CN114605546A

CN114605546A - CD3 binding molecules

Info

Publication number: CN114605546A
Application number: CN202210274738.1A
Authority: CN
Inventors: 彼得·福科·万隆
Original assignee: Merus BV
Current assignee: Merus BV
Priority date: 2019-03-29
Filing date: 2020-03-27
Publication date: 2022-06-10

Abstract

The present application relates to CD3 binding molecules. The present invention relates to heavy chain variable regions, binding domains and antibodies specific for human CD3, and CD3 binding proteins. The invention further relates to the use of a CD3 binding protein, preferably an antibody, of the invention for the treatment of cancer or autoimmune diseases.

Description

CD3 binding molecules

The present application is a divisional application of the chinese patent application having the invention title "CD 3 binding molecule" of application No. 202080019367.1, which was originally an application of PCT international application PCT/NL2020/050214 filed on year 03, month 27 of 2020, entering the chinese national stage on year 09, month 07 of 2021.

Technical Field

The present invention relates to the field of antibodies, and in particular to the field of therapeutic antibodies. Such antibodies are useful for treating humans. More specifically, the invention relates to antibodies and preferably bispecific or multispecific antibodies for the treatment of tumors.

Background

Monoclonal antibodies that bind human CD3 were the first antibodies developed for human therapeutic use. Monoclonal CD 3-binding antibodies are typically used for their immunosuppressive properties, e.g., in transplant rejection. Antibodies that are bispecific against CD3 on T cells and against surface target antigens on cancer cells can link any kind of T cells with cancer cells, independently of T cell receptor specificity, co-stimulation or peptide antigen presentation. These bispecific T cell engagement (T-cell engaging) antibodies show great promise in the treatment of various cancers and neoplastic growths.

It is an object of the present invention to provide novel antibodies with CD3 binding properties, not necessarily quantitative in nature, with improved characteristics, having relatively low affinity, having high cytotoxicity, which are suitable for use in tumor immunology applications for T cell and effector cell engagement, and conversely, to provide novel antibodies with CD3 binding, having relatively high affinity, having low cytotoxicity, which are suitable for use in T cell and effector cell down-regulated autoimmune applications. It is a further object of the present invention to provide T cell engagement CD3 binding proteins and antibodies having the above properties of binding to at least one additional membrane associated molecule.

Disclosure of Invention

The present invention provides an antigen binding protein, preferably an antibody, that binds to human CD3, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：SFGIS

CDR2：GFIPVLGTANYAQKFQG

CDR 3: RGNWNPFDP, respectively; or

Comprising the following amino acid sequence:

CDR1：SX₁TFTIS；

CDR2：GIIPX₂FGTITYAQKFQG；

CDR3：RGNWNPFDP；

wherein

X₁K or R;

X₂l or I.

In a preferred embodiment X₁K; and X₂＝L；

In another preferred embodiment, X₁R; and X₂＝I。

In preferred embodiments, the present invention provides an antigen binding protein that binds human CD3, preferably an antibody, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：SKTLTIS；

CDR2：GIIPIFGSITYAQKFQD；

CDR 3: RGNWNPFDP, respectively; or

Comprising the following amino acid sequence:

CDR1：GSGIS；

CDR2：GFIPFFGSANYAQKFRD；

CDR3：RGNWNPX₁₃DP；

wherein

X₁₃L or F.

The present invention further provides an antigen binding protein, preferably an antibody, that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence

Having 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof at one or more positions other than such CDRs.

Further provided is an antigen binding protein that binds human CD3, preferably an antibody, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：RX₃WIG；

CDR2：IIYPGDSDTRYSPSFQG；

CDR3：X₄IRYFX₅WSEDYHYYX₆DV；

wherein

X₃F or Y;

X₄h or N;

X₅d or V;

X₆l or M.

In one embodiment X₃＝F；X₄＝H；X₅(ii) D; and X₆L. In another embodiment X₃＝Y；X₄＝N；X₅V; and X₆＝M。

CDR1：SYALS；

CDR2：GISGSGRTTWYADSVKG；

CDR3：DGGYSYGPYWYFDL。

CDR1：SYALS；

CDR2：AISGSGRTTWYADSVKG；

CDR3：DGGYTYGPYWYFDL。

further provided is an antigen binding protein, preferably an antibody, that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence

Also provided is an antigen binding protein, preferably an antibody, that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：DYTMH；

CDR2：DISWSSGSIGYADSVKG；

CDR3：DHRGYGDYEGGGFDY。

CDR1：DYTMH；

CDR2：DISWSX₇GX₈X₉X₁₀YADSVKG；

CDR3：DHX₁₁GYGDYEGGGFDX₁₂；

wherein

X₇(ii) S or G;

X₈(ii) S or T;

X₉i or T;

X₁₀g or Y;

X₁₁r or M;

X₁₂h or Y.

In one embodiment, X₇、X₈、X₉And X₁₀Is S, S, I and G, and X₁₁And X₁₂R and H. In another embodiment, X₇、X₈、X₉And X₁₀Is G, S, I and Y, and X₁₁And X₁₂R and Y. In another embodiment, X₇、X₈、X₉And X₁₀S, T, T and G, and X₁₁And X₁₂M and Y.

The light chain variable regions of an antigen binding protein of the invention, preferably an antibody, preferably comprise a common light chain variable region. The common light chain variable region preferably comprises an IgV kappa 1-39 light chain variable region. The light chain variable region is preferably a reproductive series IgV kappa 1-39 x 01 variable region. The light chain variable region preferably comprises a kappa light chain IgV kappa 1-39X 01/IGJ kappa 1X 01 or IgV kappa 1-39X 01/IGJ kappa 5X 01. In one embodiment the light chain variable region comprises the human reproductive series kappa light chain IgV kappa 1-39X 01/IGJ kappa 1X 01 or IgV kappa 1-39X 01/IGJ kappa 5X 01. The light chain variable region preferably comprises an amino acid sequence

Having 0-5 amino acid variations, insertions, deletions, substitutions, additions, or combinations thereof.

The antigen binding protein is preferably an antibody, preferably a bispecific or multispecific antibody.

The antibody preferably comprises a H/L chain combination as set forth herein that binds human CD3 and a H/L chain combination that binds a tumor antigen. The H/L chain combination that binds to a tumor antigen preferably binds to human BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, FOLR1, FOLR3, HER2, HM1.24, MCSP, PD-L1, PSMA protein or a variant thereof, in preferred embodiments EGFR, PD-L1 or CLEC 12A.

The antibody, bispecific or multispecific antibody is preferably a human or humanized antibody.

The bispecific or multispecific antibody preferably comprises two different immunoglobulin heavy chains with compatible heterogeneous dimeric domains. The compatible hetero-dimeric domain is preferably a compatible immunoglobulin heavy chain CH3 hetero-dimeric domain.

The bispecific or multispecific antibody is preferably an IgG antibody with a mutated CH2 and/or lower hinge domain such that the bispecific or multispecific IgG antibody has reduced interaction with Fc-gamma receptors. The mutant CH2 and/or lower hinge domain preferably comprises an amino substitution at position 235 and/or 236 (according to EU numbering), preferably a L235G and/or G236R substitution.

The bispecific or multispecific antibody preferably comprises a common light chain.

The invention further provides an antigen binding protein or antibody as set out herein for use in the treatment of a subject in need thereof. The individual preferably has cancer or is to be treated for cancer. An antigen binding protein or antibody having the CDRs and/or VH sequences of MF8057, MF8058, MF8078 or MF8508, or a variant thereof having 0-10 amino acid substitutions, variations, insertions, additions or deletions, preferably for use in therapy, in particular for use in a therapy comprising local administration and/or local release of the antigen binding protein or antibody. An antigen binding protein or antibody having the CDRs and/or VH sequences of MF9249, MF9267, MF8397, or variants thereof having 0-10 amino acid substitutions, variations, insertions, additions or deletions, is preferably used in the treatment of an individual having an overactivated immune system, such as an autoimmune disease.

The antibody of the invention is preferably a bispecific antibody, unless explicitly specified otherwise. The bispecific antibody preferably binds at least human CD 3. Furthermore, the bispecific antibody preferably binds to at least one surface molecule that preferentially displays on human tumor cells. In preferred embodiments, the bispecific antibody binds BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, PD-L1, FOLR1, FOLR3, HER2, HM1.24, MCSP, or PSMA. In a more preferred embodiment, the bispecific antibody binds to EGFR or CLEC 12A. In a more preferred embodiment, the multispecific antibody binds to EGFR and PD-L1.

The invention further provides a pharmaceutical composition comprising an antibody according to the invention.

Further provided is an antibody according to the invention, further comprising a label, preferably a label for in vivo imaging.

The invention also provides a method for treating a subject having or at risk of having a tumor comprising administering to the subject a bispecific or multispecific antibody according to the invention. Also provided is a bispecific or multispecific antibody according to the invention for use in treating an individual having a tumor or at risk of having a tumor. Further provided is the use of an antibody of the invention for the preparation of a medicament for the treatment of an individual having or at risk of having a tumor. In preferred embodiments, the tumor is an EGFR or CLEC12A positive tumor or an EGFR and PD-L1 positive tumor.

Drawings

FIG. 1 shows a schematic view of a

Evaluation of functional Activity: BxPC3 target cells were analyzed for T cell cytotoxicity following treatment with EGFRxCD3 bispecific antibody. Each bispecific antibody includes a CD3 binding domain consisting of a heavy chain variable region designated by MF number, and an EGFR binding domain including a heavy chain variable region MF 8233. The variable regions are paired with a common light chain to form an EGFRxCD3 bispecific antibody. Affinity (HPB-ALL binding) was solubilized relative to BxPC 3. Certain antibodies of the invention exhibit a relatively low level of HPB-ALL cell binding which indicates that the CD3 binding domain of the antibody binds human CD3 with relatively low affinity. It is clear that the relatively low affinity does not necessarily prevent tumor antigen-mediated T cell cytotoxicity of BxPC3 cells (BxPC3 lysis, vertical axis). The bispecific antibodies MF8233x MF8508 and MF8233x MF8057 were able to efficiently lyse BxPC3 cells while the bispecific antibodies MF8233x MF8397 and MF8233x MF9249, although having similar binding, did not do so efficiently. Furthermore, the bispecific antibody MF8233x MF6955 was compared to bind HPB-ALL (i.e. human CD3) with higher affinity, but did not lyse BxPC3 cells more efficiently than the bispecific antibodies MF8233x MF8508 and MF8233x MF8057, which bind CD3 to a lesser extent. MF6955 is a heavy chain variable region combined with a common light chain and used as comparator sequences, and corresponds to H1H7232B (1129) VH in US2014/0088295 a 1. The comparator bispecific antibody with MF6955 and the same EGFR binding domain has a higher affinity for human CD3 than MF9267, and exhibits more effective killing than MF9267, respectively. In contrast, other antibodies incorporating the CD3 binding domain of the invention, e.g., MF8058, have about the same binding activity as MF6955, but show more effective BxPC3 cell killing, e.g., MF8233x MF 8058. Other bispecific antibodies comprising a binding domain capable of binding CD3 of the invention exhibit relatively high binding and more effective killing, for example MF8233x MF8078, which is useful for the particular applications described herein. However other bispecific antibodies comprising a binding domain of the invention capable of binding CD3 exhibit relatively low affinity and low killing, for example MF8233xMF 9249 and MF8233x MF8397, which are useful for alternative applications as described herein.

FIG. 2 is a schematic view of a display device

It represents the% killing of T cell-mediated BxPC3 target cells induced compared to antibody titration curves without antibody control. Antibodies MF8233x MF8078, MF8233x MF8397 are shown; and curves for MF8233xMF 8508.

FIG. 3

Summary of titration curve data for various bispecific antibodies in T cell cytotoxicity with BxPC3 target cells. The CD3 Fab column indicates the MF number of the CD3 binding arm. The EGFR arm has the indicated MF8233 number. This column indicates the super group number to list variants based on the same VH gene segment. Columns showing binding of CD3 reflect the results of HBP-ALL binding experiments.

Results of two independent cytotoxicity assays to determine the lytic capacity of T cell-mediated BxPC3 target cells are shown.

FIG. 4

T cell activation by T cell cytotoxicity assay on expressed CD8+ T cells using BxPC3 target cells. Antibody titration curves for various supergroup numbers listing variant CD3 binding domains. The other arm of the bispecific antibody has the heavy chain binding domain of MF 8233. To compare the bispecific antibodies MF8233x MF6955 and MF8233x MF6964 were also tested, MF6955 and MF6964 are heavy chain variable regions combined with a common light chain and used as comparator sequences, and correspond to H1H7232B (1129) VH and HH7241B (1145), respectively, within US2014/0088295 a 1.

FIG. 5

Summary of titration curve data for various antibodies in T cell activation T cell cytotoxicity assays with BxPC3 target cells. Column indicates the MF number of the CD3 binding domain. The EGFR-binding domain has the indicated MF8233 number. Super group information for various CD3 binding domain sequences is shown in the column "super group"; columns showing affinity for CD3 reflect the results of HBP-ALL binding experiments.

Results are shown for CD4+ and CD8+ cells identified as CD69 and CD 25. The indicated bispecific antibodies are exemplary of a large pool of bispecific antibodies.

FIG. 6

Evaluation of functional Activity: t cell cytotoxicity assay with BxPC3 target cells. Affinity (HPB-ALL binding) was relative to CD8+ T cell activation measured by CD69 expression. Certain antibodies of the invention exhibit a relatively low level of HPB-ALL cell binding which indicates that the CD3 binding domain of the antibody binds human CD3 with relatively low affinity. These affinities do not necessarily prevent tumor antigen-mediated T cell activation, as shown by the results of CD8 positive CD69 activation assays. The bispecific antibodies MF8233x MF8508 and MF8233x MF8057 were able to efficiently activate T cells while the bispecific antibodies MF8233xMF8397 and MF8233xMF 9249 were unable to efficiently do so. Some CD 3-binding domains that are unable to bind these cells efficiently also fail to activate T cells (see bottom left). While other CD3 binding domains, e.g., MF8508 and MF8057, bind less to HPB-ALL cells than the comparator CD3 binding domain MF6955, activating T cells to a similar extent. Other bispecific antibodies comprising a binding domain of the invention capable of binding CD3 exhibit relatively high binding and high levels of activation, for example MF8078, which is useful for the specific applications described herein. However other bispecific antibodies comprising a binding domain of the invention capable of binding CD3 exhibit relatively low affinity and low activation, such as MF9249 and MF8397, which are useful for alternative applications described herein.

FIG. 7

Functional activity evaluation: t cell cytotoxicity assay with HCT116 target cells. Affinity (HPB-ALL binding) was solubilized relative to HCT-116.

Certain bispecific antibodies of the invention exhibit low HPB-ALL cell binding levels which indicate that the CD3 binding domain of the antibody binds human CD3 with relatively low affinity. It is clear that low affinity does not necessarily prevent tumor antigen-mediated cytolysis of HCT-116 cells (vertical axis). The bispecific antibodies MF8233x MF8508 and MF8233x MF8057 were able to efficiently lyse HCT-116 cells while the bispecific antibodies MF8233xMF8397 and MF8233xMF 9249 were unable to do so efficiently. To compare the bispecific antibodies MF8233x MF6955 and MF8233x MF6964 bound HPB-ALL (i.e., human CD3) with higher affinity than, for example, MF8233x MF8508, MF8233xMF8057, and MF8233xMF 9267, but failed to lyse HCT-116 cells more effectively than MF8233x MF8508 or MF8233x MF9267, or significantly more effectively than MF8233xMF8057, relative to the difference in binding in the assay as shown herein. Another bispecific antibody comprising a binding domain of the invention capable of binding CD3 shows relatively high binding and a high level of killing, for example MF8078, which is useful for the specific applications described herein. However other bispecific antibodies comprising a binding domain of the invention capable of binding CD3 exhibit relatively low affinity and low killing, such as MF9249 and MF8397, which are useful for alternative applications described herein.

FIG. 8

In the T cell cytotoxicity assay with HCT-116 target cells, it represents% killing of HCT-116 cells compared to the antibody titration curve without antibody control. Curves for various bispecific antibodies are shown.

FIG. 9

Summary of titration curve data for various antibodies in T cell cytotoxicity assays with HCT-116 target cells. The CD3 Fab column indicates the MF number of the CD3 binding domain. The EGFR-binding domain has the indicated MF8233 number. This column indicates the super group number to list variants based on the same VH gene segment. Columns showing binding of CD3 reflect the results of HBP-ALL binding experiments.

The percent lysis of HCT-116 cells and the EC50 value (ng/mL) of lysis are shown in the following fields. The indicated bispecific antibodies are exemplary of a large pool of bispecific antibodies.

FIG. 10 shows a schematic view of a

FIGS. 10a and 10b show schematic representations of MV1624 expression vector and MV1625 expression vector.

FIG. 11

Common light chains used within mono-and bispecific IgG.

FIG. 11A: a common light chain amino acid sequence. FIG. 11B: common light chain variable domain DNA sequences and presentations (IGKV1-39/jk 1). FIG. 11C: common light chain constant region DNA sequences and translations. FIG. 11D: IGKV1-39/jk 5. FIG. 11E: v region IGKV 1-39A; FIG. 11F: CDR1, CDR2, and CDR3 of a common light chain.

FIG. 12

IgG heavy chain of bispecific molecule. FIG. 12A: CH1 region. FIG. 12B: a hinge region. FIG. 12C: CH2 region. FIG. 12D: CH2 containing L235G and G236R silent substitutions. FIG. 12E: contains the CH3 domain in place of L351K and T366K (KK). FIG. 12F; containing the CH3 domain in place of L351D and L368E (DE).

FIG. 13

Sequences of various DNAs encoding the heavy chain variable region and portions thereof described in the specification and amino acid sequences thereof.

FIG. 14

Characterization of additional clones from supergroup 1 compared to clones MF8057 and MF 8058. A: binding of selected MF clones to HPB-ALL human cells expressing the human CD3-TCR complex in FACS analysis. B: t cell cytotoxicity assay with HCT-116 cells, which represents% kill of HCT-116 cells. C-E: activation markers for T cell activation CD25 and CD69 were quantified by FACS. F-G: production of interleukins from supernatants of cytotoxicity assays.

FIG. 15 shows a schematic view of a

Characterization of clones from supergroup 4. A: binding of selected MF clones to HPB-ALL human cells. B: t cell cytotoxicity assay with BxPC3 cells, which indicates% kill of BxPC3 cells. C-E: production of interleukins from supernatants of cytotoxicity assays.

FIG. 16

CD3 functional activity assessment: additional clones from supergroup 1(MF8048, MF8101, MF8056), supergroup 3(MF8562) and supergroup 4(MF8998) had affinities (HPB-ALL) that were resolved on the X-axis relative to HCT-116 on the Y-axis. B: antibodies belonging to supergroup 1 and supergroup 4, which showed similar activity and different binding affinities in cytotoxicity assays. C: antibodies belonging to supergroup 1 and supergroup 3, which exhibit similar binding affinity and differential lytic activity.

FIG. 17

Activity of CD3 Fabs MF8998 and MF8058 in bispecific CD3xEGFR format.

FIG. 18

FACS binding data for a large set of IgGs specific for CD 3. Against antibodies MF5196, MF6955 and MF6964, by BIAcore^TMTo determine binding to the CD3 δ epsilon-Fc antigen, while showing FACS binding data for the remaining clones to HPB-ALL cells.

FIG. 19

Nucleotide sequence of human CLEC 12A.

FIG. 20

Human CD3 gamma-, delta-, epsilon-and zeta-chain amino acid sequences.

Detailed Description

An "antibody" is a protein molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind epitopes on an antigen, wherein the domains are derived from or share sequence homology with the variable region of an antibody.

Antibodies bind with different properties, including specificity and affinity. Specificity determines which antigen or epitope thereof the binding domain specifically binds. Affinity is a measure of the amount of binding to a particular antigen or epitope (measure). It may be convenient to note here: the "specificity" of an antibody means its selectivity for a particular antigen, while "affinity" means the amount of interaction between the antigen binding site of the antibody and the epitope to which it binds. Antibodies typically consist of basic building blocks-two heavy and two light chains for each basic building block. The antibody for therapeutic use is preferably as close as possible to the natural antibody of the individual to be treated (e.g. a human antibody of a human individual). The antibody according to the present invention is not limited to any particular format or production method thereof.

Thus, "binding specificity" as used herein means the ability of an individual antibody binding site to react with an epitope. Typically, the binding address of the antibodies of the invention is located within such variable domains, including the Fab portion of such variable domains, and is constructed from highly variable regions of the heavy and light chains.

The antibody of the invention is preferably an IgG antibody, preferably an IgG1 antibody. Full length IgG antibodies may be preferred because of their favorable half-life and because of reasons of immunogenicity, it is desirable to remain close to fully autologous (human) molecules. IgG1 is advantageous based on its long circulating half-life in humans. In order to prevent or avoid immunogenicity in humans, it is preferred that a bispecific full length IgG antibody according to the invention is a human IgG 1.

A "bispecific antibody" is an antibody as described herein wherein one variable domain of the antibody binds to a first antigen and a second variable domain of the antibody binds to a second antigen, wherein the first and second antigens are not identical. The term "bispecific antibody" also includes bispecific antibodies (biparatopic antibodies) in which a variable domain of the antibody binds to a first epitope on an antigen and a second variable domain of the antibody binds to a second epitope on the antigen. The term further includes antibodies wherein at least one VH is capable of specifically recognizing a first antigen and a VL paired with the at least one VH of an immunoglobulin variable domain is capable of specifically recognizing a second antigen. The resulting VH/VL pair binds antigen 1 or antigen 2 and is referred to as a "two-in-one antibody" and is described, for example, in WO 2008/027236, WO2010/108127 and Schaefer et al (Cancer Cell 20, 472-. Bispecific antibodies as in the present invention are not limited to any particular bispecific format or method of production thereof. A bispecific antibody is a multispecific antibody.

Multispecific multimers or antibodies as referred to herein encompass protein molecules belonging to the immunoglobulin class of proteins which contain two or more domains that bind epitopes on antigens, wherein the domains are derived from or share sequence homology with the variable region of an antibody, as well as protein molecules that bind three or more antigens as known in the art, including as described in WO 2019/190327.

An "antigen" is a molecule capable of eliciting an immune response (to produce an antibody) in a host organism and/or an antibody-targeted molecule. On a molecular level, an antigen is characterized by its ability to be bound by the antigen-binding site of an antibody. Also, a mixture of antigens may be considered an "antigen", i.e., it will be understood by those skilled in the art that sometimes a tumor cell lysate or virus particle may be referred to as an "antigen", and that many epitopes exist in such tumor cell lysate or virus particle preparations. An antigen includes at least one, but typically a plurality of epitopes. For binding proteins and antibodies as disclosed herein, the antigen is typically associated with the cell membrane and is present on the extracellular portion of the cell membrane.

An "epitope" or "antigenic determinant" refers to an address on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes may be formed by contiguous amino acids or by discontinuous amino acids juxtaposed by tertiary folding of the protein (so-called linear or conformational epitopes, respectively). Epitopes formed from adjacent, linear amino acids are generally retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding generally lose conformation on treatment with denaturing solvents. Epitopes typically comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial configuration.

The term "heavy chain" or "immunoglobulin heavy chain" includes an immunoglobulin heavy chain constant region sequence from any organism and, unless otherwise specified, includes a heavy chain variable domain. Unless otherwise specified, the term heavy chain variable domain will include three heavy chain CDRs and four FR regions. Fragments of the heavy chain include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has a CH1 domain, a hinge, a CH2 domain, and a CH3 domain after (from N-to C-terminus) the variable domain. A functional fragment of a heavy chain comprises a fragment capable of specifically recognizing an antigen and comprising at least one CDR.

The term "light chain" includes an immunoglobulin light chain variable domain or V from any organism_L(or functional fragments thereof; and an immunoglobulin constant domain or C_LOr a functional fragment thereof. Unless otherwise specified, the term light chain may include a light chain selected from human κ, λ, and a combination thereof. Unless otherwise specified, light chain variable (V)_L) The domain typically includes three light chain CDRs and four Framework (FR) regions. Generally, a full-length light chain includes, from N-terminus to C-terminus, a V comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4_LDomains and a light chain constant domain. Light chains that can be used in the present invention include, for example, such that they do not selectively bind an epitope that is selectively bound by such heavy chains.

Suitable light chains for use in the antibodies of the invention include a common light chain (cLC), such as may be obtained by screening existing antibody libraries [ wet libraries ] or computer simulation (in silico)]Wherein such light chains do not substantially interfere with the affinity and/or selectivity of the epitope binding domain of such heavy chains, but are also suitable for pairing with a series of heavy chains. For example, a suitable light chain includes a light chain derived from a transgenic animal, such as

The transgenic animal has a common light chain embedded within its genome and is operable to produce a plurality (large panels of) common light chain antibodies that are diverse at the heavy chain and that specifically bind an antigen when exposed thereto.

The term "common light chain" as used herein refers to light chains that may be identical or differ in some amino acid sequence without affecting the binding specificity of the antibodies of the invention, i.e., such differences do not substantially affect the formation of functional binding regions.

For example, within the definition of common light chains as used herein, it is possible to make or find variable chains that are not identical but are still functionally equivalent, e.g., by introducing and testing for conservative amino acid changes that do not contribute, or only partially contribute, to binding specificity when paired with a homologous chain or the like. These variants are therefore also capable of binding to different homologous chains and forming functional antigen binding domains. The term common light chain as used herein thus refers to light chains that may be identical or differ in some amino acid sequence, but which retain the binding specificity of the resulting antibody after pairing with a heavy chain. Combinations of certain common light chains and these functionally equivalent variants are encompassed within the term common light chain. For a detailed description of the use of a common light chain see WO 2004/009618 and WO 2009/157771.

"Fab" refers to a binding domain that includes a variable region, typically a binding domain that includes a pair of heavy and light chain variable regions. Fab may include constant region domains comprising one CH1 and VH domain paired with one constant light chain domain (CL) and VL domain. These pairings may occur, for example, as covalent linkages via a disulfide bond at the CH1 and CL domains.

"Single chain variable fragment" (scFv) refers to a binding domain that includes a VH domain and a VL domain linked via a linker, e.g., a peptide linker, of from about 10 to about 25 amino acids in length.

The term "full-length IgG" or "full-length antibody" as used herein is defined to include substantially intact IgG, although it does not necessarily have the full functionality of intact IgG. For the avoidance of doubt, full-length IgG contains two heavy and two light chains. Each chain contains constant (C) and variable (V) regions, which can be subdivided into domains designated CH1, CH2, CH3, VH and CL, VL. IgG antibodies bind to antigens via variable regions contained within the Fab portion and, upon binding, interact with molecules and cells of the immune system via constant domains, primarily via the Fc portion. Full length antibodies according to the invention include IgG molecules in which mutations may be present that provide the desired characteristics. Full-length IgG should not have any substantial portion of the domain deleted. However, the term "full length IgG" includes IgG molecules in which one or several amino acid residues are deleted without substantially altering the binding characteristics of the resulting IgG molecule. For example, the IgG molecule can have between 1 and 10 amino acid residues deleted, preferably in the non-CDR regions, wherein the deleted amino acids are not essential for binding specificity of the IgG.

When referring to nucleic acid or amino acid sequences herein, "percent (%) identity" is defined as the percentage of residues of a candidate sequence that are identical to the residues of a selected sequence after aligning the matched sequences for optimal comparison. Either of the two sequences to be compared may introduce gaps to maximize the alignment of the two sequences. This alignment can be performed over the full length sequences to be compared. Alternatively, alignment can be performed over shorter lengths, such as about 20, about 50, about 100, or more nucleic acids/base or amino acids. Sequence identity is the percentage of matches between two sequences that are identical over the reported aligned region.

Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using a mathematical algorithm. One skilled In the art will recognize that several different computer programs can be used to align two sequences and determine the identity between the two sequences (Kruskal, J.B., 1983) An overview of sequence complexity In D.Sankoff and J.B.Kruskal, (ed.), Time wars, string indexes and algorithms: the organ and practice of sequence compliance, pp.1-44 Addison Wesley).

With respect to the sequences of the present invention and described herein, the percent sequence identity between two nucleic acid sequences can be determined using the AlignX application of Vector NTI Program Advance 10.5.2 software, using presets, using a modified ClustalW algorithm (Thompson, J.D., Higgins, D.G., and Gibson T.J (1994) Nuc.acid Res.22: 4673. sup. 4680), swgapdna score matrix, a 15 gap open penalty (gap open penalty) and a 6.66 gap extension penalty (gap extension penalty). The amino acid sequences can be aligned using the AlignX application of Vector NTI Program Advance 11.5.2 software, using default alignment using a modified ClustalW algorithm (Thompson, j.d., Higgins, d.g., and Gibson t.j., 1994), blosum62mt2 scoring matrix, a 10 gap open penalty, and a 0.1 gap extension penalty.

The term "super-cluster" or "super-cluster" as used herein means a group of clones (clones) and their producible binding domains based on the use of the same VH V gene segment and having at least 70% sequence identity within HCDR3 and the same HCDR3 length.

Thus, in a preferred embodiment, the invention provides a "super-cluster" or "super-cluster" comprising a set of clones and their producible binding domains based on the use of the same VHV gene segment and having at least 70% sequence identity within HCDR3 and the same HCDR3 length. In preferred embodiments, the sequence identity is 80%, more preferably 90%, and most preferably 95%, provided that a clone comprising a nucleic acid encoding HCDR3 sequences DGGYSYGPYWYFDL and DHRGYGDYEGGGFDY, a clone comprising nucleic acids encoding HCDR2 sequences GFIPVLGTANYAQKFQG, GIIPLFGTITYAQKFQG and SIIPIFGTITYAQKFQG, or a clone comprising a nucleic acid encoding VH sequences

Or with the proviso that a clone from the group comprises nucleic acid encoding HCDR3 consisting of or designed to consist of a bispecific antibody.

The term "super-cluster 1" or "super-cluster 1" as used herein means a group of clones and their productive binding domains based on the use of the same VH V gene segment (VH1-69) as a member of the super-cluster and having at least 70% sequence identity within HCDR3 and the same length of HCDR 3. Including, for example, MF8048, MF8056, MF8057, MF8058, MF8078, and MF 8101. In another preferred embodiment, the anti-CD 3 antibodies herein are based on the use of the same VH V gene segment of VH1-69 and/or have at least 80% identity within HCDR3 and the same length of HCDR3, more preferably 90% or most preferably 95% identity within HCDR 3. In another preferred embodiment, the anti-CD 3 antibodies herein are based on the use of the same VH V gene segment of VH1-69 and/or have at least 80% identity and the same HCDR3 length within HCDR3, preferably at least 90% sequence identity and the same HCDR3 length within HCDR3, more preferably 95% or optimally 98% identity and the same HCDR3 length as compared to the encoded CDR3 segment RGNWNPFDP, with the proviso that clones comprising nucleic acids encoding HCDR2 sequences GFIPVLGTANYAQKFQG, GIIPLFGTITYAQKFQG and SIIPIFGTITYAQKFQG are excluded or with the proviso that clones comprising nucleic acids encoding VH sequences 2 and SIIPIFGTITYAQKFQG are excluded

Or with the proviso that a clone from the group comprises nucleic acid encoding HCDR3 consisting of or designed to consist of a bispecific antibody. The term "super-cluster 3" or "super-cluster 3" as used herein means a group of clones and their productive binding domains based on the use of the same VH V gene segment (VH3-23) as a member of the super-cluster and having at least 70% sequence identity within HCDR3 and the same length of HCDR 3. Including MF8397 and MF8562, for example. In another preferred embodiment, the anti-CD 3 antibodies herein are based on the use of the same VH V gene segment of VH3-23 and/or have at least 80% identity within HCDR3 and the same length of HCDR3, more preferably 90% or most preferably 95% identity within HCDR 3. In another preferred embodiment, the anti-CD 3 antibodies herein are used based on the same VH V gene segment of VH3-23 and/or have at least 80% identity and the same HCDR3 length within HCDR3, preferably at least 90% sequence identity and the same HCDR3 length within HCDR3, more preferably 95% or optimally 98% identity and the same HCDR3 length as compared to the encoded CDR3 segment DGGYSYGPYWYFDL, with the proviso that a clone comprising a nucleic acid encoding HCDR3 sequence DGGYSYGPYWYFDL is excluded or with the proviso that a clone comprising a nucleic acid encoding VH sequence 3 sequence DGGYSYGPYWYFDL is excluded

The term "super-cluster 4" or "super-cluster 4" as used herein means a group of clones and their productive binding domains based on the use of the same VH V gene segment (VH3-9) as a member of the super-cluster and having at least 70% sequence identity within HCDR3 and the same length of HCDR 3. Including, for example, MF8508, MF8998, MF10401, and MF 10428. In another preferred embodiment, the anti-CD 3 antibodies herein are based on the use of the same VH V gene segment of VH3-9 and/or have at least 80% identity within HCDR3 and the same length of HCDR3, more preferably 90% or most preferably 95% identity within HCDR 3. In another preferred embodiment, the anti-CD 3 antibodies herein are based on the use of the same VH V gene segment of VH3-9 and/or have at least 80% identity and the same HCDR3 length within HCDR3, preferably at least 90% sequence identity and the same HCDR3 length within HCDR3, more preferably 95% or optimally 98% identity and the same HCDR3 length as compared to the encoded CDR3 segment DHRGYGDYEGGGFDY, with the proviso that clones comprising nucleic acids encoding the HCDR3 sequence DHRGYGDYEGGGFDY are excluded or with the proviso that clones comprising nucleic acids encoding the VH sequence DHRGYGDYEGGGFDY are excluded

The term "super-cluster 7" or "super-cluster 7" is used herein to mean a group of clones and binding domains thereof that are capable of producing based on the use of the same VH V gene segments as the members of the super-cluster (VH5-51) and have at least 70% sequence identity within HCDR3 and the same length of HCDR 3. Including, for example, MF9249 and MF 9267. In another preferred embodiment, the anti-CD 3 antibodies herein are based on the use of the same VH V gene segment of VH5-51 and/or have at least 80% identity within HCDR3 and the same length of HCDR3, more preferably 90% or most preferably 95% identity within HCDR 3. In another preferred embodiment, the anti-CD 3 antibodies herein are based on the use of the same VH V gene segment of VH5-51 and/or have at least 80% identity and the same HCDR3 length within HCDR3, preferably at least 90% sequence identity and the same HCDR3 length within HCDR3, more preferably 95% or optimally 98% identity and the same HCDR3 length as compared to the encoded CDR3 segment HIRYFDWSEDYHYYLDV.

The invention further provides a bispecific antibody comprising a variable domain having a VH encoded by

-the V gene segment VH 1-69; or

-a variant of the V gene segment VH1-69 which comprises at least 70%, preferably at least 80%, more preferably at least 90% and more preferably at least 95% sequence identity with the sequence of the V gene segment;

wherein the VH further comprises

-an HCDR3 of MF8048, MF8056, MF8057, MF8058, MF8078 or MF 8101;

or a variant of the HCDR3 comprising at least 70% sequence identity to the HCDR3 and the same length as the HCDR 3.

In a preferred embodiment, the variant of the HCDR3 comprises the same length as the HCDR3 and at least 80% sequence identity to the HCDR3, more preferably at least 90% and more preferably at least 95% sequence identity to the sequence of the HCDR 3.

In some embodiments, the bispecific antibody does not have a VH encoded by: the V gene segment VH 1-69; or a variant of the V gene segment VH1-69 having the sequence HCDR2 GFIPVLGTANYAQKFQG, or GIIPLFGTITYAQKFQG or SIIPIFGTITYAQKFQG.

In some embodiments, the bispecific antibody does not have a VH encoded by: the V gene segment VH 1-69; or a variant of the V gene segment VH1-69 having the VH sequence

-the V gene segment VH 3-23; or

-a variant of the V gene segment VH2-23 which comprises at least 70%, preferably at least 80%, more preferably at least 90% and more preferably at least 95% sequence identity with the sequence of the V gene segment;

wherein the VH further comprises

-MF 8397; or an HCDR3 of MF 8562;

In preferred embodiments, the variant of the HCDR3 comprises the same length as the HCDR3 and at least 80% sequence identity to the HCDR3, more preferably at least 90%, more preferably at least 93% and more preferably at least 95% sequence identity to the sequence of the HCDR 3.

In some embodiments, the bispecific antibody does not have a VH encoded by: the V gene segment VH 3-23; or a variant of the V gene segment VH3-23 having the sequence HCDR3 DGGYSYGPYWYFD.

In some embodiments, the bispecific antibody does not have a VH encoded by: the V gene segment VH 3-23; or a variant of the V gene segment VH3-23 having the VH sequence

-the V gene segment VH 3-9; or

-a variant of the V gene segment VH3-9 comprising at least 70%, preferably at least 80%, more preferably at least 90% and more preferably at least 95% sequence identity with the sequence of the V gene segment;

wherein the VH further comprises

-MF 8508; MF 8998; MF 1041; or an HCDR3 of MF 10428;

In some embodiments, the bispecific antibody does not have a VH encoded by: the V gene segment VH 3-9; or a variant of the V gene segment VH3-9 having the sequence HCDR3 DHRGYGDYEGGGFDY.

In some embodiments, the bispecific antibody does not have a VH encoded by: the V gene segment VH 3-9; or a variant of the V gene segment VH3-9 having the VH sequence

-the V gene segment VH 5-51; or

-a variant of the V gene segment VH5-51 comprising at least 70%, preferably at least 80%, more preferably at least 90% and more preferably at least 95% sequence identity to the sequence of the V gene segment;

wherein the VH further comprises

-an HCDR3 of MF9249 or MF 9267;

A bispecific antibody as defined herein provided by the present invention is preferably not a bispecific antibody comprising a CD3 binding variable domain as defined in PCT/NL 2019/050199.

The invention further provides a VH encoded by

-the V gene segment VH 1-69; or

wherein the VH further comprises

-an HCDR3 of MF8048, MF8056, MF8057, MF8058, MF8078 or MF 8101;

or a variant of the HCDR3 comprising at least 70% sequence identity to the HCDR3 and the same length of the HCDR 3.

In some embodiments, the VH is not a VH encoded by: the V gene segment VH 1-69; or a variant of the V gene segment VH1-69 which has the sequence HCDR2 GFIPVLGTANYAQKFQG, or GIIPLFGTITYAQKFQG or SIIPIFGTITYAQKFQG.

In some embodiments, the VH is not a VH encoded by: the V gene segment VH 1-69; or a variant of the V gene segment VH1-69 having the VH sequence

The invention further provides a VH encoded by

-the V gene segment VH 3-23; or

wherein the VH further comprises

-MF 8397; or an HCDR3 of MF 8562;

In some embodiments, the VH is not a VH encoded by: the V gene segment VH 3-23; or a variant of the V gene segment VH3-23 having the sequence HCDR3 DGGYSYGPYWYFDL.

In some embodiments, the VH is not a VH encoded by: the V gene segment VH 3-23; or a variant of the V gene segment VH3-23 having the VH sequence

The invention further provides a VH encoded by

-the V gene segment VH 3-9; or

-a variant of the V gene segment VH3-9 comprising at least 70%, preferably at least 80%, more preferably at least 90% and more preferably at least 95% sequence identity to the sequence of the V gene segment;

wherein the VH further comprises

-MF 8508; MF 8998; MF 10401; or an HCDR3 of MF 10428;

In some embodiments, the VH is not a VH encoded by: the V gene segment VH 3-9; or a variant of the V gene segment VH3-9 having the sequence HCDR3 DHRGYGDYEGGGFDY.

In some embodiments, the VH is not a VH encoded by: the V gene segment VH 3-9; or a variant of the V gene segment VH3-9 having the VH sequence

The invention further provides a VH encoded by

-the V gene segment VH 5-51; or

wherein the VH further comprises

-an HCDR3 of MF9249 or MF 9267;

A VH as defined herein provided by the invention is preferably not a VH of a CD3 binding variable domain as defined in PCT/NL 2019/050199.

Also provided is an antigen binding protein or antibody, preferably a bispecific antibody, wherein such CDRs are 70%, preferably 80%, more preferably 90% identical to such CDRs as claimed. In a preferred embodiment, the antigen binding protein or antibody is a bispecific antibody comprising CDRs having at most 2, preferably at most 1 and more preferably at most 0 amino acid residue variations, insertions, substitutions, deletions or additions with respect to such claimed CDRs.

Unlike random, non-specific attachment of antibodies, antigen binding by antibodies is typically mediated through specific three-dimensional structures of both the complementary regions of the antibody and the antigen and variable domains, allowing these two structures to bind together precisely (similar to lock and key interactions). Since an antibody typically recognizes only one epitope of an antigen, and such an epitope may also be present in other proteins, e.g. antibodies of the invention that bind CD3 or CLEC12A may also recognize other proteins if these other proteins contain the same epitope. Thus, the term "binding" does not exclude the binding of the antibody to another protein or proteins containing the same epitope. The heavy/light chain combination in the antibodies of the invention that binds CD3 does not bind to other proteins on the cell membrane of postnatal, preferably adult, human cells. The heavy/light chain combination of the invention that binds CLEC12A, EGFR, PD-L1 or a tumor cell antigen does not bind to other proteins on the cell membrane after birth, preferably in adult humans. Suitable tumor antigen-specific arms are disclosed in PCT/NL 2019/050199.

"plural" means two or more.

"variants" of an antibody as described herein may include functional portions, derivatives and/or analogs of the antibody. This includes mimetics (antibodies), monobodies (monobodies) and aptamers (aptamers).

Variants will typically retain the binding specificity of an antibody, e.g., the specificity of a bispecific antibody. Variants can be binding domains, multimers or functional portions or derivatives of antibodies as described herein.

A functional part of a binding domain, multimer or antibody as described herein is a part that includes variable domains that bind to the same target as such binding domain, multimer or antibody binds.

A functional derivative of an antibody as described herein is a protein comprising a variable domain that binds to a target and a variable domain that binds to a second target linked by linking regions. The variable domain may be a variable domain itself or a Fab fragment or variable domain-like molecule, for example a single chain fv (scFv) fragment comprising VH and VL linked together via a linker. Antibody variable domains or antibody-like variable domain molecules can be linked to each other in different ways. Various linker and carrier structures have been described which are capable of binding one, two or more variable domains. An antigen binding protein as described herein is a protein that includes at least one such variable domain. In the case of bispecific or multispecific antigen binding proteins, such proteins include two or more variable domains, at least two of which bind different targets. The variable domains are bonded to each other via a bonding portion. This is typically a stretch of 0-15, preferably 3-12, more preferably about 5-8 amino acid residues. Other examples of variable domain-like molecules are so-called single domain antibody fragments. Single domain antibody fragments (sdabs) are antibody fragments with a single monomeric variable antibody region. Like whole antibodies, they are capable of selectively binding to specific antigens. The molecular weight is only 12-15kDa, the single domain antibody fragment is much smaller than the normal antibody composed of two heavy protein chains and two light chains (150-160kDa), and even smaller than the Fab fragment (-50 kDa, one light chain and half a light chain)Heavy chain) and single-chain variable fragments (-25 kDa, two variable regions, one from the light and one from the heavy chain). Single domain antibodies are not by themselves much smaller than normal antibodies (typically 90-100 kDa). Single domain antibody fragments are engineered mainly from heavy chain antibodies found in camelidae (camelid); these are termed VHH fragments

Some fish also have heavy chain-only antibodies (IgNAR, "immunoglobulin neo-antigen receptor") from which single domain antibody fragments, called VNAR fragments, are available. Another approach is to separate dimeric variable domains from common immunoglobulin g (igg) from human or mouse into monomers. While most studies on single domain antibodies are currently based on heavy chain variable domains, nanobodies (nanobodies) derived from light chains have been shown to bind to target epitopes. Other non-limiting examples of variable Domain-like molecules are VHH, Human Domain Antibodies (dAbs) and single Antibodies (Unibodies). Preferred functional portions are those comprising a variable domain comprising a heavy chain variable region and a light chain variable region. Non-limiting examples of such variable domains are f (ab) fragments and single chain Fv fragments. Bispecific formats for (class) variable domain linkage are e.g. Human Serum Albumin (HSA) bound to two different scfvs; bispecific miniantibodies include two different scfvs that are bound together by secondary structures such as helical bundles or coiled coils, either through a dimerization motif (motif) or self-association (self-association) to cause dimerization of the scFv fragments (Morrison (2007) nat. biotechnol. 25: 1233-34). Examples of suitable HAS linkers and methods of coupling scFv to the linkers are described in WO 2009/126920.

The functional derivative may be a mimetibody, a polypeptide, an aptamer, or a combination thereof. These proteins or aptamers typically bind to a target. The proteins of the invention bind to two or more targets. It is understood that any combination of these antibodies, mimetibodies, polypeptides, and aptamers may be linked together by methods known in the art. For example, in some embodiments, a binding molecule of the invention is a complex or fusion protein.

A mimobody is a polypeptide that, like an antibody, can specifically bind to an antigen, but is structurally unrelated to the antibody. The mimetibodies are typically artificial peptides or proteins of about 3 to 20kDa in molar mass. Non-limiting examples of mimetibodies are affibody molecules (usually based on the Z domain of protein a); affinin (affilin) (usually based on γ -B crystals or ubiquitin); adhesins (affimers) (usually based on cystatins); afttins (generally based on Sac7d from Sulfolobus acidocaldarius); alpha bodies (usually based on Triple helix coiled coil); anti-transportan (anticalin) (typically lipocalin) -based; avamer (generally based on the a domain of various membrane receptors); d ARPin (usually based on ankyrin repeat motif); fenomom (fynomers) (generally based on the SH3 domain of Fyn 7); the pornizian domain (kunitz domain) peptide (a pornizian domain that is typically based on various protease inhibitors); and unions (typically type III domains based on fibronectin).

The single-entity type is a synthetic binding protein that is constructed using the type III domain of fibronectin (FN3) as a molecular architecture. For the production of target binding proteins, single bodies are alternatives to antibodies.

Monotypes and other mimetics are typically generated from combinatorial libraries using molecular display and directed evolution techniques such as phage display, mRNA display, and yeast surface display to diversify part of the architecture.

Aptamers are oligonucleotide or peptide molecules that bind to a specific target molecule. Aptamers are usually created by selecting them from a large pool of random sequences (sequence pool), but natural aptamers are also present in riboswitches (riboswitch). Aptamers can be used as macromolecules for both basic research and clinical purposes.

Throughout the description of the invention and the claims appended hereto, the words "comprising", "including" and "having" and variations (variations) such as "comprising", "including" and "including" are to be interpreted inclusively. That is, where the context permits, these terms are intended to convey the possible inclusion of other elements or integers not specifically recited.

The articles "a" and "an" as used herein mean one or more than one (i.e., one or at least one) of the grammatical object of the article. For example, an element may represent one element or more than one element.

The antibody of the invention is preferably a bispecific or multispecific antibody. The bispecific or multispecific antibody preferably binds at least human CD 3.

The antigen binding protein or antibody of the invention is preferably a bi-or multispecific antigen binding protein or antibody. The bi-or multispecific antigen-binding protein or antibody preferably binds at least human CD3, and preferably also at least one surface molecule expressed on human tumor cells. In preferred embodiments, the bi-or multispecific antigen-binding protein or antibody binds BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, PD-L1, FOLR1, FOLR3, HER2, HM1.24, MCSP, or PSMA. In a particularly preferred embodiment, the bispecific antibody binds CLEC 12A. In a particularly preferred embodiment, the bispecific antibody binds to CD3, PD-L1 and EGFR.

As used herein, the term "CLEC 12A" refers to member a of the C-type lectin domain family 12. CLEC12A is also known as C-type lectin protein CLL-1; MICL; dendritic cell associated lectin 2; the C-type lectin superfamily; myelosuppressive C-type lectin-like receptors; c-type lectin-like molecule 1; DCAL 2; CLL 1; c-type lectin-like molecule 1; DCAL-2; killer lectin-like receptor subfamily L, member 1(KLRL 1); CD371 (differentiation group 371) (Cancer Res.2004, 64, p884350 by Bakker A. et al; GenBank TM accession No.: AY 547296; GenBank TM accession No.: AF247788 by Zhang W. et al; J Biol Chem 2004, 279, p14792-802 by A.S. Marshall et al; GenBank TM accession No.: AY 498550; Blood 2004, 104, p 285866 by Y.Han et al; GenBank TM accession No.: AY 426759; Blood 2006, 107, p145967 by C.H.Chen et al). Ids: HGNC: 31713; entrez Gene: 160364; ensembl: ENGG 00000172322; OMIM: 612088, respectively; UniProtKB: q5QGZ 9.

CLEC12A is an antigen that is expressed on leukemic blasts and leukemic Stem Cells in Acute Myeloid Leukemia (AML), including CD34 negative or CD34 low expressing leukemic Stem Cells (side population) (Cancer Res 2004, 64, p844350, Van Rheenen et al 2007 Blood 110: 2659, Moshaver et al 2008 Stem Cells 26: 3059), and in myelodysplastic syndrome (MDS) (2004, and Toff-Peterson et al, Br. J. Haematol.175 (3): 393-401, 2016). Expression of CLEC12A is additionally believed to be restricted to hematopoietic lineages, particularly peripheral blood and myeloid lineages within the bone marrow, namely granulosa spheroids, monocytes and dendritic cell precursors. More importantly, CLEC12A was not present on normal hematopoietic stem cells. Where reference is made herein to CLEC12A, it refers to human CLEC12A (SEQ ID NO: 1; FIG. 19), unless specifically noted otherwise.

The term "CLEC 12A" means all variants (e.g., splicing and mutation) described herein and their isoforms that retain a myeloid phenotype resolution (both at the level of surface expression and at the mRNA level), including, for example, those described in Bakker et al Cancer Res 2004, 64, p8443-50 and Marshall 2004-J Biol Chem 279(15), p 14792-802B. Although accession numbers are primarily provided as additional means of identification, the actual protein sequence may, for example, vary due to mutations in the encoding gene, such as occur in some cancers and the like.

The term "CD 3" (clade 3) refers to a protein complex (complex) consisting of the CD3 γ chain (SwissProt P09693), the CD3 δ chain (SwissProt P04234), the CD3 ∈ chain (SwissProt P07766), and the CD3 ζ chain homodimer (SwissProt P20963). CD3 epsilon is known as various aliases, some of which are: "molecule CD3e,. epsilon. (CD3-TCR complex)"; "CD 3e antigen, ∈ polypeptide (TiT3 complex)"; t cell surface antigen T3/Leu-4 epsilon chain; T3E; t cell antigen receptor complex, epsilon subunit of T3; CD3e antigen; CD3- ε 3; an IMD 18; TCRE. Ids of CD3E gene is HGNC: 1674; entrez Gene: 916; ensembl: ENGG 00000198851; OMIM: 186830 and UniProtKB: p07766. These chains associate with the T Cell Receptor (TCR) and zeta chains to form TCR complexes, which upon mitogenic signaling, generate activation messages within T lymphocytes. CD3 appears on T cells and NK T cells. Where reference is made herein to CD3, it refers to human CD3(SEQ ID NOS: 2-5; FIG. 20), unless specifically noted otherwise.

BCMA is also known as tumor necrosis factor receptor superfamily, member 17(TNFRSF 17); TNFRSF13a 2; b cell maturation antigen; BCM; b cell maturation factor; b cell maturation protein; CD269 or CD269 antigen. Ids: HGNC: 11913, a support base; entrez Gene: 608; ensembl: ENSG 00000048462; OMIM: 109545, respectively; UniProtKB: q02223.

CD19 is also known as CD19 molecule; the T cell surface antigen Leu-12; a CD19 antigen; CvID 3; the differentiation antigen CD 19; b4; b lymphocyte surface antigen B4; the B lymphocyte antigen CD 19. Ids: HGNC: 1633; entrez Gene: 930; ensembl: ENGG 00000177455; OMIM: 107265, respectively; UniProtKB: p15391.

CD20 is also known as transmembrane 4-domain, subfamily a, member 1(MS4a 1); MS4a 2; CD 20; s7; leukocyte surface antigen Leu-16; b lymphocyte antigen CD 20; bp 35; b lymphocyte cell surface antigen B1; a CD20 antigen; the CD20 receptor; CVID 5; b lymphocyte surface antigen B1; b1; transmembrane 4-domain subfamily a member 1; LEU-16. Ids: HGNC: 7315; entrez Gene: 931; ensembl: ENGG 00000156738; OMIM: 112210, respectively; UniProtKB: p11836.

CD30 is also known as tumor necrosis factor receptor superfamily, member 8(TNFRSF 8); a Ki-1 antigen; CD 30; ki-1; D1S 166E; the interleukin receptor CD 30; lymphocyte activation antigen CD 30; tumor necrosis factor receptor superfamily member 8; the CD30L receptor; CD30 antigen. Ids: HGNC: 11923; entrez Gene: 943; ensembl: ENGG 00000120949; OMIM: 153243, respectively; UniProtKB: p28908.

CD33 is also known as CD33 molecule; SIGLEC-3; CD33 antigen (Gp 67); bone marrow cell surface antigen CD 33; sialic acid binds to Ig-like lectin 3; siglec-3; SIGLEC 3; CD33 antigen and gp 67. Ids: HGNC: 1659; entrez Gene: 945; ensembl: ENGG 00000105383; OMIM: 159590, respectively; UniProtKB: p20138.

CD38 is also known as CD38 molecule; t10; CD38 antigen (P45); CADPr hydrolase 1; ADP-ribosyl cyclase 1; ADP-ribosyl cyclase/cyclic ADP-ribosyl hydrolase; NAD (+) nucleosidase; EC 3.2.2.5; cyclic ADP-ribose hydrolase 1; the CD38 antigen. Ids: HGNC: 1667; entrez Gene: 952; ensembl: ENSG 00000004468; OMIM: 107270, respectively; UniProtKB: p28907.

CD44 is also known as CD44 molecule (indian blood group); IN; MDU 2; CD44 antigen (Homing) function and indian blood group system); MDU 3; CDW 44; MIC 4; CSPG 8; chondroitin sulfate proteoglycan 8; HCELL; hematopoietic cell E-and L-selectin ligands; MC 56; extracellular matrix receptor III; pgp 1; heparan Sulfate (heparin Sulfate) protein polysaccharides; cell surface glycoprotein CD 44; a hyaluronic acid receptor; epidermal proteoglycans (epicans); phagocytic glycoprotein 1; homing function and indian blood group system; ECMR-III; CDw 44; HUTCH-I; epidermal proteoglycans (epicans); LHR; PGP-1; a CD44 antigen; p GP-I; CP90 lymphocyte homing/adhesion receptor; a phagocytic glycoprotein I; amanshi (Hermes) antigen. Ids: HGNC: 1681; entrez Gene: 960 (f); ensembl: ENSG 00000026508; OMIM: 107269, respectively; UniProtKB: p16070.

CD123 is also known as cell division cycle 123; cell division cycle 123 homologues; c10orf 7; cell-division cyclin 123 homologue; d123; protein D123; HT-1080; CCEP 123; PZ 32; CEP 89; cell division cycle 123 homolog (saccharomyces cerevisiae); FLJ 14640; chromosome 10 open reading frame 7. Ids: HGNC: 16827; entrez Gene: 8872; ensembl: ENSG 00000151465; OMIM: 615470, respectively; UniProtKB: and O75794.

CD138 is also known as Syndecan 1 (syndecano 1) (SCD 1); CD 138; SDC; heparan sulfate protein polysaccharide fibroblast growth factor receptor; syndecan 1(Syndecan Proteoglycan 1); syndecans; SYND 1; syndecan-1; the CD138 antigen. Ids: HGNC: 10658; entrez Gene: 6382; ensembl: ENGG 00000115884; OMIM: 186355, respectively; UniProtKB: p18827.

CEA is also known as carcinoembryonic antigen-associated cell adhesion molecule 5(CEACAM 5); 100 parts of fetal feces antigen; CD66 e; carcinoembryonic antigen; CD66e antigen. Ids: HGNC: 1817; entrez Gene: 1048; ensembl: ENGG 00000105388; OMIM: 114890; UniProtKB: p06731.

EGFR is also known as epidermal growth factor receptor; erythroblastic leukemia virus (V-Erb-B) oncogene homolog (avian); ERBB 1; PIG 61; the proto-oncogene C-ErbB-1; avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog; receptor tyrosine protein kinase ErbB-1; cytostatic protein 40; cell proliferation-inducing protein 61; HER 1; a mENA; EC 2.7.10.1; EC 2.7.10; epidermal growth factor receptor (avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog). Ids: HGNC: 3236; entrez Gene: 1956; ensembl: ENSG 00000146648; OMIM: 131550, respectively; UniProtKB: p00533.

EGFRvIII is a common EGFR variant (oncogene.2013 May 23; 32 (21): 2670-81. doi: 10.1038/onc.2012.280.Epub 2012 Jul 16).

Delta-Like 3(Delta Like 3) (DLL3) is also referred to as Delta-Like 3(Delta-Like 3)); drosophila delta homolog 3; delta 3; delta (drosophila) -like 3; SCDO 1. Ids for DLL3 is: HGNC: 2909; entrez Gene: 10683; ensembl: ENGG 00000090932; OMIM: 602768 and UniProtKB: q9NYJ 7.

LGR5 is a G Protein-Coupled Receptor 5 Containing Leucine-Rich repeats (Leucine-Rich Repeat contacting G Protein-Coupled Receptor 5). The alternative name for this gene or protein is G protein-coupled receptor 5 containing leucine-rich repeats; g protein-coupled receptor 5 containing leucine rich repeats; g protein-coupled receptor HG 38; g protein-coupled receptor 49; a G protein-coupled receptor 67; GPR 67; GPR 49; orphan G Protein-Coupled Receptor HG38(Orphan G Protein-Coupled Receptor HG 38); g protein-coupled receptor 49; GPR 49; HG38 and FEX. The LGR 5-binding protein or antibody of the invention binds human LGR 5. Due to the similarity in sequence and tertiary structure between human and other mammalian xenologues (orthologs), LGR5 binding proteins or antibodies of the invention may also, but need not, bind to such xenologues. The human LGR5 protein and the gene encoding the protein have database accession numbers (NC-000012.12; NT-029419.13; NC-018923.2; NP-001264155.1; NP-001264156.1; NP-003658.1).

MSLN or Mesothelin (Mesothelin) also known as Mesothelin (Mesothelin); Pre-Megakaryocyte potentiator (Pre-Pro-Megakaryocyte-Potentiating Factor); the CAK1 antigen; MPF; soluble MPF mesothelin-related protein; megakaryocyte potentiator and SMRP. The Ids of MSLN is: HGNC: 7371; entrez Gene: 10232; ensembl: ENGG 00000102854; OMIM: 601051, respectively; UniProtKB: q13421.

Folate receptor 1 is also known as FOLR 1; a folate receptor 1; ovarian tumor associated antigen MOv 18; adult folate binding proteins; folate receptor, adult; KB cell FBP; FR-alpha; FOLR; FBP; folate binding protein; and folate receptor 1. The Ids of FOLR1 is HGNC: 3791; entrez Gene: 2348; ensembl: ENGG 00000110195; OMIM: 136430, respectively; UniProtKB: p15328.

Folate receptor 3 is also known as FOLR 3; folate receptor 3(γ); FR-gamma; a folate receptor 3; gamma-HFR; and FR-G. The Ids of FOLR3 is HGNC: 3795; entrez Gene: 2352, a step of processing the mixture; ensembl: ENGG 00000110203; OMIM: 602469, respectively; and UniProtKB: p41439.

EPCAM is also known as epithelial cell adhesion molecule; EGP 40; M4S 1; an ESA; MIC 18; KS 1/4; Tumor-Associated Calcium Signal Transducer 1(Tumor-Associated Calcium Signal Transducer 1); MK-1; tactd 1; human epithelial glycoprotein-2; TROP 1; membrane components, chromosome 4, surface marker (35kD glycoprotein); an adenocarcinoma-associated antigen; EGP; cell surface glycoprotein Trop-1; Ep-CAM; epithelial glycoprotein 314; GA 733-2; a major gastrointestinal tumor associated protein GA 733-2; M1S 2; EGP 314; a CD326 antigen; KSA; epithelial cell surface antigens; d IAR 5; epithelial glycoproteins; HNPCC 8; hEGP 314; the antigen identified by monoclonal antibody AUA 1; a KS1/4 antigen; EGP-2; ACSTD 1. Ids: HGNC: 11529; entrez Gene: 4072; ensembl: ENGG 00000119888; OMIM: 185535; UniProtKB: p16422.

HER2 is also known as V-Erb-B2 avian erythrocytic leukemia virus oncogene homolog 2; ERBB 2; CD 340; NGL; HER-2; HER-2/neu 2; NEU 2; TKR 1; neuro/glioblastoma derived oncogene homolog; C-Erb B2/Neu protein; metastatic lymph node gene 19 protein; herstatin (herstatin); the proto-oncogene C-ErbB-2; neuroblastoma/glioblastoma derived oncogene homolog; the proto-oncogene Neu; receptor tyrosine protein kinase ErbB-2; tyrosine kinase-type cell surface receptor HER 2; V-Erb-B2 erythroblastic leukemia virus oncogene homolog 2, neuro/glioblastoma derived oncogene homolog; MLN 19; MLN 19; p185erbB 2; a CD340 antigen; EC 2.7.10.1; EC 2.7.10; V-Erb-B2 avian erythroblastoid leukemia virus oncogene homolog 2 (neuro/glioblastoma derived oncogene homolog). Ids:

HGNC：3430；Entrez Gene：2064；Ensembl：ENSG00000141736；OMIM：164870；UniProtKB：P04626。

HM1.24 is also referred to as BST 2; bone marrow stromal cell antigen 2; TETHERIN; BST-2; bone marrow stromal antigen 2; HM1.24 antigen; contact protein (Tetherin); CD 317; a CD317 antigen; NPC-A-7. Ids: HGNC: 1119; entrez Gene: 684; ensembl: ENGG 00000130303; OMIM: 600534, respectively; UniProtKB: q10589.

MCSP is also known as Sperm mitochondrion-Associated Cysteine-Rich Protein (SMCP); MCSP; MCS; mitochondrial vesicular Selenoprotein (Mitochondrial Capsule selenin); HSMCSGEN 1; sperm mitochondria are related to protein rich in cysteine. Ids: HGNC: 6962; entrez Gene: 4184; ensembl: ENSG 00000163206; OMIM: 601148, respectively; UniProtKB: p49901.

PD-L1 is a type 1 transmembrane protein that plays a role in suppressing immune responses during specific events such as pregnancy, tissue allograft, autoimmune diseases and other disease states such as hepatitis. PD-L1 bound to PD-1 or B7.1(CD80) conveying inhibitory information that reduces the proliferation of PD-1 expressing T cells. It is believed that PD-1 is able to control the accumulation of foreign antigen-specific T cells through apoptosis. A number of cancer cells display PD-L1, and their display is believed to be at least partially responsible for slowing the immune response against the cancer cells. PD-L1 is a member of the B7 family of proteins and is well known under various other names, such as the CD274 molecule; a CD274 antigen; b7 homolog 1; PDCD1 ligand 1; PDCD1LG 1; PDCD1L 1; B7H 1; a PDL 1; programmed cell death 1 ligand 1; programmed death ligand 1; B7-H1; and B7-H. The external Ids of CD274 are HGNC: 17635; entrez Gene: 29126; ensembl: ENGG 00000120217; OMIM: 605402; UniProtKB: q9NZQ 7.

PSMA is also known as folate hydrolase (prostate specific membrane antigen) 1; FOLH 1; NAALAD 1; FOLH; mGCP; glutamic acid carboxypeptidase II; n-acetylated- α -linked acidic dipeptidase I; a PSM; NAALADase I; PSMA; EC 3.4.17.21; glutamic acid carboxylase II; GCP 2; cell growth suppressor gene 27 protein; NAALAdase; folylpoly (follypoly) -gamma-glutamic acid carboxypeptidase; glutamic acid carboxypeptidase 2; a membrane glutamic acid carboxypeptidase; n-acetylated α -linked acidic dipeptidase 1; pteroylpoly (Pteroylpoly) -gamma-glutamic acid carboxypeptidase; prostate specific membrane antigen variant F; FGCP; 1, folic acid hydrolase; GCPII; prostate specific membrane antigen. Ids: HGNC: 3788; entrez Gene: 2346; ensembl: ENSG 00000086205; OMIM: 600934; UniProtKB: and Q04609.

PSMA is not to be confused with the proteasome (precursor, macroprotein) subunit, alpha-type, 1, a alias of which is also known as PSMA 1.

Accession numbers are given primarily to provide additional methods of target identification, and the actual sequence of the bound protein may change, for example, due to mutations in the coding gene, such as occurs in some cancers and the like. Antigen binding sites bind to antigens and their various variants, such as those exhibited by some antigen-positive immune or tumor cells.

Where reference is made herein to a gene, protein, it preferably refers to the human form of the gene or protein. Where reference is made herein to a gene, protein, it is intended to refer to the native gene or protein as well as variant forms of the gene or protein as detectable in tumors, cancers and the like, preferably in human tumors, cancers and the like.

The bispecific or multispecific antibodies of the invention preferably bind to human BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, FOLR1, FOLR3, HER2, HM1.24, MCSP, PD-L1, PSMA protein, or a variant thereof. The antigen binding heavy/light chain combination preferably binds to the extracellular portion of the antigen. Bispecific antibodies according to the invention preferably bind to human CLEC12A or a variant thereof. Preferred bispecific antibodies according to the invention bind to human CD3 and human CLEC12A or a variant thereof. In preferred embodiments, the multispecific antibody binds to CD3, PD-L1, and EGFR.

HGNC stands for the HUGO gene naming committee. The abbreviated number is a registration number by which information on the gene and the protein encoded by the gene can be retrieved from the HGNC database. Entrez Gene provides an accession number or Gene ID by which information about the Gene or the protein encoded by the Gene can be retrieved from the NCBI (national center for Biotechnology information) database. Ensemble provides an accession number with which information about the gene or the protein encoded by the gene can be obtained from the Ensemble database. Ensembl is a common program between EMBL-EB and the Wellcome Trust Sanger Institute (Wellcome Trust Sanger Institute) to develop a software system that generates automated annotations to selected eukaryotic genomes and is maintained.

The present invention provides an antigen binding protein, preferably an antibody, that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3

The CDR1, CDR2 and CDR3 comprise the following amino acid sequences:

CDR1：SFGIS；CDR2：GFIPVLGTANYAQKFQG；CDR3：RGNWNPFDP

or

Comprising the following amino acid sequence:

CDR1：SX₁TFTIS；

CDR2：GIIPX₂FGTITYAQKFQG；

CDR3：RGNWNPFDP；

wherein

X₁K or R; x₂L or I.

In a preferred embodiment, X₁K; and X₂L. In another preferred embodiment, X₁R; and X₂＝I。

CDR 1: SKTLTIS; CDR 2: GIIPIFGSITYAQKFQD, respectively; CDR 3: RGNWNPFDP, respectively; or

Comprising the following amino acid sequence:

CDR1：GSGIS；CDR2：GFIPFFGSANYAQKFRD；CDR3：RGNWNPX₁₃DP；

wherein

X₁₃Or L or F.

The present invention further provides an antigen binding protein that binds human CD3, preferably an antibody, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：RX3WIG；CDR2：IIYPGDSDTRYSPSFQG；CDR3：X₄IRYFX₅WSEDYHYYX₆DV；

wherein

X₃F or Y; x₄H or N; x₅D or V; and X₆L or M.

In a preferred embodiment, X₃＝F；X₄＝H；X₅D; and X₆L; or X₃＝Y；X₄＝N；X₅V; and X₆＝M。

CDR1：SYALS；CDR2：GISGSGRTTWYADSVKG；CDR3：DGGYSYGPYWYFDL。

CDR1：SYALS；CDR2：AISGSGRTTWYADSVKG；CDR3：DGGYTYGPYWYFDL。

CDR1：DYTMH；CDR2：DISWSSGSIGYADSVKG；CDR3：DHRGYGDYEGGGFDY。

CDR1：DYTMH；

CDR2：DISWSX₇GX₈X₉X₁₀YADSVKG；

CDR3：DHX₁₁GYGDYEGGGFDX₁₂；

wherein

X₇(ii) S or G;

X₈(ii) S or T;

X₉i or T;

X₁₀g or Y;

X₁₁r or M;

X₁₂either of the two or more of (i) H or Y,

preferably X₇、X₈、X₉And X₁₀S, S, I and G or G, S, I and Y or S, T, T and G, and preferably X₁₁And X₁₂Is R and H, or R and Y, or M and Y, more preferably X₇、X₈、X₉、X₁₀、X₁₁And X₁₂S, S, I, G, R and H or G, S, I, Y, R and Y or S, T, T, G, M and Y or, in other words, X is preferred₇、X₈、X₉And X₁₀S, S, I and G, and X₁₁And X₁₂R and H; or X₇、X₈、X₉And X₁₀G, S, I and Y and X₁₁And X₁₂Are R and Y; or X₇、X₈、X₉And X₁₀Is S, T, T and G, and X₁₁And X₁₂M and Y.

In a preferred embodiment, the light chain variable region comprises the IgV κ 1-39 x 01 gene segment as depicted in fig. 11A, having an amino acid sequence of 0-10, preferably 0-5, amino acid variations, insertions, deletions, substitutions, additions or combinations thereof. IgV is depicted in FIG. 11A_K1-39 x 01. IgV kappa 1-39 is an abbreviation for the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39; IGKV 139; IGKV 1-39. The external Ids of the gene is HGNC: 5740; entrez Gene: 28930, respectively; ensembl: ENGG 00000242371. Preferred amino acid sequences for IgV κ 1-39 are provided in FIG. 11A. This gives the sequence of the V region. The V region can be combined with one of the five J regions. FIGS. 11B and 11D depict two preferred sequences of IgV κ 1-39 sequence combinations as a J region. The sequences of linkage are shown as IGKV1-39/jk1 and IGKV1-39/jk 5; the alternative names are IgV κ 1-39 × 01/IGJ κ 1 × 01 or IgV κ 1-39 × 01/IGJ κ 5 × 01 (named according to IMGT database web of IMGT.

IgV κ 1-39 × 01, which includes the light chain variable region, is preferred for the reproductive series of sequences. It is more preferred that the IGJkappa 1X 01 or/IGJkappa 5X 01 comprising the light chain variable region is a reproductive series sequence. In preferred embodiments, the light chain variable regions of IGKV1-39/jk1 or IGKV1-39/jk5 are germline sequences.

In a preferred embodiment the light chain variable region comprises the reproductive series IgV kappa 1-39 x 01. In preferred embodiments the light chain variable region comprises a kappa light chain IgV kappa 1-39X 01/IGJ kappa 1X 01 or IgV kappa 1-39X 01/IGJ kappa 5X 01. In a preferred embodiment IgV κ 1-39 × 01/IGJ κ 1 × 01. The light chain variable region preferably comprises the reproductive series kappa light chain IgV kappa 1-39X 01/IGJ kappa 1X 01 or the reproductive series kappa light chain IgV kappa 1-39X 01/IGJ kappa 5X 01, preferably the reproductive series IgV kappa 1-39X 01/IGJ kappa 1X 01.

Mature B cells producing antibodies with a light chain typically produce a light chain that has undergone one or more mutations associated with the reproductive-series sequences, i.e., the normal sequences of non-lymphoid cells of the organism. The process responsible for these mutations is commonly referred to as somatic (hyper) mutation. The resulting light chain is referred to as affinity matured light chain. These light chains, when derived from the reproductive series IgV kappa 1-39X 01 sequences, are IgV kappa 1-39X 01 derived light chains. In this specification, the term "IgV κ 1-39 x 01" will include IgV κ 1-39 x 01-derived light chains, and mutations introduced by somatic hypermutation may also be introduced manually in the laboratory. Other mutations or variations can also be introduced into the light chain in the laboratory without affecting the light chain's nature in kind, not necessarily in quantity. A light chain is anyway an IgV κ 1-39 x 01 light chain if it comprises a sequence as depicted in fig. 11A, fig. 11 or fig. 11 with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof. In preferred embodiments, the IgV κ 1-39 x 01 light chain is a light chain comprising a sequence as depicted in fig. 11A, 11B or 11C having 0-9, 0-8, 0-7, 0-6, 0-5, 0-4 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof. In preferred embodiments, the IgV κ 1-39 x 01 light chain is a light chain comprising a sequence as depicted in fig. 11A, 11B or 11C having 0-5, preferably 0-4, more preferably 0-3 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof. In preferred embodiments, the IgV κ 1-39 × 01 light chain is a light chain comprising a sequence as depicted in fig. 11A, 11B or 11C having 0-2, more preferably 0-1, and most preferably 0 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof. In preferred embodiments, the IgV κ 1-39 x 01 light chain is a light chain comprising a sequence as depicted in fig. 11A or fig. 11B with the mentioned amino acid variations, insertions, deletions, substitutions, additions or combinations thereof. In a preferred embodiment, the light chain comprises the sequence of fig. 11B.

The light chains preferably comprise a common light chain variable region. The common light chain variable region preferably comprises an IgV kappa 1-39 light chain variable region. The light chain variable region is preferably the reproductive series IgV kappa 1-39 x 01 variable region. The light chain variable region preferably comprises a kappa light chain IgV kappa 1-39X 01/IGJ kappa 1X 01 or IgV kappa 1-39X 01/IGJ kappa 5X 01. The light chain variable region preferably comprises the reproductive series kappa light chain IgV kappa 1-39X 01/IGJ kappa 1X 01 or IgV kappa 1-39X 01/IGJ kappa 5X 01. The light chain variable region preferably comprises amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK having 0-5 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof.

The light chain variable region preferably comprises the CDR1, CDR2 and CDR3 regions which comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTP, i.e.the CDRs of IGKV1-39 (according to IMGT). The amino acid variation, insertion, deletion, substitution, addition, or combination thereof is preferably not within the CDR3 region, preferably not within the CDR1 or CDR2 region, of the light chain variable region. In a preferred embodiment, the light chain variable region does not include deletions, additions or variations with respect to the indicated sequence. In this embodiment, the light chain variable region may have 0-5 amino acid substitutions with respect to the amino acid sequence shown. The amino acid substitution is preferably a conservative amino acid substitution. The CDR1, CDR2 and CDR3 of the light chain of the antibody of the invention preferably comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTP, respectively, i.e.the CDRs of IGKV1-39 (according to IMGT).

The antigen binding protein is preferably an antibody, preferably a bispecific or multispecific antibody. The antibody preferably comprises a common light chain comprising a common light variable region as defined herein and a light chain constant region as defined herein.

The antibody of the invention as described above is preferably a bispecific antibody. A "bispecific antibody" is an antibody as described herein in which one domain of the antibody binds to a first antigen and a second domain of the antibody binds to a second antigen, wherein the first and second antigens are not identical. The term "bispecific antibody" also includes antibodies in which a heavy chain variable region/light chain variable region (VH/VL) combination binds a first epitope on an antigen and a second VH/VL combination binds a second epitope. The term further includes antibodies wherein a VH is capable of specifically recognizing a first antigen and a VL paired with a VH of an immunoglobulin variable region is capable of specifically recognizing a second antigen. The resulting VH/VL pair will bind to antigen 1 or antigen 2. These so-called "two-in-one antibodies" are described, for example, in WO 2008/027236, WO2010/108127 and Schaefer et al (Cancer Cell 20, 472-. Bispecific antibodies as in the present invention are not limited to any particular bispecific format or method of production thereof.

The bispecific antibody preferably has a one heavy chain variable region/light chain variable region (VH/VL) combination that binds CD3 and a second VH/VL combination that binds an antigen other than that on CD 3. In a preferred embodiment, the antigen is a tumor antigen. In a preferred embodiment, the VL in the first VH/VL combination is similar to the VL in the second VH/VL combination. In a more preferred embodiment, the VLs in the first and second VH/VL combinations are identical. In preferred embodiments, the bispecific antibody is a full length antibody having a heavy/light (H/L) chain combination that binds CD3 and an H/L chain combination that binds another antigen, preferably a tumor antigen. In a preferred embodiment, the light chain of the first H/L chain combination is similar to the light chain of the second H/L chain combination. In a more preferred embodiment, the light chains of the first and second H/L chain combinations are identical, i.e. a similar or identical human light chain is a so-called "common light chain", which is a light chain of an antibody that can be combined with different heavy chains to form functional antigen binding domains. In preferred embodiments, the light chain in the first H/L chain combination includes a light chain variable region that is similar to the light chain variable region in the second H/L chain combination. In a more preferred embodiment, the light chain variable regions within the first and second H/L chain combinations are identical, i.e., a similar or identical human light chain variable region is a so-called "common light chain variable region", which is a light chain variable region of an antibody that can be combined with different heavy chain variable regions to form functional antigen binding domains. The light chains comprising a common light chain variable region are preferably a common light chain.

The common light chain in bispecific antibodies is preferably an IgV kappa 1-39 light chain as indicated above.

The invention also provides Alternative bispecific antibody formats, such as those described in Spiess, C.et al (Alternative molecular formats and therapeutic applications for bispecific antibodies. mol. immunol. (2015) http: dx. doi. org/10.1016/j. mollim. 2015.01.003). Bispecific antibody formats-not classical antibodies with two H/L combinations-have at least one variable domain of the invention comprising a heavy chain variable region and a light chain variable region. This variable domain can link a single chain Fv fragment, a monobody (monobody), a VH and a Fab fragment to provide a second binding activity.

In the context of bispecific antibodies of the present invention, the light chain within the H/L chain combination that CD3 binds to is preferably similar to the light chain within the H/V chain combination that binds to an antigen other than CD3, preferably a tumor antigen. In a more preferred embodiment, the light chains within two H/L chain combinations are identical, i.e. the human light chain is a so-called "common light chain", which is a light chain of an antibody that can be combined with different heavy chains to form a functional antigen-binding domain. Preferably, the common light chain has a reproductive series of sequences. The preferred reproductive series sequences are the light chain variable regions commonly used in the human repertoire (human recombinant) and have good thermodynamic stability, yield and solubility. Preferred reproductive series sequences are IgV κ 1-39, preferably rearranged reproductive series human κ light chain IgV κ 1-39 x 01/IGJ κ 1 x 01 or a fragment or functional equivalent thereof (i.e. identical IgV κ 1-39 gene segments but different IGJ κ gene segments) (named according to IMGT database web of IMGT.

The term "abnormal cells" as used herein includes tumor cells, more specifically blood-derived tumor cells, and also pre-leukemic cells such as those responsible for myelodysplastic syndrome (MDS) and leukemic cells such as Acute Myelogenous Leukemia (AML) tumor cells or Chronic Myelogenous Leukemia (CML) cells.

As used herein, the term "immune effector cell" or "effector cell" refers to a cell within the natural pool of cells within the mammalian immune system that can be activated to affect the viability of a target cell. Immune effector cells include lymphoid lineage cells such as Natural Killer (NK) cells, T cells including cytotoxic T cells, or B cells, and include myeloid lineage cells such as monocytes or macrophages, dendritic cells, and neutrophils. Thus, the effector cell is preferably an NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell or a neutrophil. Recruitment of effector cells to abnormal cells means bringing immune effector cells into proximity with abnormal target cells so that the effector cells can directly kill the abnormal cells or indirectly initiate killing of the abnormal cells.

As used herein, the terms "subject" and "patient" are used interchangeably and refer to a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc. (e.g., a patient with cancer, such as a human patient).

As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or procedure performed on a subject or administration of an active agent or combination of active agents to the subject with the purpose of reversing, alleviating, ameliorating, inhibiting, or slowing or preventing the progression, severity, or recurrence of the symptoms, complications, conditions, or biochemical indicators associated with a disease.

As used herein, "effective treatment" or "positive therapeutic response" refers to a treatment that produces a beneficial effect, such as, for example, alleviation of at least one symptom of a disease or disorder, such as cancer. Beneficial effects can be manifested in a manner that is improved over baseline, including improvements over measurements or observations made prior to initiation of therapy according to the method. For example, a beneficial effect can be manifested in a manner that slows, stabilizes, halts or reverses the progression of cancer in an individual at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease or cancer markers. An effective treatment may, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, slow or arrest tumor growth, and/or prevent or delay tumor recurrence or recurrence.

The term "therapeutic amount" refers to the amount of an agent or combination of agents that provides the desired biological, therapeutic, and/or prophylactic result. The result can be a reduction, amelioration, palliation, attenuation, delay, and/or alleviation of one or more signs, symptoms, or causes of disease, or any other desired alteration of a biological system. In some embodiments, the therapeutic amount is an amount sufficient to delay tumor development. In some embodiments, the therapeutic amount is an amount sufficient to prevent or delay tumor recurrence. The therapeutic amount can be administered in one or more administrations. The therapeutic amount of the drug or composition may be: (i) reducing the number of cancer cells; (ii) reducing the size of the tumor; (iii) inhibit, slow down and stop cancer cell infiltration into peripheral organs to a certain extent; (iv) inhibiting tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying tumorigenesis and/or recurrence; and/or (vii) alleviate one or more symptoms associated with cancer to some extent. In one example, a "therapeutic amount" is an amount of CLEC12A/CD3 bispecific antibody that reduces cancer (e.g., a reduction in the number of cancer cells) or slows progression of cancer, such as acute myelogenous leukemia, myelodysplastic syndrome, or chronic myelogenous leukemia.

The present invention also provides an antigen binding protein, preferably an antibody, that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence

Also provided is an antigen binding protein, preferably an antibody, that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence

The amino acid variation, insertion, deletion, substitution, addition or combination thereof is preferably not within the CDR3 region, preferably not within the CDR1 and/or CDR2 region of the heavy chain variable region. In a preferred embodiment, the heavy chain variable region does not include deletions, additions, or variations, insertions with respect to the indicated sequence. In one embodiment, the heavy chain variable region may have 0-10, preferably 0-5 amino acid substitutions with respect to the amino acid sequence shown. In a preferred embodiment, the heavy chain variable region comprises 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, preferably 0-3, preferably 0-2, preferably 0-1 and preferably 0 amino acid variations, insertions, deletions, substitutions, additions, or combinations thereof, for the amino acid sequences shown, at positions other than the CDRs. A combination of one insertion, addition, deletion or substitution is a claimed combination if the aligned sequences do not differ by more than 10 positions, preferably by more than 5 positions. A gap located within one of the aligned sequences would count as many amino acids as there are skips (skippeds) in the other sequence. An amino acid substitution, if any, is preferably a conservative amino acid substitution.

The present invention further provides an antigen binding protein, preferably an antibody, that binds to human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises MF8057 as depicted in fig. 13; MF8058 or MF 8078.

The present invention further provides an antigen binding protein, preferably an antibody, that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of MF8397 as depicted in figure 13.

The present invention further provides an antigen binding protein, preferably an antibody, that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence MF8508 as depicted in figure 13.

The present invention further provides an antigen binding protein, preferably an antibody, that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of MF9249 or MF9267 as depicted in figure 13.

The light chain preferably includes CDRs 1, CDR2 and CDR3 as defined elsewhere herein. Preferably comprising a common light chain variable region and preferably a common light chain as defined elsewhere herein. The bispecific antibody preferably further comprises a combination of a heavy chain and a light chain that binds to another antigen, preferably a tumor antigen. The light chains of the heavy and light chain combination that bind to another antigen are preferably a common light chain as defined elsewhere herein. The heavy chain of the heavy and light chain combination that binds to another antigen preferably comprises a heavy chain variable region comprising an amino acid sequence: MF8233(EGFR)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNANTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDYWGQGTLVTVSS, having 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof at one or more positions other than such CDRs; or MF4327(CLEC12A)

Variable domains that bind CLEC12A as defined herein, having a heavy chain variable region and a common light chain region, are described in WO2014/051433 and WO2017/010874 and the like, specifically mentioned for this purpose herein and incorporated herein by reference. The heavy chain variable region of the heavy/light chain combination that will bind to human EGFR or CLEC12A can have 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof with respect to the amino acid sequence shown. In a preferred embodiment, the heavy chain variable region comprises 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, preferably 0-3, preferably 0-2, preferably 0-1 and preferably 0 amino acid variations, insertions, deletions, substitutions, additions, or combinations thereof, with respect to the amino acid sequence shown. Let it be assumed that a combination of insertions, deletions, additions or substitutions is a claimed combination if the permuted sequences do not differ by more than 5 positions. A gap located in one of the aligned sequences would count as many amino acids as are skipped in the other sequence.

An amino acid variation, insertion, deletion, substitution, addition, or combination thereof is preferably not performed/present in the binding interface (interface) of the heavy and light chains.

Given that there is a change in the amino acid in the interface of the H/L chain interaction, it is preferred that the corresponding amino acid in the other chain also be changed to accommodate the change. Preferably, the insertion or addition of an amino acid does not require the insertion or addition of proline.

The addition of amino acids can in principle be considered as identical to the insertion. The addition of amino acids to one end of the polypeptide chain is sometimes not considered an insertion but a strict addition (elongation). For the purposes of the present invention, both incorporation into a strand or addition to a terminus are generally considered insertions.

The amino acid variation, insertion, deletion, substitution, addition, or combination thereof is preferably not within the CDR3 region, preferably not within the CDR1 or CDR2 region, of the heavy chain variable region. In a preferred embodiment, the heavy chain variable region does not include deletions, additions or variations with respect to the indicated sequence. In this embodiment, the heavy chain variable region may have 0-5 amino acid substitutions with respect to the amino acid sequence shown. The amino acid substitution is preferably a conservative amino acid substitution. The CD3 VH binding CDRs 1, CDR2 and CDR3 of the invention preferably comprise the combination of CD3 VH binding CDRs 1, CD2 and CDR3 depicted in fig. 13, preferably MF 8057; MF 8058; MF 8078; MF 8397; MF 8508; VH of one of MF9249 or MF 9267.

The constant region of the antibodies of the invention, including bispecific or multispecific antibodies, is preferably a human constant region. The constant region may contain one or more, preferably no more than 10, preferably no more than 5 amino acid differences from the constant region of a naturally occurring human antibody. The various variable regions of the antibodies produced herein are derived from a repertoire of human antibody variable domains. These variable domains are therefore human. The unique CDR regions may be derived from a human, synthetic, or derived from another organism. The antibody or bispecific antibody of the invention is preferably a human or humanized antibody. Suitable heavy chain constant regions are illustrated, without limitation, in FIG. 12.

There are a variety of methods for producing antibodies in the art. Antibodies are typically produced by cells expressing nucleic acids encoding the antibodies. Suitable antibody-producing cells are hybridoma cells, Chinese Hamster Ovary (CHO) cells, NS0 cells, or PER-C6 cells. In a particularly preferred embodiment, the cell is a CHO cell.

Various organizations and companies have developed cell lines for large-scale production of antibodies, such as for clinical use. Non-limiting examples of these cell lines are CHO cells, NS0 cells or per.c6 cells. These cells are also used for other purposes such as protein production. Cell lines developed for the production of proteins and antibodies on an industrial scale are further referred to herein as industrial cell lines. In a preferred embodiment, the present invention provides an industrial cell line producing the antibody of the present invention.

In one embodiment the invention provides a cell comprising an antibody according to the invention and/or a nucleic acid according to the invention. The cell is preferably an animal cell, more preferably a mammalian cell, more preferably a primate cell, most preferably a human cell. For the purposes of the present invention, a suitable cell is any cell which is capable of comprising and preferably producing an antibody according to the invention and/or a nucleic acid according to the invention.

The invention further provides a cell comprising an antibody according to the invention. Preferably the cell (typically an in vitro, isolated or recombinant cell) produces the antibody. In a preferred embodiment, the cell is a hybridoma cell, a Chinese Hamster Ovary (CHO) cell, NS0 cell, or PER-C6 cell. In a particularly preferred embodiment the cell is a CHO cell. Further provided is a cell culture comprising a cell according to the invention. Various organizations and companies have developed cell lines for large-scale production of antibodies, such as clinical use. Non-limiting examples of these cell lines are CHO cells, NS0 cells or per.c6 cells. These cells are also used for other purposes such as protein production. Cell lines developed for the production of proteins and antibodies on an industrial scale are further referred to herein as industrial cell lines. Thus in a preferred embodiment, the invention provides the use of a cell line developed for large scale production of antibodies, for use in the production of antibodies of the invention. The invention further provides a cell for the production of an antibody comprising a nucleic acid molecule encoding the VH, VL and/or heavy and light chains of an antibody as claimed. The nucleic acid molecule preferably encodes the VH as shown in figure 13, a nucleic acid molecule encoding the VH as shown by the numeral 4327 or by the numeral 8233 or a combination thereof.

The invention further provides a method for producing an antibody comprising culturing the cell of the invention and harvesting the antibody from the culture. The cells are preferably cultured in serum-free medium. The cells are preferably adapted for growth in suspension. Further provided is an antibody obtainable by a method for producing an antibody according to the invention. The antibody is preferably purified from the culture medium of the culture. The antibody ratio is preferably affinity purified.

The cells of the invention are for example hybridoma cell lines, CHO cells, 293F cells, NS0 cells or another cell type known to be suitable for antibody production for clinical purposes. In a particularly preferred embodiment the cell is a human cell. Preferably a cell transformed by the adenovirus E1 region or a functional equivalent thereof. C6 cell line or an equivalent thereof is a preferred example of such a cell line. In a particularly preferred embodiment the cell is a CHO cell or a variant thereof. Preferably a variant for expressing antibodies using the Glutaminase Synthase (GS) vector system.

Bispecific antibodies are also typically produced by cells expressing nucleic acids encoding the antibodies. In this case the cells exhibit different light and heavy chains which make up the bispecific antibody. For this purpose the cell displays two different heavy chains and at least one light chain. Since unmodified heavy chains can pair with each other to form dimers, these cells typically produce two monospecific antibodies (homodimers) in addition to bispecific antibodies (heterodimers). This principle also applies to unmodified heavy chains, which include a first heavy chain having a heavy chain variable region and a second heavy chain having at least two heavy chain variable regions, such that cells expressing these two heavy chains produce a monospecific antibody (two first heavy chain paired homodimers), tetravalent (quadravaent) antibody (two second heavy chain paired homodimers), and trispecific antibody (heterodimers of the first and second heavy chains). When the cell exhibits two or more light chains, the number of possible heavy/light chain combinations in the antibody produced increases. In order to reduce the number of different antibody species (different heavy and light chain combinations) it is preferred to produce the aforementioned "common light chain".

An antibody-producing cell that exhibits a common light chain and equal amounts of both heavy chains typically produces 50% bispecific antibody and 25% of each monospecific antibody (i.e., with identical heavy and light chain combinations). Optionally, in the above examples relating to a first heavy chain having one variable region and a second heavy chain having two variable regions, the two heavy chains typically produce 50% trispecificity, 25% monospecificity, and 25% tetraspecificity (quadrospecific).

Several approaches have been disclosed to facilitate the production of bispecific antibodies or conversely monospecific antibodies, which can be further used to facilitate multispecific antibody production. It is preferred in the present invention that the cell facilitates the production of the bispecific antibody over the corresponding monospecific antibody. This is usually achieved by modifying the constant region of the heavy chain such that it favours heterogeneous dimerization (i.e. dimerization of the heavy chain with another heavy/light chain combination) over homogeneous dimerization. In a preferred embodiment the bispecific antibody of the invention comprises two different immunoglobulin heavy chains with compatible heterogeneous dimeric domains. Various compatible heterogeneous binary aggregation domains have been described in the art. The compatible hetero-dimeric domain is preferably a compatible immunoglobulin heavy chain CH3 hetero-dimeric domain. Various methods are described in the art to accomplish the heterodimerization of these heavy chains, including the use of "knob-to-hole (knob-to-hole)" bispecific antibodies.

From US13/866, 747 (now published as US 9,248,181), US14/081,848 (now published as US 9,358,286) and PCT/NL2013/050294 (published as WO 2013/157954); incorporated herein by reference) discloses methods and means for producing bispecific antibodies using compatible hetero-polymeric dibodies. These components and methods may also be advantageously used in the present invention. In particular, preferred mutations which produce essentially only bispecific full length IgG molecules are the amino acid substitutions L351K and T366K (according to EU numbering) ("KK-variant" heavy chain) of the first CH3 domain and the amino acid substitutions L351D and L368E of the second domain ("DE-variant" heavy chain), or vice versa. It was previously demonstrated in our US 9,248,181 and US 9,358,286 patents and WO2013/157954 PCT application that DE-variants and KK-variants preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Due to the strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains, little homodimerization of the DE-variant heavy chains (DE homodimers) or KK-variant heavy chains (kkkkkk homodimers) occurs. In one embodiment, a heavy chain/light chain combination comprising a variable domain that binds CD3 includes KK variants of the heavy chain. Included in this embodiment are heavy chain/light chain combinations that bind variable domains of antigens other than CD3, including DE variants of the heavy chain. In a preferred embodiment, the antigen other than CD3 is CLEC 12A. In a preferred embodiment, the VH that will bind the variable domain of CLEC12A is MF4327 depicted in fig. 13.

Some antibodies are modified at the CH 2/lower hinge region, for example to reduce Fc receptor interaction or to reduce C1q binding. In some embodiments, the antibodies of the invention are IgG antibodies with a mutated CH2 and/or a lower hinge domain such that the bispecific IgG antibody has reduced interaction with Fc-gamma receptors. Such a mutant CH2 and/or lower hinge domain preferably comprises amino substitutions at positions 235 and/or 236 (according to EU numbering), preferably L235G and/or G236R substitutions.

Optionally, some antibodies are modified, for example, to promote Fc receptor interaction or to promote binding of C1 q. With respect to specific embodiments directed to autoimmune indications, such a modification may be preferred.

The invention further provides a method of treating a subject comprising administering to the subject in need thereof an antigen binding protein, preferably an antibody, of the invention. The invention further provides an antigen binding protein, preferably an antibody, of the invention for use in the treatment of a subject in need thereof. The individual preferably has cells to be removed from the body. Such cells may be abnormal immune cells or cancer cells, etc., which direct an autoimmune response. Variable domains suitable for this purpose are these and others having a common light chain and VH of MF9249 and MF 8397. These have suitably low affinity and low cell killing activity but are functional under autoimmune reaction conditions. Variable domains suitable for this purpose are these and others having a common light chain and VH of MF9267, MF8057, MF8058, MF8078 and MF 8508. These variable domains have appropriate affinity and appropriate cell killing activity for anti-cancer purposes.

Furthermore, an invention listed herein includes an antigen binding protein or antibody having high affinity variable domains with high cell killing activity, which can be administered locally or expressed locally, comprising the binding domain MF 8078. The invention further provides a method of treating a subject comprising administering an antigen binding protein of the invention, preferably an antibody, to the subject in need thereof by means of an administered localised member, comprising, for example, an oncolytic virus, as known to those skilled in the art, for local treatment of melanoma or other cancers in isolated compartments, such as the brain.

The invention further provides an antigen binding protein of the invention, preferably an antibody, for use in a subject in need of treatment, preferably with moderate to high affinity and relatively high cytotoxicity such as those described herein. Variable domains suitable for this purpose are these and others having a common light chain and VH of MF8057, MF8058, MF9267, MF8508, and MF 8078.

The invention further provides a method of treating cancer or a risk of cancer in an individual comprising administering to the individual in need thereof an antigen binding protein that binds human CD3, preferably an antibody comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：SFGIS

CDR2：GFIPVLGTANYAQKFQG

CDR 3: RGNWNPFDP, respectively; or

Comprising the following amino acid sequence:

CDR1：SX₁TFTIS；

CDR2：GIIPX₂FGTITYAQKFQG；

CDR3：RGNWNPFDP；

wherein

X₁K or R;

X₂l or I.

The invention further provides an antigen binding protein, preferably an antibody, of the invention for use in a subject in need of treatment, preferably with moderate to high affinity and relatively high cytotoxicity such as those described herein. Variable domains suitable for this purpose are these and others having a common light chain and VH of MF8048, MF8056 and MF 8101.

The present invention further provides a method of treating cancer or a risk of cancer in an individual comprising administering to the individual in need thereof an antigen binding protein that binds human CD3, preferably an antibody comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3 comprising the amino acid sequences:

CDR1：SKTLTIS；

CDR2：GIIPIFGSITYAQKFQD；

CDR 3: RGNWNPFDP, respectively; or

Comprising the following amino acid sequence:

CDR1：GSGIS；

CDR2：GFIPFFGSANYAQKFRD；

CDR3：RGNWNPX₁₃DP；

wherein

X₁₃Or L or F.

The invention further provides a method of treating cancer or a risk of cancer in an individual comprising administering to the individual in need thereof an antigen binding protein that binds human CD3, preferably an antibody comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence

CDR1：RX₃WIG；

CDR2：IIYPGDSDTRYSPSFQG；

CDR3：X₄IRYFX₅WSEDYHYYX₆DV；

wherein

X₃F or Y;

X₄h or N;

X₅d or V;

X₆l or M.

In one embodiment, X₃＝F；X₄＝H；X₅D; and X₆L. In another embodiment, X₃＝Y；X₄＝N；X₅V; and X₆＝M。

CDR1：SYALS；

CDR2：GISGSGRTTWYADSVKG；

CDR3：DGGYSYGPYWYFDL。

CDR1：SYALS；

CDR2：AISGSGRTTWYADSVKG；

CDR3：DGGYTYGPYWYFDL。

QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSAISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDGGYTYGPYWYFDLWGRGTLVTVSS, respectively; having 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof at one or more positions other than such CDRs.

CDR1：DYTMH；

CDR2：DISWSSGSIGYADSVKG；

CDR3：DHRGYGDYEGGGFDY。

CDR1：DYTMH；

CDR2：DISWSX₇GX₈X₉X₁₀YADSVKG；

CDR3：DHX₁₁GYGDYEGGGFDX₁₂；

wherein

X₇(ii) S or G;

X₈(ii) S or T;

X₉i or T;

X₁₀g or Y;

X₁₁r or M;

X₁₂(ii) either H or Y, or a combination thereof,

preferably X₇、X₈、X₉And X₁₀S, S, I and G or G, S, I and Y or S, T, T and G, and preferably X₁₁And X₁₂Is R and H, or R and Y, or M and Y, more preferably X₇、X₈、X₉、X₁₀、X₁₁And X₁₂S, S, I, G, R and H or G, S, I, Y, R and Y or S, T, T, G, M and Y or, in other words, X is preferred₇、X₈、X₉And X₁₀S, S, I and G, and X₁₁And X₁₂R and H; or X₇、X₈、X₉And X₁₀G, S, I and Y and X₁₁And X₁₂R and Y; or X₇、X₈、X₉And X₁₀Is S, T, T and G, and X₁₁And X₁₂M and Y.

The antigen binding protein in the treatment as described above is preferably an antibody, preferably comprising a heavy chain-light chain (H/L) combination that binds to a tumor antigen.

The antibody is preferably a human or humanized antibody. The antibody preferably comprises two different immunoglobulin heavy chains with compatible heterogeneous dimeric domains. The compatible hetero-dimeric domain is preferably a compatible immunoglobulin heavy chain CH3 hetero-dimeric domain. The bispecific antibody is preferably an IgG antibody with a mutated CH2 and/or lower hinge domain for tumor immunology applications such that the bispecific or multispecific IgG antibody has reduced interaction with Fc-gamma receptors. The mutant CH2 and/or lower hinge domain preferably comprises an amino substitution at position 235 and/or 236 (according to EU numbering), preferably a L235G and/or G236R substitution. Optionally, for autoimmune applications, the bispecific or multispecific interaction with Fc-gamma receptors is promoted by modifying the CH2 and/or CH3 domains. For example, by engineering (by introducing amino acid substitutions) Fc regions with greater selectivity for binding to activated receptors, antibodies can be created that target the binding arms of anti-cancer mabs or CD3 with the desired ability to have greater mediating cytotoxic activity for use in the treatment of autoimmune related diseases. For example, one reported technique for enhancing ADCC of antibodies is afucosylation (afucosylation). (see, e.g., Junttla, T.T., K.Parsons, et al (2010), "Superior In vivo Efficacy of oxygenated transfuzumab In the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70 (11): 4481-4489). Thus further provided are bispecific antibodies according to the invention which are afucosylated. Alternatively, or in addition, a variety of other strategies have been reported to achieve ADCC improvements, including, for example, glycoengineering (Kyowa Hakko/Biowa, glycart (roche) and Eureka Therapeutics) and mutagenesis (xenocor and macrogenetics), all seeking to improve Fc binding to low affinity activated Fc γ RIIIa and/or to reduce binding to low affinity inhibitory Fc γ RIIb.

The antibodies preferably comprise a common light chain.

The present invention further provides a bispecific antigen binding protein, preferably a bispecific antibody, comprising a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the variable domains each comprise a different heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds to the variable domain of human CD3 comprises a CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 comprising the amino acid sequences:

CDR1：SFGIS

CDR2：GFIPVLGTANYAQKFQG

CDR 3: RGNWNPFDP, respectively; or

Comprising the following amino acid sequence:

CDR1：SX₁TFTIS；

CDR2：GIIPX₂FGTITYAQKFQG；

CDR3：RGNWNPFDP；

wherein

X₁K or R;

X₂l or I.

CDR1：SKTLTIS；

CDR2：GIIPIFGSITYAQKFQD；

CDR 3: RGNWNPFDP, respectively; or

Comprising the following amino acid sequence:

CDR1：GSGIS；

CDR2：GFIPFFGSANYAQKFRD；

CDR3：RGNWNPX₁₃DP；

wherein

X₁₃Or L or F.

The present invention further provides a bispecific antigen binding protein, preferably a bispecific antibody, comprising a variable domain that binds to a tumor antigen and a variable domain that binds to human CD3, wherein the variable domains each comprise a different heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region of the variable domain that binds to human CD3 comprises the amino acid sequence

CDR1：RX₃WIG；

CDR2：IIYPGDSDTRYSPSFQG；

CDR3：X₄IRYFX₅WSEDYHYYX₆DV；

wherein

X₃F or Y;

X₄h or N;

X₅d or V;

X₆l or M.

CDR1：SYALS；

CDR2：GISGSGRTFWYADSVKG；

CDR3：DGGYSYGPYWYFDL。

the present invention further provides a bispecific antigen binding protein, preferably a bispecific antibody, comprising a variable domain that binds tumor antigen and a variable domain that binds human CD3, the variable domain that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：SYALS；

CDR2：AISGSGRTTWYADSVKG；

CDR3：DGGYTYGPYWYFDL。

CDR1：DYTMH；

CDR2：DISWSSGSIGYADSVKG；

CDR3：DHRGYGDYEGGGFDY。

CDR1：DYTMH；

CDR2：DISWSX₇GX₈X₉X₁₀YADSVKG；

CDR3：DHX₁₁GYGDYEGGGFDX₁₂；

wherein

X₇(ii) S or G;

X₈(ii) S or T;

X₉i or T;

X₁₀g or Y;

X₁₁r or M;

X₁₂(ii) either H or Y, or a combination thereof,

In one embodiment, the heavy chain variable region of the variable domain that binds to a tumor antigen preferably comprises MF8233(EGFR)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNANTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDYWGQGTLVTVSS amino acid sequence

From 0 to 10, preferably from 0 to 5, amino acid variations, insertions, deletions, substitutions, additions or combinations thereof at one or more positions other than the CDRs.

In another embodiment, the heavy chain variable region that binds to a variable domain of a tumor antigen preferably comprises

Amino acid sequence of MF4327(CLEC12A)

The invention further provides an antibody of the invention or a derivative thereof or a pharmaceutical composition of the invention for use in the treatment of a subject in need thereof. For treating an individual having a tumor or at risk of having a tumor, it is preferred that the antibody is a bispecific antibody of the invention. Preferably wherein the CD3 binding antibody comprises a heavy/light chain combination that binds to a tumor antigen.

CD 3/tumor antigen bispecific antibodies and pharmaceutical compositions comprising these bispecific antibodies are provided for use in the treatment of solid or hematological tumors. Preferred solid tumors are of epithelial origin; gynecological cancers such as ovarian cancer and intimal cancer; prostate cancer, brain cancer and any other solid tumor.

Also provided are CD 3/tumor antigen bispecific antibodies or derivatives thereof of the invention or pharmaceutical compositions comprising these bispecific antibodies or derivatives thereof for use in the treatment of various myeloid-derived leukemias and pre-leukemic diseases but additionally B-cell lymphomas. Diseases that can be treated according to the invention include myeloid leukemia or pre-leukemic diseases such as, for example, Acute Myeloid Leukemia (AML), myelodysplastic syndrome (MDS) and Chronic Myeloid Leukemia (CML), and Hodgkin's (Hodgkin) lymphoma and most non-Hodgkin's lymphoma. And B-ALL; T-ALL, adventitial cell lymphoma, is also a preferred therapeutic target for the antibodies of the invention. Accordingly, the present invention provides a bispecific full length IgG antibody according to the invention for use as an agent for the treatment of myelodysplastic syndrome (MDS), Chronic Myelogenous Leukemia (CML), Multiple Myeloma (MM) or preferably Acute Myelogenous Leukemia (AML). Also provided is the use of a bispecific IgG antibody according to the invention for the preparation of a medicament for the treatment or prevention of MDS, CML, MM or preferably AML.

The amount of antibody according to the invention to be administered to a patient typically falls within the therapeutic range (therapeutic window), meaning that an amount sufficient to obtain a therapeutic effect is used, while not exceeding a threshold that would result in an unacceptable degree of side effects. The lower the amount of antibody required to achieve the desired therapeutic effect, the greater the therapeutic range will typically be. Thus, it is preferred that the antibody of the present invention exerts sufficient therapeutic effect at a low dose.

Approximately 30.000 patients are diagnosed with AML each year in europe and the united states. Most of these patients are over 60 years old. Older age is the major negative determinant of AML outcome and the long-term (five-year) survival of critically aged AML patients is probably 10%. Disease progression was observed within 3 years for almost all patients who had remitted after induced chemotherapy. Current post-remission treatments show limited, if any, value in elderly patients with AML. Thus, the burden of residual resistant leukemia remains large and the surviving drug resistant leukemia cell sub-population rapidly recurs. These chemotherapy-unresponsive AML tumor cells require new drugs with completely different modes of action in order to induce and support complete remission. Even though Complete Remission (CR) can be achieved in more than 50% of elderly AML patients and approximately 80% of younger patients with many aggressive chemotherapeutic combinations, the progression of response and survival remains a significant research challenge. In a recent published network-integrated analysis of 65 randomized clinical trials in older patients with AML (15.110 patients), most of the modified study induced regimens had similar or even worse efficacy resolution than the traditional 3+7 induced regimens with daunorubicin (daunorubicin) and cytarabine (cytarabine). This standard of AML treatment is associated with high morbidity and even mortality. Most patients with CR recurrence are due to leukemic stem cells remaining after chemotherapy. Further dose escalation is limited due to unacceptable toxicity. The urgent need to prefer new less toxic treatment modalities has therefore emerged, especially in elderly patients with AML.

Treatment of AML in which chemotherapy does not respond can be achieved by using bispecific antibodies to recruit T cells from the patient's own immune system to the AML tumor cells and subsequently activate tumor-specific T cells. This method is also referred to as "T-cell engagement (T-cell engaging) method". In this way, the patient's immune system is strengthened and retargeted to attack and eradicate AML tumor cells. For example, a CD3xCLEC12A bispecific IgG antibody effectively reverts to T cells to AML tumor cells, thereby inducing AML tumor cell lysis.

Examples

Cell line

bxPC3 human pancreatic cancer cell line.

HCT-116 human colon cancer cell line.

Immunization with CD3

Mouse

To generate human antibodies that bind CD3, mice genetically transformed for the human common light chain and a human Heavy Chain (HC) minilocus (minimus) including a selected population of human V gene segments, all human Ds, and all human Js (see W02009/157771, incorporated herein by reference) were used with TCR/CD 3-containing antibodiesLipid globules (TCR/CD3 stabilizing lipids) (Intergral Molecular) were immunized. These mice are called

A mouse. Specific heavy chain variable regions, or trivalent multimers having the sequences disclosed herein, can be generated by methods known to those of ordinary skill in the art.

Mice were immunized with lipid globules containing Hek 293T-derived human 5D5M TCR/CD3 followed by human T cells to generate an anti-TCR/CDR 3 immune response and a panel of anti-TCR/CD 3 antibodies.

Lipid globules concentrate (concentrate) topographically intact membrane proteins directly from the cell surface, allowing these complex proteins to be manipulated (manipulated) as soluble high-concentration proteins for antibody immunization and screening.

The lipid globules used for immunization in this study contained a 5D5M TCR α β combination. Vectors containing the 5D5M TCR α β combination were synthesized, transformed and used to generate lipid globules containing this TCR/CD3 combination by transient transfection into HEK293T cells (intersgral Molecular).

5D5M TCRα

5D5M TCRβ

Mice were used for immunization using TCR/CD3 lipid globules and primary human T cells.

The immunization schedule (immunization schedule) included time points (points) at days 35, 56, 77 and 98, at which antigen-specific Ig serum titers were determined by ELISA using QTG-derived 3SDX TCR/CD3 positive and negative lipid globules detected with anti-mouse IgG and by ELISA using CD3c5E-Fc fusion protein as a positive control. The reactivity observed in sera drawn at day 35 will determine which mice developed the relevant anti-TCR/CD 3 response.

For all immunized mice, lymphoid material was collected and stored for antibody exploration when there were the following:

a titer of 1/300 for human TCR/CD3 (in ELISA using lipid globules); or:

the titer for human TCR/CD3 was < 1/300 and > 1/100 and did not increase during the last booster immunization (boost immunization) period.

Activated immunization with lipid globules (Priming immunization)

To activate (prime)

Humoral immune response of mice to TCR/CD3, lipospheres containing a combination of human 5D5M TCR α β were used for immunization. Lipid globules were used with Gerbu adjuvant for the first and second injections.

Enhanced injection immunization using multiple T cell lines

Mice were immunized by subcutaneous injection of a cell suspension. The first booster immunization (day 28) included a mixture of cells and adjuvant in PBS and all subsequent injections consisted of cells only in PBS. Mice that had developed a serum IgG titer of 1/300 (as determined by ELISA using lipid globules) to human TCR/CD3 at day 35 received additional injections with cells on days 42, 43, and 44. Mice that did not meet these criteria received booster injections using cells (day 42 and day 49). All subsequent immunizations were given as subcutaneous injections of cells in PBS. After the final immunization, mice were sacrificed, sera were bled and spleen and left inguinal lymph node were collected.

Screening of sera from immunized mice by ELISA

Serum IgG titers in phase (intercerim) were screened by ELISA using TCR/CD3 containing lipid globules and empty (null) lipid globules. Anti-mouse IgG staining was used to determine serum IgG titers, as this staining proved to be the most sensitive.

Generation of "immune" phage antibody libraries by RT-PCR transfer of VH genes

The inguinal lymph node from a successfully immunized mouse was used to construct an "immune" phage antibody library. Trizol LS was used to extract RNA from lymphoid tissues and 1. mu.g of total RNA was used in an RT reaction with an IgG-CH1 specific primer. The resulting cDNA was then used to amplify a polyclonal pool (polyclonal pool) of VH-encoding cDNAs using internally developed VH-specific primers, essentially as described in Marks et al (J Mol biol.1991 Dec 5; 222 (3): 581-97) B. The resulting PCR product was then cloned into a phagemid (phagemid) vector for displaying Fab fragments on phage as described in Haard et al (J Biol chem.1999 Jun 25; 274 (26): 18218-30) B, with the exception that the light chain was identical for each antibody and was encoded by the vector. After connection, such granulocyte macrophage used to transform Escherichia coli TG1 bacteria and transformed bacteria were plate culture in the presence of ampicillin and glucose LB-agar plate. All phage pools contained > 10e6 transformants and had an insert frequency of > 80% (insert frequency). The bacteria were harvested after overnight growth and used to prepare phage according to established procedures (de Haard et al, Jbiol chem.1999 Jun 25; 274 (26): 18218-30).

Phage carrying Fab fragments that specifically bind to human CD3 were selected.

Phage libraries were rescued (rescued) (J Mol biol.1991 Dec 5; 222 (3): 581-97; J Biol chem.1999 Jun 25; 274 (26): 18218-30) according to standard procedures and phages were selected with one or more rounds of selection of immune phage antibody libraries. In round 1, recombinant CD3 protein was coated on maxisorp^TMELISA plate wells or NUNC immune tubes, and round 2, cells overexpressing human CD3 protein or recombinant CD3 protein were used. Blocking maxisorp with 4% ELK^TMELISA plates or immune tubes. Phage antibody pools were also blocked with 4% ELK and also blocked with excess human IgG to deplete Fc region binders (binders) before phage pools were added to the coated antigen.

Incubation of the phage library with the coated proteins was performed for 2hrs at room temperature under shaking conditions. The plate or tube was then washed with 0.05% Tween-20 (Tween-20) in PBS, followed by 5 to 10 washes with PBS. The bound phage were eluted using 50mM glycine (pH2.2) and added to E.coli TG-1 and incubated at 37 ℃ for phage infection.

Subsequently, the infected bacteria were plated onto agar plates containing ampicillin and glucose and incubated overnight at 37 ℃. After round 1 selection, colonies were scraped from the plates and pooled and then rescued and amplified to prepare enriched round 1 phage pools (pool) for use in synthetic pools (reteroaire). For the immune repertoire, single colonies were screened for target binding after round 1 phage selection.

Antibody cloning and production

Bispecific antibodies as used herein differ from each other usually only in the specific amino acid sequence of the heavy chain variable region of one or both variable domains. Antibody production is used to express heavy and light chains by cloning the heavy chain variable region into an expression vector. Methods for producing bispecific antibodies are known in the art.

Briefly, DNA encoding the heavy chain variable region of a CD 3-targeted variable domain was cloned into MV1624 vector (see fig. 10A) encoding KK residues (L351K, T366K) in the CH3 region for use in the production of IgG heavy chain heterodimers (WO2013/157954 and WO 2013/157953). The Fc constant region contains a mutation within CH2 to silence Fc effector function. The DNA encoding the heavy chain variable region replaces the stuffer (stuffer) region within the construct. The variable region is preceded by an encoded HC signal peptide (not shown). DNA encoding the heavy chain variable region of the EGFR-targeted variable domain was cloned into MV1625 vector (fig. 10B) which represents the second heavy chain of the bispecific antibody-based antibody portion with L351D-L368E mutations in the CH3 region (WO2013/157954 and WO 2013/157953). A filling region in the DNA substitute construct encoding the heavy chain variable region. The variable region is preceded by an encoded HC signal peptide (not shown). Both constructs also contained an expression cassette for expression of IGKV1-39/jk1 light chain. The simultaneous expression of the double chain and the mentioned light chain leads to the production of bispecific antibodies.

293-F cells were used for expression of the designed antibodies in a 24-well plate format. Two days prior to transfection, the 293-F cell stock was separated in 293-F medium at a ratio of 1: 1 (split) and at 37 ℃ and 8% CO at a rotary shaking speed (orbital shaking speed) of 155rpm₂Incubate overnight. Cells were diluted to 5x10 the day before transfection⁵Density of individual cells/mL. 4mL of suspension cells were seeded (seed) into 24 deep well culture plates, covered with a gas permeable seal and shaken in a rotary shaker at 285rpm at 37 ℃ and 8% CO₂Incubate overnight. On the day of transfection, 4.8mL of 293-F medium was mixed with 240. mu.g of linear Polyethyleneimine (PEI) (MW 25,000). For each IgG to be produced, 200. mu.L of 293F medium-PEI mixture was supplemented with 8. mu.L of DNA (4. mu.L of DNA encoding each heavy chain for heterodimers). The mixture was incubated at room temperature for 20 minutes before being gently added to the cells. On the day after transfection, penicillin-streptomycin (Pen Strep) diluted in 500 μ L of 293F medium was added to each well. Such a flat disc was set at 37 ℃ and 8% CO at a rotary oscillation speed of 285rpm₂Incubations were continued until 7 days after transfection to harvest. The plates were centrifuged at 500g for 5min and the IgGs-containing supernatant was applied to 10-12 μm melt-blown polypropylene filter plates (melt blown polypropyle)ne filter plates) were filtered and stored at-20 ℃ before purification.

Purification of antibodies from culture supernatants

The antibody-containing medium was harvested and centrifuged to remove cell debris. Protein a Sepharose beads were then added to the medium. The medium and protein a Sepharose beads were incubated with antibodies to allow binding.

After incubation, the beads were isolated from the medium by passing through a vacuum filter and washed. Such antibodies are eluted from the beads by incubation with elution buffer.

Optionally, the buffer of purified IgG is exchanged/desalted (desalted).

Buffer exchange

To desalt the purified antibody, the antibody fraction (fraction) is centrifuged using a filter plate or column (filter column). The filter plate or column is centrifuged to reduce the volume of the antibody fraction. Subsequently, PBS or the required buffer is added to the fraction to replace the buffer with a low salt buffer. Optionally, this centrifugation step is repeated followed by a buffer addition step to further desalt the antibody storage buffer.

Antibody tumor antigen-specific T cell activation and lysis of BxPC3 cells or HTC-116 cells.

Specific CD3x tumor antigen bispecific IgG combinations induced tumor antigen specific T cell activation and lysis of tumor antigen positive tumor cells were tested in a cytotoxicity assay. The effector cells were healthy donor-derived resting T cells and the target cells were BxPC3 cells or HTC-116 cells.

Dormant T cells were isolated from whole blood from healthy donors according to standard techniques using Ficoll and EasySep human T cell isolation kits, > 95% T cell purity was checked by anti-CD 3 antibody using flow cytometry analysis and subsequently cryopreserved. For cytotoxicity assays, cryopreserved T cells were thawed and used if their viability, as determined by standard trypan blue staining, was > 90% at the time of thawing. Cytotoxicity assay briefly, thawed resting T cells and BxPC3 or HCT116 target cells were co-cultured at an E: T ratio of 5: 1 for 48 hours. Antibodies were tested at a dilution range. A CD3 monospecific antibody and an EGFR monospecific antibody, as well as an unrelated IgG1 isotype control mAb are included in the assay as controls (e.g., an antibody that binds CD3 and another antigen such as Tetanus Toxoid (TT)). T cell activation was quantified using flow cytometry; CD8T cells were selected (gated) according to CD8 expression and subsequently analyzed for their activation status by measuring CD69 expression on T cells. Target cell lysis was determined by measuring the fraction of viable cells (fraction), which was measured by ATP levels assessed by celltiterglo (promega). ATP levels, measured by luminescence on an Envision microplate reader, were Relative Light Unit (RLU) values, which were analyzed using GraphPad Prism.

The target cell lysis for each sample was calculated as follows:

kill ═ 100 (RLU sample/RLU no IgG) × 100).

In this assay, the bispecific antibody has two binding domains. One targeting EGFR and the other targeting CD 3. Both binding domains have the same (common) light chain variable region (VL) and a different heavy chain variable region (VH). The EGFR-targeting binding domain has a VH with the amino acid sequence MF 8233. The CD 3-targeting binding domain has a VH with an amino acid sequence directed to one of the indicated MFs of CD 3. The bispecific antibody contains a mutation in CH2 to disrupt Fc effector function.

The antibody MF8233xMF8397 induced up-regulation of CD69(FIG 4-6) and CD25(FIG 4-6) on CD4 and CD8T cells, which were judged after 48 hours of co-culture at an E + -T ratio of 5: 1. T cell mediator lysis was measured after 48 hours.

CD3 bispecific antibody characterization

The binding of a candidate EGFR/CD3 IgG bispecific antibody can be tested using any suitable assay. For example, CD3 binding to membrane expression on HPB-ALL cells (DSMZ, ACC 483) can be assessed by mobilization cytometry (according to FACS program as described previously in WO 2014/051433). In one embodiment, binding of a candidate EGFR/CD3 bispecific antibody to CD3 on HPB ALL cells was confirmed by flow cytometry, performed according to standard procedures known in the art. Binding to cell-expressed CD3 can be determined using CHO cells transfected with CD 3. delta./epsilon. or CD 3. gamma./epsilon. Binding of the candidate bispecific IgG1 to EGFR can be determined using BxPC3 and HCT-116 transfected EGFR expression constructs and CHO cells; a CD3 monospecific antibody and an EGFR monospecific antibody, as well as an unrelated IgG1 isotype control mAb are included in the assay as controls (e.g., an antibody that binds CD3 and another antigen such as Tetanus Toxoid (TT)).

Generation of additional clones from

superpopulations

1, 3 and 4

From the immunophage library screen (as described in section "select phage carrying Fab fragment that specifically binds human CD 3"), additional clones carrying Fab fragments that specifically bind human CD3 were characterized. Additional clones were identified from supergroup 1, including MF8048, MF8101, and MF 8056. Additional clones were identified from supergroup 3 and supergroup 4, including MF8562 for supergroup 3 and MF8998 for supergroup 4.

Additional new clones were identified from supergroup 4 using Next Generation Sequencer (NGS) analysis. NGS is against the group from which anti-CD 3 was generated

A mouse-presented VH gene pool (pool) was performed. For this purpose, sequence datasets obtained from different mice were compared with MF sequences belonging to supergroup 4 MF. This resulted in the identification of clones MF10401 and MF10428, which belong to supergroup 4 sequence variants. Several different mutations were found within HCDR1 and HCDR2 of different sequences.

All VH sequences from additional clones from

superpopulations

1, 3 and 4 were cloned into MV1624(DM-KK) vector and presented in CD3xEGFR bispecific format for further characterization as described in the "antibody cloning and production" section above.

Characterization of additional clones from

supergroups

1 and 4

Additional clones from supergroup 1 were characterized for their functional activity in a bispecific format. The EGFR-binding arm of the bispecific CD3xEGFR antibody has the amino acid sequence encoded by MF 8233. As a control, these CD3 clones were also tested with another antigen of the amino acid sequence encoded by MF1337 (e.g., tetanus toxoid). Reference MFs from supergroup 1(MF 8057 and MF8058) are included to follow, as above: the direct comparison of the affinity of sequence variants with the affinity from MF clones already characterized by supergroup 1 was described in section "activation of antibody tumor antigen specific T cells and lysis of BxPC3 cells or HTC-116 cells" and "characterization of CD3 bispecific antibodies". Binding affinity of HPB-ALL cells expressing the human CD3-TCR complex was performed using portable cytometry (fig. 14A and 18) and T cell activation and lysis of tumor antigen positive tumor cells (HCT-116) was performed with cytotoxicity assays (fig. 14B-E). No target cell lysis was observed with the bispecific antibody with MF1337 control arm. Target cell lysis was observed in a dose-dependent manner for the different CD3 clones tested. MF8048 observed low target cell lysis. Activation on CD4 and CD8T cells identifies CD69 and CD25 expression levels measured with FACS staining for assessment of T cell activation. Dose-dependent T cell activation was observed for all clones, and no T cell activation was observed for the negative control. Finally, use after 48hrs

Assays(eBiosciences^TM) IFN-. gamma.and TNF-. alpha.cytokine production in the derived supernatants was determined from EGFRxCD3 cytotoxicity assays with HCT-116 cells following the standard manufacturer's instructions (FIGS. 14F-G).

Binding affinity to HPB-ALL cells was determined by FACS for characterization of additional clones belonging to supergroup 4 (fig. 15). PG1337, a monovalent antibody with two identical MF1337 arms specific for tetanus toxoid, was used as a negative control. For cytotoxicity assays, use is made ofHCT-116 cells and BxPC3 cells were used as target cells to test the activity of MF8998 and BxPC3 target cells were used to test the activity of MF10401 and MF 10428. A positive control was included containing a bispecific antibody against CD3xTAA with known high activity. Target cell lysis was quantified using cell viability measurements. Using supernatant from cytotoxicity assay with

Assays to measure the interleukin levels of IL-6, IFN-gamma and TNF-alpha.

It was thus found that the three supergroup 4 clones tested exhibited different binding but similar lytic activity. Even though the lytic activities of these clones were similar, reduced cytokine production was observed.

TABLE 1

MF combination	AUC
		MF8233xMF8998	13630
MF8233xMF10428	2700
		MF8233xMF10401	9646
MF1337xMF1337	255

Table 1: FIG. 15A refers to the AUC values for binding of the indicated CD3 clones.

TABLE 2

MF combination	Target	AUC	EC50(ng/mL)
				MF8233xMF8998	EGFR	253	6.9
MF8233xMF10428	EGFR	216	10.7
				MF8233xMF10401	EGFR		241	4.6
CD3x false: -ctrl	False	11	-
				CD3xTAA：+ctrl	TAA	369	4.5

Table 2: the AUC and EC50 values for the target cell lysis of the indicated CD3 clone referenced in fig. 15A.

TABLE 3

MF combination	Interleukin	AUC	EC50(ng/mL)
				MF8233xMF8998	IL-6	858	51.2
MF8233xMF10428	IL-6	659	8.6
				MF8233xMF10401	IL-6	657	3.1
MF8233xMF8998	IFNγ	311	＞400
				MF8233xMF10428	IFNγ	107	＞4000
MF8233xMF10401	IFNγ	114	＞4000
				MF8233xMF8998	TNFα	50	-
MF8233xMF10428	TNFα	50	-
				MF8233xMF10401	TNFα	50	-

Table 3: the indicated amounts of the interleukins and the AUC and EC50 values for the CD3 clones referred to in FIG. 15A.

All additionally identified MFs were observed to be functional as assessed by cytotoxicity assays. Next, a plot is plotted (fig. 16) between dissolution on the Y-axis and binding affinity on the X-axis to understand the relationship between dissolution and affinity within and across the supergroup. Overall, a diverse group of anti-CD 3 Fabs was generated that consisted of diverse superpopulations and covered a range of affinities. Interestingly, superpopulations 1 and 4 resulted in clones that exhibited similar activity but different CD3 binding (fig. 16B). Comparison of

superpopulations

1 and 3 revealed clones exhibiting similar CD3 binding and differential activity (fig. 16C).

TABLE 4

MF combination	AUC	EC50(ng/mL)
			MF8233xMF8057	117	28.5
MF8233xMF8058	283	4.4
			MF8233xMF8101	355	1.0
MF8233xMF8508	267	6.4
			MF8233xMF8998	232	8.0
MF8233xMF8397	62	＞400
			MF8233xMF8562	57	＞400

Table 4: the AUC and EC50 values for the target cell lysis of the indicated CD3 clone are presented in fig. 16B and 16C.

CD3 antigen characterization

As described above, it was found that two clones from supergroup 1 and supergroup 4, MF8058 and MF8998, respectively, had similar lytic activity in case of bispecific format together with clone MF8233, which is the Fab arm binding to EGFR as tumor cell antigen (fig. 17). For this experiment, lytic activity on HCT-116 cells was measured as described above. MF9257xMF8233 was used as positive control and MF9257xMF1337 was used as negative control. As can be seen in figure 17 and table 5, dose dependence and high percent kill were observed for the various bispecific antibodies tested.

TABLE 5

Table 5: the AUC and EC50 values for target cell lysis for the indicated bispecific antibodies mentioned in figure 17. The negative control showed no significant measurable activity.

Binding affinity

The additional CD3 clone was analyzed by FACS for binding affinity to HPB-ALL cells expressing human CD3, as described in the section "CD 3 bispecific antibody characterization". Affinity of MF6955 and MF6964 for CD3 by Surface Plasmon Resonance (SPR) technique using BIAcore^TMT100. Anti-human IgG mouse monoclonal antibody (Becton and Dickinson, cat. Nr.555784) was coupled to the surface of a CM5 sensor chip using free amine chemistry (NHS/EDC). Followed by capture of the CD3xTAA bispecific antibody on this sensor surface. A range of concentrations of the recombinant purified antigen human CD3 δ epsilon-Fc protein was subsequently mobilized on the sensor surface to measure association and dissociation rates. After each cycle, the sensor surface was regenerated by HCl pulses and the CD3xTAA bispecific antibody was captured again. From the resulting sensorgrams, association and dissociation rates were determined using BIAevaluation software. Fig. 18 illustrates the binding affinity range of the resulting CD3 group.

Claims

1. An antigen binding protein that binds human CD3, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：SFGIS

CDR2：GFIPVLGTANYAQKFQG

CDR 3: RGNWNPFDP, respectively; or

Comprising the following amino acid sequence:

CDR1：SX₁TFTIS；

CDR2：GIIPX₂FGTITYAQKFQG；

CDR3：RGNWNPFDP；

wherein

X₁K or R;

X₂l or I.

2. The antigen binding protein of claim 1, wherein

X₁K; and X₂＝L；

X₁R; and X₂＝I。

3. An antigen binding protein that binds human CD3, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：SKTLTIS；

CDR2：GIIPIFGSITYAQKFQD；

CDR 3: RGNWNPFDP, respectively; or

Comprising the following amino acid sequence:

CDR1：GSGIS；

CDR2：GFIPFFGSANYAQKFRD；

CDR3：RGNWNPX₁₃DP；

wherein

X₁₃Or L or F.

4. An antigen binding protein that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence

5. An antigen binding protein that binds human CD3, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：RX₃WIG；

CDR2：IIYPGDSDTRYSPSFQG；

CDR3：X₄IRYFX₅WSEDYHYYX₆DV；

wherein

X₃F or Y;

X₄h or N;

X₅d or V;

X₆l or M.

6. An antigen binding protein according to claim 5, wherein

X₃＝F；X₄＝H；X₅D; and X₆L; or

X₃＝Y；X₄＝N；X₅V; and X₆＝M。

7. An antigen binding protein that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence

8. An antigen binding protein that binds human CD3, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：SYALS；

CDR2：GISGSGRTTWYADSVKG；

CDR3：DGGYSYGPYWYFDL。

9. an antigen binding protein that binds human CD3, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：SYALS；

CDR2：AISGSGRTTWYADSVKG；

CDR3：DGGYTYGPYWYFDL。

10. an antigen binding protein that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence

11. An antigen binding protein that binds human CD3, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：DYTMH；

CDR2：DISWSSGSIGYADSVKG；

CDR3：DHRGYGDYEGGGFDY。

12. an antigen binding protein that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence

13. An antigen binding protein that binds human CD3, comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 comprise the amino acid sequences:

CDR1：DYTMH；

CDR2：DISWSX₇GX₈X₉X₁₀YADSVKG；

CDR3：DHX₁₁GYGDYEGGGFDX₁₂；

wherein

X₇(ii) S or G;

X₈(ii) S or T;

X₉i or T;

X₁₀g or Y;

X₁₁r or M;

X₁₂(ii) either H or Y, or a combination thereof,

preferably X₇、X₈、X₉And X₁₀S, S, I and G, and X₁₁And X₁₂R and H; or preferably X₇、X₈、X₉And X₁₀G, S, I and Y, and X₁₁And X₁₂R and Y; or preferably X₇、X₈、X₉And X₁₀S, T, T and G, and X₁₁And X₁₂M and Y.

14. The antigen binding protein of any one of claims 1-13, wherein said light chain variable regions comprise a common light chain variable region.

15. The antigen binding protein of any one of claims 1-14, wherein said common light chain variable region comprises an IgV κ 1-39 light chain variable region.

16. The antigen binding protein of any one of claims 1-15, wherein the light chain variable region is a reproductive series IgV κ 1-39 x 01 variable region.

17. The antigen binding protein of any one of claims 1-16, wherein said light chain variable region comprises a kappa light chain igvk 1-39 x 01/igjk 1 x 01 or igvk 1-39 x 01/igjk 5x 01.

18. The antigen binding protein of any one of claims 1-17, wherein the light chain variable region comprises a reproductive series kappa light chain IgV κ 1-39 x 01/igjκ 1 x 01 or IgV κ 1-39 x 01/igjκ 5x 01.

19. The antigen binding protein of any one of claims 1-18, wherein the light chain variable region comprises an amino acid sequence

Having 0-5 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof.

20. The antigen binding protein of any one of claims 1-19, which is an antibody, preferably a bispecific antibody.

21. The bispecific antibody of claim 20, comprising an H/L chain combination according to any one of claims 1-19, and an H/L chain combination that binds a tumor antigen.

22. The bispecific antibody of claim 21, wherein the H/L chain combination that binds to a tumor antigen binds to human BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, FOLR1, FOLR3, HER2, HM1.24, MCSP, PD-L1, PSMA protein, or a variant thereof.

23. The antibody or bispecific antibody of any one of claims 20-22, which is a human or humanized antibody.

24. The bispecific antibody of any one of claims 20-23, comprising two different immunoglobulin heavy chains with compatible heterogeneous dimeric domains.

25. The bispecific antibody of claim 24, wherein the compatible heterogeneous dimeric domain is a compatible immunoglobulin heavy chain CH3 heterogeneous dimeric domain.

26. The bispecific antibody of any one of claims 20-25, wherein the bispecific antibody is an IgG antibody with a mutated CH2 and/or lower hinge domain such that the bispecific IgG antibody has reduced interaction with an Fc-gamma receptor.

27. The bispecific antibody of claim 26, wherein the mutant CH2 and/or lower hinge domain comprises an amino substitution at position 235 and/or 236 (according to EU numbering), preferably an L235G and/or G236R substitution.

28. The bispecific antibody of any one of claims 20-27, comprising a common light chain.

29. The antigen binding protein or antibody of any one of claims 1-28, for use in treating a subject in need thereof.

30. The antigen binding protein or antibody for use according to claim 29, wherein the individual has cancer or is to be treated for cancer.

31. The antigen binding protein or antibody for use according to claim 29 or claim 30, wherein the treatment comprises local administration and/or local release of an antigen binding protein or antibody according to any one of claims 1-3, 11-13.

32. The antigen binding protein or antibody for use according to claim 29, wherein the treatment comprises administering an antigen binding protein or antibody according to any one of claims 4-10 to a subject having an overactive immune system.

33. The antigen binding protein or antibody for use according to claim 32, wherein said individual has an autoimmune disease.