAA1 is CLL1 and TAA2 is CD23 (or vice versa). TAA1 is CLL1 and TAA2 is CD24 (or vice versa). In some embodiments, TAA1 is CLL1 and TAA2 is CD40L (or In one embodiment, TAA1 is CLL1 and TAA2 is , CD72 (or vice versa). In one embodiment, TAA1 is a CLL 1 and TAA2 is CD79a (or vice versa). where TAA1 is CLL1 and TAA2 is CD79b (or vice versa). In one embodiment, TAA1 is CD138 and TAA2 is CS1. (or vice versa). In one embodiment, TAA1 is CD138 and TA A2 is CD38 (or vice versa). CD138 and TAA2 is CD133 (or vice versa). In this embodiment, TAA1 is CD138 and TAA2 is FLT3 (or vice versa). In one embodiment, TAA1 is CD138 and TAA2 is CD 52 (or vice versa). In one embodiment, TAA1 is CD138. and TAA2 is TNFRSF13C (or vice versa). In this case, TAA1 is CD138 and TAA2 is TNFRSF13B (or In one embodiment, TAA1 is CD138 and TAA2 is , CXCR4 (or vice versa). 138 and TAA2 is PD-L1 (or vice versa). In this case, TAA1 is CD138 and TAA2 is LY9 (or vice versa). In one embodiment, TAA1 is CD138 and TAA2 is CD200. In one embodiment, TAA1 is CD138 (or vice versa). , TAA2 is FCGR2B (or vice versa). AA1 is CD138 and TAA2 is CD21 (or vice versa). In some embodiments, TAA1 is CD138 and TAA2 is CD23 (or In one embodiment, TAA1 is CD138 and TAA2 is CD138. is CD24 (or vice versa). 138 and TAA2 is CD40L (or vice versa). In this case, TAA1 is CD138 and TAA2 is CD72 (or vice versa). In one embodiment, TAA1 is CD138 and TAA2 is CD79 In one embodiment, TAA1 is CD138. and TAA2 is CD79b (or vice versa). AA1 is CS1 and TAA2 is CD38 (or vice versa). In an embodiment, TAA1 is CS1 and TAA2 is CD133 (or vice versa). In one embodiment, TAA1 is CS1 and TAA2 is FLT. 3 (or vice versa). In one embodiment, TAA1 is CS1, TAA2 is CD52 (or vice versa). is CS1 and TAA2 is TNFRSF13C (or vice versa). In some embodiments, TAA1 is CS1 and TAA2 is TNFRSF13B. In one embodiment, TAA1 is CS1 and TAA 2 is CXCR4 (or vice versa). In one embodiment, CS1 is TAA2 and PD-L1 is TAA2 (or vice versa). wherein TAA1 is CS1 and TAA2 is LY9 (or vice versa). In one embodiment, TAA1 is CS1 and TAA2 is CD200 ( In one embodiment, TAA1 is CS1 and TAA2 is , FCGR2B (or vice versa). In one embodiment, TAA2 is S1 and TAA2 is CD21 (or vice versa). Thus, TAA1 is CS1 and TAA2 is CD23 (or vice versa). In one embodiment, TAA1 is CS1 and TAA2 is CD24 (or In one embodiment, TAA1 is CS1 and TAA2 is C In one embodiment, TAA1 is CS1 and D40L (or vice versa). and TAA2 is CD72 (or vice versa). AA1 is CS1 and TAA2 is CD79a (or vice versa). In an embodiment, TAA1 is CS1 and TAA2 is CD79b (or vice versa). In one embodiment, TAA1 is CD38 and TAA2 is C In one embodiment, TAA1 is CD38. and TAA2 is FLT3 (or vice versa). TAA1 is CD38 and TAA2 is CD52 (or vice versa). R In an embodiment, TAA1 is CD38 and TAA2 is TNFRSF13C. In one embodiment, TAA1 is CD38 and TA A2 is TNFRSF13B (or vice versa). AA1 is CD38 and TAA2 is CXCR4 (or vice versa). In some embodiments, TAA1 is CD38 and TAA2 is PD-L1 (or In one embodiment, TAA1 is CD38 and TAA2 is , LY9 (or vice versa). In one embodiment, TAA1 is CD38 and TAA2 is CD200 (or vice versa). , TAA1 is CD38 and TAA2 is FCGR2B (or vice versa) In one embodiment, TAA1 is CD38 and TAA2 is CD21. (or vice versa). In one embodiment, TAA1 is CD38 and TAA 2 is CD23 (or vice versa). In one embodiment, TAA2 is CD38 and TAA2 is CD24 (or vice versa). In this case, TAA1 is CD38 and TAA2 is CD40L (or vice versa). In one embodiment, TAA1 is CD38 and TAA2 is CD72. In one embodiment, TAA1 is CD38 and TA A2 is CD79a (or vice versa). In one embodiment, TAA1 is , CD38 and TAA2 is CD79b (or vice versa). In this embodiment, TAA1 is CD133 and TAA2 is FLT3 (or vice versa). In one embodiment, TAA1 is CD133 and TAA2 is CD 52 (or vice versa). In one embodiment, TAA1 is CD133. and TAA2 is TNFRSF13C (or vice versa). In this case, TAA1 is CD133 and TAA2 is TNFRSF13B (or In one embodiment, TAA1 is CD133 and TAA2 is , CXCR4 (or vice versa). 133 and TAA2 is PD-L1 (or vice versa). In this case, TAA1 is CD133 and TAA2 is LY9 (or vice versa). In one embodiment, TAA1 is CD133 and TAA2 is CD200. In one embodiment, TAA1 is CD133 (or vice versa). , TAA2 is FCGR2B (or vice versa). AA1 is CD133 and TAA2 is CD21 (or vice versa). In some embodiments, TAA1 is CD133 and TAA2 is CD23 (or In one embodiment, TAA1 is CD133 and TAA2 is CD133. is CD24 (or vice versa). 133 and TAA2 is CD40L (or vice versa). In this case, TAA1 is CD133 and TAA2 is CD72 (or vice versa). In one embodiment, TAA1 is CD133 and TAA2 is CD79 a (or vice versa). In one embodiment, TAA1 is CD133. and TAA2 is CD79b (or vice versa). AA1 is FLT3 and TAA2 is CD52 (or vice versa). In an embodiment, TAA1 is FLT3 and TAA2 is TNFRSF13C. In one embodiment, TAA1 is FLT3 and TA A2 is TNFRSF13B (or vice versa). AA1 is FLT3 and TAA2 is CXCR4 (or vice versa). In some embodiments, TAA1 is FLT3 and TAA2 is PD-L1 (or In one embodiment, TAA1 is FLT3 and TAA2 is , LY9 (or vice versa). In one embodiment, TAA1 is FLT3 and TAA2 is CD200 (or vice versa). , TAA1 is FLT3 and TAA2 is FCGR2B (or vice versa) In one embodiment, TAA1 is FLT3 and TAA2 is CD21. (or vice versa). In one embodiment, TAA1 is FLT3 and TAA 2 is CD23 (or vice versa). LT3 and TAA2 is CD24 (or vice versa). In this case, TAA1 is FLT3 and TAA2 is CD40L (or vice versa). In one embodiment, TAA1 is FLT3 and TAA2 is CD72. In one embodiment, TAA1 is FLT3 and TA A2 is CD79a (or vice versa). In one embodiment, TAA1 is , FLT3 and TAA2 is CD79b (or vice versa). In this state, TAA1 is CD52 and TAA2 is TNFRSF13C (also In one embodiment, TAA1 is CD52 and TAA2 is , TNFRSF13B (or vice versa). In some embodiments, TAA2 is CD52 and TAA2 is CXCR4 (or vice versa). In some embodiments, TAA1 is CD52 and TAA2 is PD-L1 (or vice versa). In one embodiment, TAA1 is CD52 and TAA2 is LY. 9 (or vice versa). In one embodiment, TAA1 is CD52 , TAA2 is CD200 (or vice versa). A1 is CD52 and TAA2 is FCGR2B (or vice versa). In some embodiments, TAA1 is CD52 and TAA2 is CD21 (or In one embodiment, TAA1 is CD52 and TAA2 is In one embodiment, TAA1 is CD52. and TAA2 is CD24 (or vice versa). TAA1 is CD52 and TAA2 is CD40L (or vice versa). In one embodiment, TAA1 is CD52 and TAA2 is CD72 (or In one embodiment, TAA1 is CD52 and TAA2 is , CD79a (or vice versa). 52 and TAA2 is CD79b (or vice versa). In this case, TAA1 is TNFRSF13C and TAA2 is TNFRSF13B. (or vice versa). In one embodiment, TAA1 is TNFRSF13C. and TAA2 is CXCR4 (or vice versa). AA1 is TNFRSF13C and TAA2 is PD-L1 (or vice versa). In one embodiment, TAA1 is TNFRSF13C and TAA2 is In one embodiment, TAA1 is TNFRS. F13C and TAA2 is CD200 (or vice versa). In this case, TAA1 is TNFRSF13C and TAA2 is FCGR2B ( In one embodiment, TAA1 is TNFRSF13C. , TAA2 is CD21 (or vice versa). 1 is TNFRSF13C and TAA2 is CD23 (or vice versa) In one embodiment, TAA1 is TNFRSF13C and TAA2 is CD2 4 (or vice versa). In one embodiment, TAA1 is TNFRSF1 3C and TAA2 is CD40L (or vice versa). In this case, TAA1 is TNFRSF13C and TAA2 is CD72 (or vice versa). In one embodiment, the TAA1 is TNFRSF13C, and the TAA 2 is CD79a (or vice versa). TNFRSF13C and TAA2 is CD79b (or vice versa). In some embodiments, TAA1 is TNFRSF13B and TAA2 is CXCR4 In one embodiment, TAA1 is TNFRSF13 B and TAA2 is PD-L1 (or vice versa). TAA1 is TNFRSF13B and TAA2 is LY9 (or vice versa) In one embodiment, TAA1 is TNFRSF13B and TAA2 is , CD200 (or vice versa). FRSF13B and TAA2 is FCGR2B (or vice versa). In an embodiment, TAA1 is TNFRSF13B and TAA2 is CD21. In one embodiment, TAA1 is TNFRSF13B. and TAA2 is CD23 (or vice versa). AA1 is TNFRSF13B and TAA2 is CD24 (or vice versa). In one embodiment, TAA1 is TNFRSF13B and TAA2 is C In one embodiment, TAA1 is TNFR D40L (or vice versa). SF13B and TAA2 is CD72 (or vice versa). In this study, TAA1 is TNFRSF13B and TAA2 is CD79a (also In one embodiment, TAA1 is TNFRSF13B, and vice versa. TAA2 is CD79b (or vice versa). 1 is CXCR4 and TAA2 is PD-L1 (or vice versa). In an embodiment, TAA1 is CXCR4 and TAA2 is LY9 (or vice versa). In one embodiment, TAA1 is CXCR4 and TAA2 is In one embodiment, TAA1 is a C-X-C R4 and TAA2 is FCGR2B (or vice versa). In this case, TAA1 is CXCR4 and TAA2 is CD21 (or vice versa). In one embodiment, TAA1 is CXCR4 and TAA2 is CD23 In one embodiment, TAA1 is CXCR4 (or vice versa). , TAA2 is CD24 (or vice versa). TAA1 is CXCR4 and TAA2 is CD40L (or vice versa). In an embodiment, TAA1 is CXCR4 and TAA2 is CD72 (or In one embodiment, TAA1 is CXCR4 and TAA2 is , CD79a (or vice versa). In one embodiment, TAA2 is CR4 and TAA2 is CD79b (or vice versa). In this case, TAA1 is PD-L1 and TAA2 is LY9 (or vice versa). In one embodiment, TAA1 is PD-L1 and TAA2 is CD200. In one embodiment, TAA1 is PD-L1 (or vice versa). , TAA2 is FCGR2B (or vice versa). A1 is PD-L1 and TAA2 is CD21 (or vice versa). In an embodiment, TAA1 is PD-L1 and TAA2 is CD23 (or In one embodiment, TAA1 is PD-L1 and TAA2 is , CD24 (or vice versa). In one embodiment, TAA2 is CD40L and L1 is CD40L (or vice versa). In this case, TAA1 is PD-L1 and TAA2 is CD72 (or vice versa). In one embodiment, TAA1 is PD-L1 and TAA2 is CD79a In one embodiment, TAA1 is PD-L1 (or vice versa). , TAA2 is CD79b (or vice versa). A1 is LY9 and TAA2 is CD200 (or vice versa). In an embodiment, TAA1 is LY9 and TAA2 is FCGR2B (or vice versa). In one embodiment, TAA1 is LY9 and TAA2 is CD 21 (or vice versa). In one embodiment, TAA1 is LY9 , TAA2 is CD23 (or vice versa). 1 is LY9 and TAA2 is CD24 (or vice versa). In some embodiments, TAA1 is LY9 and TAA2 is CD40L (or vice versa). In one embodiment, TAA1 is LY9 and TAA2 is CD72. In one embodiment, TAA1 is LY9 and TA A2 is CD79a (or vice versa). In one embodiment, TAA1 is , LY9 and TAA2 is CD79b (or vice versa). In this example, TAA1 is CD200 and TAA2 is FCGR2B (or vice versa). In one embodiment, TAA1 is CD200 and TAA2 is C In one embodiment, TAA1 is CD200. and TAA2 is CD23 (or vice versa). TAA1 is CD200 and TAA2 is CD24 (or vice versa). In one embodiment, TAA1 is CD200 and TAA2 is CD40L (or vice versa). In one embodiment, TAA1 is CD200 and TA A2 is CD72 (or vice versa). CD200 and TAA2 is CD79a (or vice versa). In some embodiments, TAA1 is CD200 and TAA2 is CD79b (or vice versa). In one embodiment, TAA1 is CD21 and TAA2 is CD 23 (or vice versa). In one embodiment, TAA1 is CD21. and TAA2 is CD24 (or vice versa). A1 is CD21 and TAA2 is CD40L (or vice versa). In an embodiment, TAA1 is CD21 and TAA2 is CD72 (or vice versa). In one embodiment, TAA1 is CD21 and TAA2 is C In one embodiment, TAA1 is CD21 and TAA2 is CD79b (or vice versa). , TAA1 is CD23 and TAA2 is CD24 (or vice versa). In one embodiment, TAA1 is CD23 and TAA2 is CD40L ( In one embodiment, TAA1 is CD23 and TAA2 is CD24. is CD72 (or vice versa). 23 and TAA2 is CD79a (or vice versa). In this case, TAA1 is CD23 and TAA2 is CD79b (or vice versa). In one embodiment, TAA1 is CD24 and TAA2 is CD40L. In one embodiment, TAA1 is CD24 and T AA2 is CD72 (or vice versa). In one embodiment, TAA1 is , CD24 and TAA2 is CD79a (or vice versa). In this embodiment, TAA1 is CD24 and TAA2 is CD79b (or vice versa). In one embodiment, TAA1 is CD40L and TAA2 is CD 72 (or vice versa). In one embodiment, TAA1 is CD40L. and TAA2 is CD79a (or vice versa). TAA1 is CD40L and TAA2 is CD79b (or vice versa) In one embodiment, TAA1 is CD72 and TAA2 is CD79a. (or vice versa). In one embodiment, TAA1 is CD72 and TAA 2 is CD79b (or vice versa). CD79a and TAA2 is CD79b (or vice versa).

[0624] The TAA-binding ABM can include, for example, an anti-TAA antibody or an antigen-binding fragment thereof. Anti-TAA antibodies or antigen-binding fragments can be, for example, those listed in Table 15A or Table 15B. In one embodiment, the anti-TAA antibody or its antigen-binding The domains have the heavy and light chain variable region sequences of the antibodies set forth in Table 15A. In some embodiments, the anti-TAA antibody or antigen-binding domain thereof is an antibody of any of the antibodies listed in Table 15B. It has heavy and light chain variable region sequences.

[0625] [Table 127]

[0626] [Table 128]

[0627] [Table 129]

[0628] [Table 130]

[0629] [Table 131]

[0630] [Table 132]

[0631] [Table 133]

[0632] In a specific embodiment, TAA1 and TAA2 are CD19, CD20 and BCMA In another embodiment, TAA1 and TAA2 are selected from BCMA and CD1 9. Exemplary BCMA and CD19 binding sequences are selected from Sections 7.10.1 and 7.10.2 below. and 7.10.2.

[0633] 7.10.1.BCMA In certain aspects, the present disclosure provides MBMs in which ABM1 or ABM2 is BCMA. (Such an ABM may be conveniently referred to as a "BCMA ABM"). , a tumor necrosis family receptor (TNFR) member expressed in cells of the B-cell lineage BCMA expression is expressed in plasma cells, plasmablasts, and subpopulations of activated and memory B cells. It is highest in terminally differentiated B cells, including the B population, which assume a long-lived plasma cell fate. CMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. CMA expression has recently been associated with a number of cancers, autoimmune diseases, and infectious diseases. Cancers with increased expression of MA include some hematological cancers, such as multiple myeloma, rhesus mammary gland cancer, and rhesus mammary gland cancer. These include Hodgkin's and non-Hodgkin's lymphoma, various leukemias, and glioblastoma.

[0634] MBMs containing ABMs that bind BCMA can be, for example, anti-BCMA antibodies or their antigen-binding The anti-BCMA antibody or antigen-binding domain thereof may comprise, for example, any of the antibodies listed in Tables 16A-16C. The fragment may comprise the CDR, VH, VL or scFV sequences described in 16G.

[0635] [Table 134]

[0636] [Table 135]

[0637] [Table 136]

[0638] [Table 137]

[0639] [Table 138]

[0640] [Table 139]

[0641] [Table 140]

[0642] [Table 141]

[0643] [Table 142]

[0644] Table 143

[0645] Table 144

[0646] Table 145

[0647] Table 146

[0648] Table 147

[0649] Table 148

[0650] Table 149

[0651] Table 150

[0652] Table 151

[0653] Table 152

[0654] Table 153

[0655] [Table 154]

[0656] [Table 155]

[0657] [Table 156]

[0658] In one embodiment, the BCMA ABM is any one of BCMA-1 to BCMA-40. In one embodiment, the ABM comprises one or more CDR sequences of BCMA-1. In one embodiment, the ABM comprises the CDR sequences of BCMA-2. In embodiments, the ABM comprises the CDR sequences of BCMA-3. The ABM comprises the CDR sequences of BCMA-4. In one embodiment, the ABM comprises the CDR sequences of BCMA-6. In one embodiment, the ABM comprises the CDR sequences of BCMA-7. In embodiments, the ABM comprises the CDR sequences of BCMA-8. The ABM comprises the CDR sequences of BCMA-9. In one embodiment, the ABM comprises the CDR sequences of BCMA-10. In one embodiment, the ABM comprises the CDR sequences of BCMA-12. In some embodiments, the ABM comprises the CDR sequences of BCMA-13. In one embodiment, the ABM comprises the CDR sequences of BCMA-14. comprises the CDR sequences of BCMA-15. In one embodiment, the ABM comprises 16 CDR sequences of BCMA-17. In one embodiment, the ABM comprises the CDR sequences of BCMA-18. In an embodiment, the ABM comprises the CDR sequences of BCMA-19. In one embodiment, the ABM comprises the CDR sequences of BCMA-20. In one embodiment, the ABM comprises the CDR sequences of BCMA-21. In one embodiment, the ABM comprises the CDR sequences of BCMA-23. In one embodiment, the ABM comprises the CDR sequences of BCMA-24. In one embodiment, the ABM comprises the CDR sequences of BCMA-25. The ABM comprises the CDR sequences of BCMA-26. In one embodiment, the ABM comprises the CDR sequences of BCMA-27. In one embodiment, the ABM comprises the CDR sequences of BCMA-29. In some embodiments, the ABM comprises the CDR sequences of BCMA-30. In one embodiment, the ABM comprises the CDR sequences of BCMA-31. M comprises the CDR sequences of BCMA-32. In one embodiment, ABM is In one embodiment, the ABM comprises the CDR sequences of BCMA-34. In one embodiment, the ABM comprises the CDR sequences of BCMA-35. In some embodiments, the ABM comprises the CDR sequences of BCMA-36. wherein the ABM comprises the CDR sequences of BCMA-37. In one embodiment, the ABM comprises the CDR sequences of BCMA-3, BCMA-38, and BCMA-39. In one embodiment, the ABM comprises the CDR sequences of BCMA-40. Includes:

[0659] In one embodiment, the CDRs are selected from the group consisting of Kaba, as set forth in Tables 16B and 16E. In other embodiments, the CDRs are defined by the t numbering in Tables 16C and 16F. Further embodiments are defined by Chothia numbering as described in In the above, the CDRs are selected from the Kabat and Chot It is defined by the combination of hia numbering.

[0660] In one embodiment, the MBM (e.g., MBM) comprising a BCMA ABM is selected from the group consisting of: A. A method for producing a BCMA-1 antibody comprising administering to a subject a BCMA-1 antibody or a BCMA-2 ...2 antibody or a BCMA-3 antibody comprising administering to a subject a BCMA-1 antibody or a BCMA-4 antibody comprising administering to a subject a BCMA-2 antibody or a BCMA-4 antibody comprising administering to a subject a BC obtain.

[0661] In one embodiment, the ABM comprises the heavy and light chain variable sequences of BCMA-1. In embodiments, the ABM comprises the heavy a...

Claims

1. CDR-H that specifically binds to human CD3 and is described in Table 1A, Table 1B or Table 1C 1 sequence, CDR-H2 sequence, CDR-H3 sequence, CDR-L1 sequence, CDR-L2 sequence, and and a CDR-L3 sequence.

2. The CDR-H1 sequence, CDR-H2 sequence, CDR-H3 sequence, and CDR The CD3 binding molecule according to claim 1, comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence. Combined molecules.

3. The CDR-H1 sequence, CDR-H2 sequence, CDR-H3 sequence, and CDR The CD3 binding molecule according to claim 1, comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence. Combined molecules.

4. The CDR-H1 sequence, CDR-H2 sequence, CDR-H3 sequence, and CDR The CD3 binding molecule according to claim 1, comprising a CDR-L1 sequence, a CDR-L2 sequence and a CDR-L3 sequence. Combined molecules.

5. Table 1D-1, Table 1E-1, Table 1F-1, Table 1G-1, Table 1H-1 or Table 1I- 1 and the CDR-H1, CDR-H2 and CDR-H3 sequences described in Table 1D-2, Table 1E-2, Table 1F-2, Table 1G-2, Table 1H-2 or Table 1I-2 2. The CD3 binding molecule of claim 1, comprising CDR-L1, CDR-L2 and CDR-L3 sequences. child.

6. The CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1D-1 and Table 1 D-2, comprising the corresponding CDR-L1, CDR-L2 and CDR-L3 sequences set forth therein; The CD3 binding molecule of claim 5.

7. The CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1E-1 and Table 1 E-2, comprising the corresponding CDR-L1, CDR-L2 and CDR-L3 sequences set forth therein; The CD3 binding molecule of claim 5.

8. The CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1F-1 and Table 1 F-2, comprising the corresponding CDR-L1, CDR-L2 and CDR-L3 sequences set forth therein; The CD3 binding molecule of claim 5.

9. The CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1G-1 and Table 1 G-2, comprising the corresponding CDR-L1, CDR-L2 and CDR-L3 sequences set forth therein; The CD3 binding molecule of claim 5.

10. The CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1H-1 and Table 1 H-2, comprising the corresponding CDR-L1, CDR-L2 and CDR-L3 sequences set forth therein; The CD3 binding molecule of claim 5.

11. The CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1I-1 and Table 1 I-2, comprising the corresponding CDR-L1, CDR-L2 and CDR-L3 sequences described therein; The CD3 binding molecule of claim 5.

12. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 133, 136, 139, and 142. CDR-H1; (ii) a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 134, 137, 140, and 143; CDR-H2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 135, 138, 141, and 144 Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 149, 152, 155, and 158. CDR-L1; (ii) a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 150, 153, 156, and 159; CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 151, 154, 157, and 160 Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

13. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 165, 168, 171, and 174. CDR-H1; (ii) a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 166, 169, 172, and 175; CDR-H2; (iii) any one of the amino acid sequences of SEQ ID NOs: 167, 170, 173, and 176 Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 181, 184, 187, and 190. CDR-L1; (ii) a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 182, 185, 188, and 191; CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 183, 186, 189, and 192 Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

14. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 197, 200, 203, and 206. CDR-H1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 198, 201, 204, and 207. CDR-H2; (iii) any one of the amino acid sequences of SEQ ID NOs: 199, 202, 205, and 208 Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 213, 216, 219, and 222. CDR-L1, (ii) a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 214, 217, 220, and 223; CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 215, 218, 221, and 224. Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

15. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 229, 232, 235, and 238. CDR-H1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 230, 233, 236, and 239. CDR-H2, and (iii) any one of the amino acid sequences of SEQ ID NOs: 231, 234, 237, and 240 Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 245, 248, 251, and 254. CDR-L1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 246, 249, 252, and 255. CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 247, 250, 253, and 256 Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

16. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 261, 264, 267, and 270. CDR-H1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 262, 265, 268, and 271. CDR-H2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 263, 266, 269, and 272 Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 277, 280, 283, and 286. CDR-L1; (ii) any one of the amino acid sequences of SEQ ID NOs: 278, 281, 284, and 287. CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 279, 282, 285, and 288 Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

17. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 293, 296, 299, and 302. CDR-H1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 294, 297, 300, and 303. CDR-H2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 295, 298, 301, and 304. Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 309, 312, 315, and 318. CDR-L1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 310, 313, 316, and 319. CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 311, 314, 317, and 320 Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

18. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 325, 328, 331, and 334. CDR-H1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 326, 329, 332, and 335. CDR-H2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 327, 330, 333, and 336 Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 341, 344, 347, and 350. CDR-L1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 342, 345, 348, and 351. CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 343, 346, 349, and 352 Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

19. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 357, 360, 363, and 366. CDR-H1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 358, 361, 364, and 367. CDR-H2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 359, 362, 365, and 368 Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 373, 376, 379, and 382. CDR-L1; (ii) a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 374, 377, 380, and 383; CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 375, 378, 381, and 384. Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

20. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 389, 392, 395, and 398. CDR-H1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 390, 393, 396, and 399. CDR-H2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 391, 394, 397, and 400; Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 405, 408, 411, and 414. CDR-L1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 406, 409, 412, and 415. CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 407, 410, 413, and 416 Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

21. specifically binds to human CD3, and (a) a heavy chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 421, 424, 427, and 430. CDR-H1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 422, 425, 428, and 431. CDR-H2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 423, 426, 429, and 432. Contains CDR-H3 a heavy chain variable region comprising: (b) a light chain variable region, (i) comprising any one of the amino acid sequences of SEQ ID NOs: 437, 440, 443, and 446. CDR-L1; (ii) comprising any one of the amino acid sequences of SEQ ID NOs: 438, 441, 444, and 447. CDR-L2; and (iii) any one of the amino acid sequences of SEQ ID NOs: 439, 442, 445, and 448 Contains CDR-L3 A light chain variable region comprising A CD3 binding molecule comprising:

22. The heavy chain variable sequences listed in Table 1J-1 and the corresponding light chain variable sequences listed in Table 1J-2. The CD3 binding molecule of claim 5, comprising a sequence.

23. Antibody, antibody fragment, scFv, dsFv, Fv, Fab, scFab, (Fab ')2 or a single domain antibody (SDAB) according to any one of claims 1 to 22. CD3 binding molecule.

24. 24. The CD3 binding molecule of claim 23, comprising an antibody or an antibody fragment.

25. The CD3 binding molecule of claim 23 comprising an scFv.

26. A CD3 binding molecule according to any one of claims 1 to 25, which is a multispecific binding molecule.

27. The CD3 binding molecule of claim 26, which is a bispecific binding molecule (BBM).

28. The BBM is (a) a CD3 binding molecule that specifically binds to CD3 and is described in any one of claims 1 to 25; Antigen binding module 1 (ABM1) comprising the heavy and light chain variable regions of the molecule; and (b) an antigen-binding module 2 (ABM2) that specifically binds to a tumor-associated antigen (TAA); The CD3 binding molecule of claim 27, comprising:

29. ABM1 can bind to CD3 at the same time that ABM2 binds to its target molecule. The CD3 binding molecule of claim 28.

30. A CD3 binding molecule according to any one of claims 27 to 29, which is bivalent.

31. A CD3 binding molecule according to any one of claims 27 to 29, which is trivalent.

32. 30. The CD3 binding molecule of any one of claims 27 to 29, which is tetravalent.

33. The CD3 binding molecule of claim 26, which is a trispecific binding molecule (TBM).

34. The TBM is (a) a CD3 binding molecule that specifically binds to CD3 and is described in any one of claims 1 to 25; Antigen binding module 1 (ABM1) comprising the heavy and light chain variable regions of the molecule; and (b) an antigen binding module 2 (ABM2) that specifically binds to a tumor-associated antigen; and (c) antigen binding module 3 (ABM3), (i) a tumor-associated antigen other than the tumor-associated antigen bound by ABM2; or (ii) CD2 Antigen binding module 3 (ABM3) that specifically binds to The CD3 binding molecule of claim 33, comprising:

35. ABM1 binds to CD3 at the same time that ABM2 and ABM3 bind to their target molecules. The CD3 binding molecule of claim 34 .

36. A CD3 binding molecule according to any one of claims 33 to 35, which is trivalent.

37. A CD3 binding molecule according to any one of claims 33 to 35, which is tetravalent.

38. A CD3 binding molecule according to any one of claims 33 to 35, which is pentavalent.

39. A CD3 binding molecule according to any one of claims 33 to 35, which is hexavalent.

40. ABM2 is a transcription factor that regulates CD19, BCMA, TSHR, CD171, CS-1, CLL-1, and G D3, Tn Ag, FLT3, CD38, CD44v6, B7H3, KIT, IL-13 Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD 24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o -Acetyl-GD2, GD2, folate receptor alpha, folate receptor beta, TEM1 / CD 248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD17 9a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAA RP, WT1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MA D-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma Translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CY P1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4 , SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2 , CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCR L5, IGLL1, CD20, CD30, ERBB2, ROR1, FLT3, TAAG7 2, CD22, CD33, GD2, gp100Tn, FAP, tyrosinase, EPCAM , CEA, Igf-I receptor, EphB2, mesothelin, cadherin 17, CD32b, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG 2D, SLC34A2, SLC39A6, SLITRK6 or TACSTD2 A CD3 binding molecule according to any one of claims 28 to 39, which specifically binds to A.

41. The CD3 binding antibody according to any one of claims 34 to 40, wherein ABM3 binds to CD2. molecule.

42. ABM2 and ABM3 are TAA ("TAA1" and "TAA2", respectively).

36. The CD3 binding molecule of claim 34 or 35, which specifically binds to

43. TAA1 and TAA2 are each independently CD19, CD20, CD22, CD1 23, BCMA, CD33, CLL1, CD138, CS1, CD38, CD133, F LT3, CD52, TNFRSF13C, TNFRSF13B, CXCR4, PD-L1 , LY9, CD200, FCGR2B, CD21, CD23, CD24, CD40L, C 43. The CD3 binding molecule of claim 42, selected from CD72, CD79a and CD79b. 。

44. A conjugate comprising a CD3 binding molecule according to any one of claims 1 to 43 and a drug. 。

45. The CD3 binding molecule according to any one of claims 1 to 43 or the conjugate according to claim 44. A pharmaceutical composition comprising a jugate and a pharma- ceutically acceptable excipient.

46. A method of activating T cells in a subject in need thereof, optionally comprising: The subject has a proliferative disease or an autoimmune disorder, 44. A CD3 binding molecule according to any one of claims 43.

47. One or more nucleic acids encoding the CD3 binding molecule according to any one of claims 1 to 43. acid.

48. A cell line modified to express a CD3 binding molecule according to any one of claims 1 to 43. Cell.

49. 1. A method for producing a CD3 binding molecule, comprising: (a) culturing the cell of claim 48 under conditions in which the CD3 binding molecule is expressed. And, (b) recovering the CD3 binding molecule from the cell culture; and The method includes:

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