CN114605546A - CD3 binding molecules - Google Patents

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 present invention further relates to the use of the CD3 binding proteins of the present invention, preferably antibodies, for the treatment of cancer or autoimmune diseases.

Description

CD3 binding molecule

This application is a divisional application of a Chinese patent application entitled "CD3 binding molecule" with the application number 202080019367.1. The original application is the PCT international application PCT/NL2020/050214 filed on March 27, 2020 and filed on September 07, 2021 Applications for entering the Chinese national phase from Japan.

technical field

The present invention relates to the field of antibodies, particularly to the field of therapeutic antibodies. Such antibodies can be used to treat humans. More specifically, the present invention relates to antibodies and preferably bispecific or multispecific antibodies for use in the treatment of tumors.

Background technique

Monoclonal antibodies that bind human CD3 were the first antibodies developed for therapeutic use in humans. Monoclonal CD3-binding antibodies are typically used for their immunosuppressive properties, eg, in transplant rejection. Bispecific antibodies directed against CD3 on T cells and against surface target antigens on cancer cells, capable of linking any kind of T cells to cancer cells, independent of T cell receptor specificity, costimulation or peptide antigen presentation. These bispecific T-cell engaging antibodies show great promise in the treatment of various cancers and neoplastic growths.

The object of the present invention is to provide novel antibodies, which have CD3 binding properties, not necessarily quantitative properties in terms of species, have improved characteristics, have relatively low affinity, and have high cytotoxicity, which are suitable for use in Tumor immunology applications of T cell and effector cell engagement, and conversely, to provide new antibodies that bind to CD3, have relatively high affinity, and have low cytotoxicity, suitable for use in T cells and effector Autoimmune applications of daughter cell downregulation. A further object of the present invention is to provide T cell engaging CD3 binding proteins and antibodies having the above properties of binding to at least one additional membrane-associated molecule.

SUMMARY OF THE INVENTION

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

CDR1: SFGIS

CDR2: GFIPVLGTANYAQKFQG

CDR3: RGNWNPFDP; or

Includes the following amino acid sequences:

CDR1: SX ₁ TFTIS;

CDR2: GIIPX ₂ FGTITYAQKFQG;

CDR3: RGNWNPFDP;

in

X ₁ =K or R;

X ₂ =L or I.

In a preferred embodiment, X ₁ =K; and X ₂ =L;

In another preferred embodiment, X ₁ =R; and X ₂ =I.

In a preferred embodiment, the present invention provides an antigen-binding protein that binds to human CD3, preferably an antibody, comprising an antibody variable domain, the antibody variable domain comprising a heavy chain variable region and a light chain Variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 include the following amino acid sequence:

CDR1: SKTLTIS;

CDR2: GIIPIFGSITYAQKFQD;

CDR3: RGNWNPFDP; or

Includes the following amino acid sequences:

CDR1: GSGIS;

CDR2: GFIPFFGSANYAQKFRD;

CDR3: RGNWNPX _13DP ;

in

X ₁₃ =L or F.

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

There are 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 provide an antigen-binding protein that can bind to human CD3, preferably an antibody, comprising an antibody variable domain, the antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is provided. The chain variable region includes a CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 include the following amino acid sequences:

CDR1: RX ₃ WIG;

CDR2: IIYPGDSDTRYSPSFQG;

CDR3: X ₄ IRYFX ₅ WSEDYHYYX ₆ DV;

in

X ₃ =F or Y;

X ₄ =H or N;

X ₅ =D or V;

X ₆ =L or M.

In a specific example X ₃ =F; X ₄ =H; X ₅ =D; and X ₆ =L. In another specific example, X ₃ =Y; X ₄ =N; X ₅ =V; and X ₆ =M.

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

There are 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 provide an antigen-binding protein that can bind to human CD3, preferably an antibody, comprising an antibody variable domain, the antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is provided. The chain variable region includes a CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 include the following amino acid sequences:

CDR1:SYALS;

CDR2:GISGSGRTTWYADSVKG;

CDR3: DGGYSYGPYWYFDL.

Further provide an antigen-binding protein that can bind to human CD3, preferably an antibody, comprising an antibody variable domain, the antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is provided. The chain variable region includes a CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 include the following amino acid sequences:

CDR1:SYALS;

CDR2: AISGSGRTTWYADSVKG;

CDR3: DGGYTYGPYWYFDL.

Further provide an antigen-binding protein that can bind to human CD3, preferably an antibody, comprising an antibody variable domain, the antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is provided. chain variable region including amino acid sequence

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

Also 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 is The chain variable region includes a CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 include the following amino acid sequences:

CDR1: DYTMH;

CDR2: DISWSSGSIGYADSVKG;

CDR3: DHRGYGDYEGGGFDY.

Also 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 is The chain variable region includes a CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 include the following amino acid sequences:

CDR1: DYTMH;

CDR2: DISSWX ₇ GX ₈ X ₉ X ₁₀ YADSVKG;

CDR3: DHX ₁₁ GYGDYEGGGFDX ₁₂ ;

in

X ₇ =S or G;

X ₈ =S or T;

X ₉ =I or T;

X ₁₀ =G or Y;

X ₁₁ =R or M;

X ₁₂ =H or Y.

In a specific example, X ₇ , X ₈ , X ₉ and X ₁₀ are S, S, I and G, and X ₁₁ and X ₁₂ are R and H. In another specific example, X ₇ , X ₈ , X ₉ and X ₁₀ are G, S, I and Y, and X ₁₁ and X ₁₂ are R and Y. In another specific example, X ₇ , X ₈ , X ₉ and X ₁₀ are S, T, T and G, and X ₁₁ and X ₁₂ are M and Y.

Further provide an antigen-binding protein that can bind to human CD3, preferably an antibody, comprising an antibody variable domain, the antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is provided. chain variable region including amino acid sequence

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

The light chain variable region of an antigen binding protein of the present invention, preferably an antibody, preferably includes a common light chain variable region. The common light chain variable region preferably includes an IgVκ1-39 light chain variable region. The light chain variable region is preferably a germline IgVκ1-39*01 variable region. The light chain variable region preferably comprises a kappa light chain IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01. In one embodiment, the light chain variable region comprises a human germline kappa light chain IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01. The light chain variable region preferably comprises an amino acid sequence

With 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 includes an H/L chain combination that binds human CD3 as shown herein and an H/L chain combination that binds a tumor antigen. The H/L chain combination that binds tumor antigens preferably binds 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 a preferred embodiment, EGFR, PD-L1 or CLEC12A.

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 heterodimeric domains. The compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain.

The bispecific or multispecific antibody is preferably an IgG antibody with a mutated CH2 and/or lower hinge domain such that the interaction of the bispecific or multispecific IgG antibody with the Fc-gamma receptor is reduced. The 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.

The bispecific or multispecific antibodies preferably comprise a common light chain.

The present invention further provides an antigen binding protein or antibody as shown herein for use in the treatment of an individual in need thereof. The individual preferably has cancer or is to be treated for cancer. A kind of CDRs and/or VH sequence with MF8057, MF8058, MF8078 or MF8508, or its variant with 0-10 amino acid substitution, variation, insertion, addition or deletion, the antigen binding protein or antibody is preferably used for treatment, In particular for a treatment involving local administration and/or local release of the antigen binding protein or antibody. A kind of CDRs and/or VH sequence with MF9249, MF9267, MF8397, or its variant with 0-10 amino acid substitution, variation, insertion, addition or deletion, the antigen binding protein or antibody is preferably used for the treatment of hyperactivation immune system, such as an individual with an autoimmune disease.

Unless expressly stated otherwise, the antibody of the invention is preferably a bispecific antibody. The bispecific antibody preferably binds at least human CD3. Furthermore, the bispecific antibody preferably binds to at least one surface molecule preferentially expressed on human tumor cells. In a preferred embodiment, the bispecific antibody binds to 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 CLEC12A. In a more preferred embodiment, the multispecific antibody binds to EGFR and PD-L1.

The present invention further provides a pharmaceutical composition comprising the antibody of the present invention.

There is further provided an antibody according to the invention, which further comprises a label, preferably a label for in vivo imaging.

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

Description of drawings

figure 1

Functional activity assessment: T cell cytotoxicity analysis of BxPC3 target cells after treatment with EGFRxCD3 bispecific antibody. Each bispecific antibody includes a CD3 binding domain consisting of a heavy chain variable region designated by the MF number, and an EGFR binding domain including a heavy chain variable region MF8233. These variable regions are paired with a common light chain to form an EGFRxCD3 bispecific antibody. Affinity (HPB-ALL binding) relative to BxPC3 solubilization. Certain antibodies of the present invention exhibit relatively low levels of binding to HPB-ALL cells indicating that the CD3 binding domain of the antibody binds human CD3 with relatively low affinity. It is clear that relatively low affinity does not necessarily prevent tumor antigen-mediated T cell cytotoxicity of BxPC3 cells (BxPC3 lysis, vertical axis). The bispecifics MF8233 x MF8508 and MF8233 x MF8057 efficiently lysed BxPC3 cells whereas the bispecifics MF8233 x MF8397 and MF8233 x MF9249, although having similar binding, did not do so efficiently. Furthermore, the bispecific antibodies MF8233 x MF6955 were compared to bind HPB-ALL (i.e. human CD3) with a higher affinity, but not more efficiently than the bispecific antibodies MF8233 x MF8508 and MF8233 x MF8057, which bound CD3 to a lesser extent Lyse BxPC3 cells. MF6955 is a heavy chain variable region combined with a common light chain and used as comparator sequences, and corresponds to the H1H7232B(1129) VH in US2014/0088295 A1. This comparator bispecific antibody with MF6955 and the same EGFR binding domain has a higher affinity for human CD3 than MF9267 and exhibits more potent killing than MF9267, respectively. In contrast, other antibodies incorporating the CD3 binding domains of the invention, eg, MF8058, have approximately the same binding activity as MF6955, but show more efficient killing of BxPC3 cells, eg, MF8233 x MF8058. Other bispecific antibodies of the invention comprising binding domains capable of binding CD3 exhibit relatively high binding and more efficient killing, eg MF8233 x MF8078, which are useful for the specific applications described herein. However other bispecific antibodies of the invention comprising binding domains capable of binding CD3 exhibit relatively low affinity and low killing, eg MF8233 x MF9249 and MF8233 x MF8397, which are useful for any of the applications described herein.

figure 2

Antibody titration curve expressed as a % kill of T cell mediated BxPC3 target cells it induces compared to no antibody control. Curves are shown for antibodies MF8233 x MF8078, MF8233 x MF8397; and MF8233 x MF8508.

image 3

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

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

Figure 4

T Cell Activation by T Cell Cytotoxicity Assay with BxPC3 Target Cells on Expressing CD8+ T Cells. Antibody titration curves for various supergroup numbers listing variant CD3 binding domains. The other arm of this bispecific antibody has the heavy chain binding domain of MF8233. To compare the bispecific antibodies MF8233 x MF6955 and MF8233 x MF6964 were also tested, MF6955 and MF6964 are heavy chain variable regions combined with a common light chain and used as comparison subsequences, and correspond to US2014/0088295 A1, respectively H1H7232B(1129) VH and HH7241B(1145) inside.

Figure 5

Summary of titration curve data for various antibodies in T cell activation T cell cytotoxicity assays with BxPC3 target cells. The column MFnr. indicates the MF number of the CD3 binding domain. The EGFR binding domain has the assigned MF8233 number. Supergroup information for various CD3 binding domain sequences is presented in the column "Supergroup"; the column indicating CD3 affinity reflects the results of HBP-ALL binding experiments.

Results are shown for CD4+ and CD8+ cells that label CD69 and CD25. The indicated bispecific antibodies are examples in a large pool of bispecific antibodies.

Image 6

Functional activity assessment: T cell cytotoxicity assay with BxPC3 target cells. Affinity (HPB-ALL binding) relative to CD8+ T cell activation measured by CD69 expression. Certain antibodies of the present invention exhibit relatively low levels of binding to HPB-ALL cells indicating 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 MF8233 x MF8508 and MF8233 x MF8057 were effective in activating T cells whereas the bispecific antibodies MF8233 x MF8397 and MF8233 x MF9249 were not. Certain CD3-binding domains that do not efficiently bind these cells also do not activate T cells (see bottom left). While other CD3 binding domains such as MF8508 and MF8057, which bound less to HPB-ALL cells than the comparator CD3 binding domain MF6955, activated T cells to a similar extent. Other bispecific antibodies of the invention comprising binding domains capable of binding CD3 exhibit relatively high binding and high levels of activation, such as MF8078, which are useful for the specific applications described herein. However other bispecific antibodies comprising binding domains of the invention capable of binding CD3 exhibit relatively low affinity and low activation, eg MF9249 and MF8397, which are useful for the optional applications described herein.

Figure 7

Functional activity assessment: T cell cytotoxicity assay with HCT116 target cells. Affinity (HPB-ALL binding) relative to HCT-116 solubilization.

Certain bispecific antibodies of the invention exhibit low levels of HPB-ALL cell binding indicating 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 MF8233 x MF8508 and MF8233 x MF8057 were able to effectively lyse HCT-116 cells while the bispecific antibodies MF8233 x MF8397 and MF8233 x MF9249 were not. To compare the bispecific antibodies MF8233 x MF6955 and MF8233 x MF6964 bind HPB-ALL (i.e. human CD3) with higher affinity than, for example, MF8233 x MF8508, MF8233 x MF8057 and MF8233 x MF9267, but not MF8233 x MF8508 or MF8233 x MF9267 lysed HCT-116 cells more efficiently, or significantly more efficiently than MF8233xMF8057, relative to the difference in binding as in the assay shown here. Another bispecific antibody comprising a binding domain of the invention capable of binding CD3 exhibits relatively high binding and high levels of killing, eg, MF8078, which is useful for the specific applications described herein. However other bispecific antibodies of the invention comprising binding domains capable of binding CD3 exhibit relatively low affinity and low killing, eg MF9249 and MF8397, which are useful for the optional applications described herein.

Figure 8

Antibody titration curves representing % killing of HCT-116 cells compared to no antibody controls in T cell cytotoxicity assays with HCT-116 target cells. Curves are shown for various bispecific antibodies.

Figure 9

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

Percent lysis of HCT-116 cells and EC50 values for lysis (ng/mL) are presented in the next fields. The bispecific antibodies indicated are examples in a large pool of bispecific antibodies.

Figure 10

Figures 10a and 10b list schematic diagrams of the MV1624 expression vector and the MV1625 expression vector.

Figure 11

A common light chain used within mono- and bispecific IgGs.

Figure 11A: Common light chain amino acid sequence. Figure 11B: Common light chain variable domain DNA sequence and representation (IGKV1-39/jk1). Figure 11C: Common light chain constant region DNA sequence and translation. Figure 1 ID: IGKV1-39/jk5 common light chain variable domain translation. Figure 11E: V region IGKV1-39A; Figure 11F: CDR1, CDR2 and CDR3 of the common light chain.

Figure 12

IgG heavy chains of bispecific molecules. Figure 12A: CH1 region. Figure 12B: Hinge area. Figure 12C: CH2 region. Figure 12D: CH2 containing silent substitutions of L235G and G236R. Figure 12E: CH3 domain containing substitutions L351K and T366K (KK). Figure 12F; CH3 domain containing substitutions L351D and L368E (DE).

Figure 13

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

Figure 14

Characterization of additional clones from supergroup 1 compared to clones MF8057 and MF8058. A: Binding of selected MF clones in FACS analysis to HPB-ALL human cells expressing human CD3-TCR complexes. B: T cell cytotoxicity assay with HCT-116 cells, which represents % killing of HCT-116 cells. C-E: Quantification of activation markers CD25 and CD69 representing T cell activation by FACS. F-G: Cytokinin production from supernatants of cytotoxicity assays.

Figure 15

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 represents % killing of BxPC3 cells. C-E: Cytokinin production from supernatants from cytotoxicity assays.

Figure 16

Assessment of CD3 functional activity: Additional clones from supergroup 1 (MF8048, MF8101, MF8056), supergroup 3 (MF8562) and supergroup 4 (MF8998) Affinity (HPB-ALL) relative to HCT-116 lysis on the X-axis on the Y axis. B: Antibodies belonging to supergroup 1 and supergroup 4 showing similar activities and different binding affinities in cytotoxicity assays. C: Antibodies belonging to supergroup 1 and supergroup 3, which exhibit similar binding affinities and different lytic activities.

Figure 17

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

Figure 18

FACS binding data for a large set of IgGs specific for CD3. For the antibodies MF5196, MF6955 and MF6964, the binding to the CD3δε-Fc antigen was determined by BIAcore ^™ , and the FACS binding data of the remaining clones to HPB-ALL cells were also shown.

Figure 19

Nucleotide sequence of human CLEC12A.

Figure 20

Amino acid sequences of human CD3 gamma-, delta-, epsilon- and zeta-chains.

Detailed ways

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 the variable regions of an antibody or associated with an antibody. The variable regions share sequence homology.

Antibodies bind with different properties, including specificity and affinity. Specificity determines which antigen or epitope the binding domain specifically binds to. Affinity is a measure of the amount of binding for a particular antigen or epitope. It is convenient to note here that "specificity" of an antibody means its selectivity for a particular antigen, and "affinity" means the interaction between the antigen binding address of the antibody and the epitope it binds Action value. Antibodies are typically composed of basic building blocks - each with a double chain and a double light chain. Antibodies for therapeutic use are preferably as close as possible to native antibodies of the subject to be treated (eg, human antibodies of a human subject). Antibodies such as the present invention are not limited to any particular format or method of production thereof.

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

The antibody of the present invention is preferably an IgG antibody, preferably an IgG1 antibody. Full length IgG antibodies may be preferred due to their favorable half-life and due to the desire to remain close to fully autologous (human) molecules for immunogenicity reasons. 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 be a human IgG1.

A "bispecific antibody" is an antibody as described herein wherein a variable domain of the antibody binds a first antigen and a second variable domain of the antibody binds a second antigen, wherein the first and The second antigens are not identical. The term "bispecific antibody" also includes biparatopic antibodies wherein a variable domain of the antibody binds a first epitope on an antigen and a second variable domain of the antibody binds the antigen the second epitope. The term further includes antibodies wherein at least one VH specifically recognizes a first antigen and a VL paired with the at least one VH of an immunoglobulin variable domain specifically recognizes a second antigen. The resulting VH/VL pairs will bind antigen 1 or antigen 2 and are referred to as "two-in-one antibodies" as described in, eg, WO 2008/027236, WO 2010/108127 and Schaefer et al. (Cancer Cell 20, 472-486, 2011 October 2009) is described in text B. Bispecific antibodies such as 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 containing two or more domains that bind epitopes on an antigen, wherein these domains are derived from an antibody The variable regions of or share sequence homology with the variable regions of an antibody, and include protein molecules that bind three or more antigens as known in the art, including as described in WO2019/190327.

An "antigen" is a molecule capable of eliciting an immune response (to generate an antibody) in a host organism and/or an antibody-targeted molecule. At the molecular level, an antigen is characterized by its ability to be bound by the antigen-binding address of an antibody. Again, a mixture of antigens can be considered an "antigen", ie those skilled in the art will understand that sometimes tumor cell lysates or viral particles can be represented as "antigens" and such tumor cell lysates A number of epitopes are present in the product or virus particle preparation. An antigen includes at least one, but usually multiple, epitopes. For the 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 "epitope" refers to the address on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from adjacent amino acids or discrete amino acids juxtaposed by the tertiary fold of the protein (so-called linear or conformational epitopes, respectively). Epitopes formed from adjacent, linear amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed from tertiary folds typically lose their conformation upon treatment with denaturing solvents. Epitopes typically include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in unique spatial configurations.

The term "heavy chain" or "immunoglobulin heavy chain" includes an immunoglobulin heavy chain constant region sequence from any organism and, unless otherwise specified, 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 heavy chains 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 the variable domain (from N-terminus to C-terminus). A functional fragment of a heavy chain includes a fragment that specifically recognizes an antigen and includes at least one CDR.

The term "light chain" includes the sequence of an immunoglobulin light chain variable domain or _VL (or functional fragment thereof; and an immunoglobulin constant domain or _CL or functional fragment thereof, from any organism. Unless otherwise specified, the term light chain may include a light chain selected from human kappa, lambda, and a combination thereof. Unless otherwise specified, a light chain variable ( _VL ) domain typically includes three light chains CDRs and four framework (FR) regions. Generally, a full-length light chain includes a _VL domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus and A light chain constant domain. Light chains useful in the present invention include, for example, such that do not selectively bind an epitope that is selectively bound by such heavy chains.

的物，该基因转殖动物具有嵌入至其基因体的内的共同的轻链且其可用来产生大量的(large panels of)共同的轻链抗体其在重链处具有多样性的且在暴露于一抗原的时能特异性地结合该抗原。Suitable light chains for use in the antibodies of the invention include a common light chain (cLC), such as can be most commonly used by screening existing antibody libraries [wet libraries or in silico] Such light chains have been identified as light chains that do not substantially interfere with the affinity and/or selectivity of the epitope binding domains of such heavy chains, but are also suitable for pairing with a range of heavy chains. For example, a suitable light chain includes one from a transgenic animal such as

The transgenic animals have a common light chain embedded within their gene bodies and which can be used to generate large panels of common light chain antibodies that are diverse at the heavy chain and are exposed to In the presence of an antigen, it can specifically bind to the antigen.

The term "common light chain" as used in the present invention refers to those which may be identical or have some amino acid sequence differences without affecting the binding specificity of the antibodies of the present invention, ie such differences do not substantially affect the formation of a functional binding region light chain.

For example, within the definition of a common light chain as used herein, it is possible to make or find variable chains that are not identical but still functionally equivalent, such as by introducing and testing retained amino acid changes, when Amino acid changes within a region have no or only partial effect on binding specificity when paired with homologous chains and the like. These variants are thus also able to bind different homologous chains and form functional antigen binding domains. The term common light chain as used herein thus refers to light chains that may be identical or have some amino acid sequence differences but 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. Detailed descriptions of the use of common light chains can be found in WO 2004/009618 and WO 2009/157771.

"Fab" refers to a binding domain comprising a variable domain, typically a binding domain comprising a paired heavy chain variable domain and light chain variable domain. Fabs can include constant region domains that include a CH1 and VH domain paired with a constant light chain domain (CL) and VL domains. These pairings can occur as covalent linkages, eg, via a disulfide bond at the CH1 and CL domains.

A "single-chain variable fragment" (scFv) refers to a binding domain comprising the VH and VL domains linked by, for example, a linker, eg, a peptide bond, of from about 10 to about 25 amino acids in length.

The term "full-length IgG" or "full-length antibody" as used in the present invention is defined to include substantially intact IgG, which however do not necessarily have all the functions of intact IgG. For the avoidance of doubt, full-length IgG contains a double chain and a double light chain. 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 are able to interact with molecules and cells of the immune system via the constant domains, primarily via the Fc portion. Full-length antibodies as in the present invention include IgG molecules in which mutations that provide the desired characteristics may be present. A full-length IgG should not have deletions of substantial portions of any domains. 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 residue deletions, preferably within non-CDR regions, where the deleted amino acids are not essential for the 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 residues of a selected sequence after matching sequences have been aligned for optimal comparison. Either of the two sequences to be compared can introduce a gap to maximize alignment of the two sequences. This alignment can be performed on the full-length sequences to be compared. Optionally, alignments can be performed over shorter lengths, eg, about 20, about 50, about 100, or more nucleic acids/bases or amino acids. Sequence identity is the percentage of matches that are identical between two sequences over the reported aligned region.

Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using mathematical algorithms. Those skilled in the art will know 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 comparison In D.Sankoff and J.B.Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley).

With regard to the present invention and the sequences described herein, the percent sequence identity between two nucleic acid sequences can be determined using the AlignX application of the 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. AcidRes. 22:4673-4680), swgapdnarnt score matrix, gap open penalty of 15 and gap extension penalty of 6.66 ). Amino acid sequences can be aligned using the AlignX application of the Vector NTI ProgramAdvance 11.5.2 software using the default alignment using a modified ClustalW algorithm (Thompson, J.D., Higgins, D.G., and Gibson T.J., 1994), blosum62mt2 score matrix, out of 10 Gap opening penalty and gap extension penalty of 0.1.

The term "super-cluster" or "supercluster" is used herein to mean, based on the same VH V gene segment and having at least 70% sequence identity within HCDR3 and identical HCDR3 A group of clones of length , and the binding domains they can produce.

Thus, in a preferred embodiment, the present invention provides a "super-cluster" or "supercluster" comprising, based on the same VHV gene segments used and having at least 70% in HCDR3 The sequence identity and the same length of HCDR3, a set of clones and the binding domains they can produce. In a preferred embodiment, the sequence identity is 80%, more preferably 90%, and optimally 95%, provided that a clone comprising a nucleic acid encoding the HCDR3 sequence DGGYSYGPYWYFDL and DHRGYGDYEGGGFDY is excluded, Colonies that include nucleic acids encoding HCDR2 sequences GFIPVLGTANYAQKFQG, GIIPLFGTITYAQKFQG and SIIPIFGTITYAQKFQG, or conditionally exclude sequences encoding VH

A clone of nucleic acid, or conditionally a clone from the group, comprises nucleic acid encoding HCDR3 consisting of or designed to consist of a bispecific antibody.

The terms "super-cluster 1" or "supercluster 1" are used herein to mean, based on the use of the same VH V gene segment as a member of the super-cluster (VH1-69) and in HCDR3 A group of clones with at least 70% sequence identity and the same HCDR3 length, and their producible binding domains. These include, for example, MF8048, MF8056, MF8057, MF8058, MF8078, and MF8101. In another preferred embodiment, the anti-CD3 antibody herein is based on the use of the same VH V gene segment of VH1-69 and/or has at least 80% identity within HCDR3 and the same length of HCDR3, more preferably in 90% or optimally 95% identity within HCDR3. In another preferred embodiment, the anti-CD3 antibodies herein are based on the use of the same VH V gene segment of VH1-69 and/or have at least 80% within HCDR3 compared to the encoded CDR3 segment RGNWNPFDP Identity and identical HCDR3 length, preferably at least 90% sequence identity within HCDR3 and identical HCDR3 length, more preferably 95% or optimally 98% identity and identical HCDR3 length, provided that Exclude clones that include nucleic acids encoding HCDR2 sequences GFIPVLGTANYAQKFQG, GIIPLFGTITYAQKFQG and SIIPIFGTITYAQKFQG, or conditionally include sequences encoding VH

A clone of nucleic acid, or conditionally a clone from the group, comprises nucleic acid encoding HCDR3 consisting of or designed to consist of a bispecific antibody. The terms "super-cluster 3" or "supercluster 3" are used herein to mean that the use of the same VH V gene segment as a member of the super-cluster (VH3-23) and in HCDR3 A group of clones with at least 70% sequence identity and the same HCDR3 length, and their producible binding domains. Including, for example, MF8397 and MF8562. In another preferred embodiment, the anti-CD3 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 in 90% or optimally 95% identity within HCDR3. In another preferred embodiment, the anti-CD3 antibody herein is based on the use of the same VH V gene segment of VH3-23 and/or has at least 80% in HCDR3 compared to the encoded CDR3 segment DGGYSYGPYWYFDL Identity and identical HCDR3 length, preferably at least 90% sequence identity within HCDR3 and identical HCDR3 length, more preferably 95% or optimally 98% identity and identical HCDR3 length, provided that Excludes clones that include nucleic acid encoding the HCDR3 sequence DGGYSYGPYWYFDL, or conditionally excludes the VH-encoding sequence

A clone of nucleic acid, or conditionally a clone from the group, comprises nucleic acid encoding HCDR3 consisting of or designed to consist of a bispecific antibody.

The term "super-cluster 4" or "supercluster 4" is used herein to mean that the use of the same VH V gene segment as a member of the super-cluster (VH3-9) and in HCDR3 A group of clones with at least 70% sequence identity and the same HCDR3 length, and their producible binding domains. Including, for example, MF8508, MF8998, MF10401 and MF10428. In another preferred embodiment, the anti-CD3 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 in 90% or optimally 95% identity within HCDR3. In another preferred embodiment, the anti-CD3 antibodies herein are based on the use of the same VH V gene segment of VH3-9 and/or have at least 80% within HCDR3 compared to the encoded CDR3 segment DHRGYGDYEGGGFDY Identity and identical HCDR3 length, preferably at least 90% sequence identity within HCDR3 and identical HCDR3 length, more preferably 95% or optimally 98% identity and identical HCDR3 length, provided that Excludes clones that include nucleic acid encoding the HCDR 3 sequence DHRGYGDYEGGGFDY, or conditionally excludes the sequence encoding VH

A clone of nucleic acid, or conditionally a clone from the group, comprises nucleic acid encoding HCDR3 consisting of or designed to consist of a bispecific antibody.

The term "super-cluster 7" or "supercluster 7" is used herein to mean that the use of the same VH V gene segment as a member of the super-cluster (VH5-51) and in HCDR3 A group of clones with at least 70% sequence identity and the same HCDR3 length, and their producible binding domains. Including, for example, MF9249 and MF9267. In another preferred embodiment, the anti-CD3 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 in 90% or optimally 95% identity within HCDR3. In another preferred embodiment, the anti-CD3 antibodies herein are based on the use of the same VH V gene segment of VH5-51 and/or have at least 80% in HCDR3 compared to the encoded CDR3 segment HIRYFDWSEDYHYYLDV Identity and identical HCDR3 length, preferably at least 90% sequence identity and identical HCDR3 length within HCDR3, more preferably 95% or optimally 98% identity and identical HCDR3 length.

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

- V gene segment VH1-69; or

- a variant of the V gene segment VH1-69 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 sex;

wherein the VH further comprises

- HCDR3 for MF8048, MF8056, MF8057, MF8058, MF8078 or MF8101;

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

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

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

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

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

- V gene segment VH3-23; or

- a variant of the V gene segment VH2-23 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 sex;

wherein the VH further comprises

-MF8397; or -HCDR3 of MF8562;

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

In a preferred embodiment, the variant of the HCDR3 comprises the same length as the HCDR3 and at least 80% sequence identity with the HCDR3, more preferably at least 90% with the sequence of the HCDR3, more preferably at least 93% and more preferably at least 95% sequence identity.

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

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

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

- V gene segment VH3-9; 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 sex;

wherein the VH further comprises

- MF8508; MF8998; MF1041; or a HCDR3 of MF10428;

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

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

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

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

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

- V gene segment VH5-51; 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 V gene segment sex;

wherein the VH further comprises

- HCDR3 of MF9249 or MF9267;

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

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

A bispecific antibody provided by the invention as defined herein is preferably not a bispecific antibody comprising a CD3 binding variable domain as defined in PCT/NL2019/050199.

The present invention further provides a VH encoded by

- V gene segment VH1-69; or

- a variant of the V gene segment VH1-69 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 sex;

wherein the VH further comprises

- HCDR3 for MF8048, MF8056, MF8057, MF8058, MF8078 or MF8101;

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

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

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

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

The present invention further provides a VH encoded by

- V gene segment VH3-23; or

- a variant of the V gene segment VH2-23 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 sex;

wherein the VH further comprises

-MF8397; or -HCDR3 of MF8562;

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

In a preferred embodiment, the variant of the HCDR3 comprises the same length as the HCDR3 and at least 80% sequence identity with the HCDR3, more preferably at least 90% with the sequence of the HCDR3, more preferably at least 93% and more preferably at least 95% sequence identity.

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

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

The present invention further provides a VH encoded by

- V gene segment VH3-9; 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 sex;

wherein the VH further comprises

- MF8508; MF8998; MF10401; or a HCDR3 of MF10428;

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

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

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

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

The present invention further provides a VH encoded by

- V gene segment VH5-51; 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 V gene segment sex;

wherein the VH further comprises

- HCDR3 of MF9249 or MF9267;

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

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

A VH provided by the invention as defined herein is preferably not a CD3 binding variable domain VH as defined in PCT/NL2019/050199.

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 of such CDRs as claimed amino acid residue variation, insertion, substitution, deletion or addition.

Unlike the random, nonspecific attachment of antibodies, antigen binding via antibodies is typically mediated through the complementary regions of the antibody and through the specific three-dimensional structure of both the antigen and variable domains allowing these two structures to bind together precisely (similar to lock and key interaction). Because an antibody usually only recognizes one epitope of an antigen, and this epitope may also exist in other proteins, such as the antibody of the present invention that binds to CD3 or CLEC12A can also recognize other proteins, if these other proteins contain the same epitope. Thus, the term "binding" does not exclude binding of the antibody to another protein or proteins containing the same epitope. The CD3-binding heavy/light chain combinations within the antibodies of the invention do not bind to other proteins on postnatal, preferably adult human cell membranes. The heavy/light chain combinations of the invention that bind CLEC12A, EGFR, PD-L1 or tumor cell antigens do not bind to other proteins on the cell membranes of postnatal, preferably adult human beings. Suitable tumor antigen-specific arms are disclosed in PCT/NL2019/050199.

"Plural" means two or more.

"Variants" of antibodies as described herein may include functional portions, derivatives and/or analogs of antibodies. This includes antibody mimetic, monobody and aptamer.

Variants typically retain the binding specificity of the antibody, eg, the specificity of a bispecific antibody. Variants may be functional parts or derivatives of binding domains, multimers or antibodies as described herein.

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

一些鱼类也具有仅有重链的抗体(IgNAR，“免疫球蛋白新抗原受体”)，从中可获得称为VNAR片段的单域抗体片段。另一方法是将从来自人类或小鼠的常见免疫球蛋白G(IgG)的二聚可变结构域分成单体。尽管对单域抗体的大多数研究目前基于重链可变结构域，但是衍生自轻链的纳米抗体(nanobody)已经显示出能结合至标靶表位。类可变结构域分子的其他非限制性实例是VHH、人类域抗体(Human Domain Antibodies，dAbs)和单抗体(Unibodies)。优选的功能部分为包括可变结构域的部分，该可变结构域包括重链可变区及轻链可变区。这些可变结构域的非限制性实例为F(ab)片段及单链Fv片段。(类)可变结构域键联的双特异性格式是例如与两个不同的scFv结合的人类血清白蛋白(HSA)；双特异性迷你抗体其包括透过二聚合模体(motif)或自我缔合(self-associating)的二级结构例如螺旋束或卷曲螺旋，以引起scFv片段的二聚合而结合在一起的两个不同scFv(Morrison(2007)Nat.Biotechnol.25：1233-34)。在WO2009/126920中描述合适的HAS键接子及使scFv偶合至键接子的方法的实例。A functional derivative of an antibody as described herein is a protein comprising a variable domain that binds a target and a variable domain that binds a second target linked by a linking region. The variable domains may be the variable domains themselves or Fab fragments or variable domain-like molecules, eg, single-chain Fv (scFv) fragments comprising VH and VL linked together by 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 that can bind one, two or more variable domains. An antigen binding protein as described herein is a protein comprising at least one such variable domain. In the case of bispecific or multispecific antigen binding proteins, such proteins comprise two or more variable domains, at least two of which bind different targets. The variable domains are linked to each other via a linking moiety. This is typically 0-15, preferably 3-12, more preferably a stretch of 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 that have a single monomeric variable antibody region. Like intact antibodies, they are capable of selectively binding to specific antigens. The molecular weight is only 12-15kDa, and single-domain antibody fragments are much smaller than ordinary antibodies (150-160kDa) composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~50kDa, one light chain and half a heavy chain) and a single chain variable fragment (~25 kDa, two variable domains, one from the light and one from the heavy chain). Single domain antibodies themselves are not much smaller than normal antibodies (usually 90-100 kDa). Single domain antibody fragments are engineered primarily from heavy chain antibodies found in the family camelid; these are called VHH fragments

Some fish also have heavy chain-only antibodies (IgNAR, "immunoglobulin neoantigen receptor"), from which single-domain antibody fragments called VNAR fragments can be obtained. Another approach is to separate dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers. Although most research on single domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have been shown to bind to target epitopes. Other non-limiting examples of variable domain-like molecules are VHHs, Human Domain Antibodies (dAbs), and Unibodies. Preferred functional moieties are those comprising a variable domain comprising a heavy chain variable region and a light chain variable region. Non-limiting examples of these variable domains are F(ab) fragments and single chain Fv fragments. A bispecific format for (class) variable domain linkage is, for example, human serum albumin (HSA) bound to two different scFvs; bispecific mini-antibodies which include via dimerization motifs (motifs) or self Self-associating secondary structures such as helical bundles or coiled-coils, two different scFvs bound together to cause dimerization of the scFv fragments (Morrison (2007) Nat. Biotechnol. 25:1233-34). Examples of suitable HAS linkers and methods of coupling scFvs to linkers are described in WO2009/126920.

Functional derivatives can be mimetibodies, polypeptides, aptamers, or combinations thereof. These proteins or aptamers typically bind to a target. The proteins of the invention bind to two or more targets. It will be appreciated that any combination of these antibodies, mimetibodies, polypeptides and aptamers can be linked together by methods known in the art. For example, in some embodiments, the binding molecules of the invention are conjugates or fusion proteins.

Mimetic antibodies are polypeptides that, like antibodies, can specifically bind to an antigen but are not structurally related to antibodies. Mimetic antibodies are typically artificial peptides or proteins with a molar mass of about 3 to 20 kDa. Non-limiting examples of mimetics are affibody molecules (usually based on the Z domain of protein A); affinins (usually based on gamma-B crystals or ubiquitin); affimers (usually based on gamma-B crystals or ubiquitin) Cystatin based); affitins (usually based on Sac7d from Sulfolobus acidocaldarius); alphabody (usually based on Triple helix coiled coil) ); anticalins (usually based on lipocalins); avimers (usually based on the A domains of various membrane receptors); D ARPins (usually based on ankyrin repeat motifs ( ankyrin repeat motif)); fynomers (usually based on the SH3 domain of Fyn 7); kunitz domain peptides (usually based on the kunitz domain of various protease inhibitors); and monosomy ( Usually based on the type III domain of fibronectin).

Monoforms are synthetic binding proteins constructed using the fibronectin type III domain (FN3) as the molecular framework. Monotypes are surrogates for antibodies for generating target binding proteins.

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

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

Throughout the present specification and the appended claims, the words "comprise", "include" and "having" as well as words such as "comprises", "comprising" , "includes" and "including" variations are to be interpreted inclusively. That is, where the context allows, these terms are intended to express the possible inclusion of other elements or integers that are not specifically recited.

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

The antibody of the present invention is preferably a bispecific or multispecific antibody. The bispecific or multispecific antibody preferably binds at least human CD3.

The antigen binding protein or antibody of the present invention is preferably a bi- or multispecific antigen binding protein or antibody. The bi- or multispecific antigen binding protein or antibody preferably binds to at least human CD3, and further preferably binds to at least one surface molecule expressed on human tumor cells. In a preferred embodiment, the bi- or multispecific antigen-binding protein or antibody binds to 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 CLEC12A. In a particularly preferred embodiment, the bispecific antibody binds CD3, PD-L1 and EGFR.

As used in the text, the term "CLEC12A" refers to C-type lectin domain family 12 member A. CLEC12A is also known as C-type lectin protein CLL-1; MICL; dendritic cell-associated lectin 2; C-type lectin superfamily; myelosuppressive C-type lectin-like receptor; C-type lectin-like molecule 1; DCAL2; CLL1; C-type lectin-like molecule 1; DCAL-2; Killer lectin-like receptor subfamily L, member 1 (KLRL1); CD371 (cluster of differentiation 371) (Bakker A. et al. Cancer Res. 2004 , 64, p884350; GenBankTM Accession No.: AY547296; Zhang W. et al. GenBankTM Accession No.: AF247788; A.S. Blood 2004, 104, p2858 66; H. Floyd et al. GenBankTM accession number: AY426759; C.H. Chen et al. Blood 2006, 107, p145967). Ids: HGNC: 31713; Entrez Gene: 160364; Ensembl: ENSG00000172322; OMIM: 612088; UniProtKB: Q5QGZ9.

CLEC12A is an antigen expressed on leukemia bud cells and leukemia stem cells in acute myeloid leukemia (AML), including CD34-negative or CD34-low expressing leukemia stem cells (sidepopulation) (Cancer by A.B. Bakker et al. Res 2004, 64, p844350; Van Rhenen et al. 2007 Blood 110:2659; Moshaver et al. 2008 Stem Cells 26:3059), and in myelodysplastic syndrome (MDS) (ibid. Bakker et al. 2004, and Toff-Peterson et al., Br. J. Haematol. 175(3):393-401, 2016). The expression of CLEC12A is additionally believed to be restricted to the hematopoietic lineage, particularly the peripheral blood and myeloid lineage within the bone marrow, ie granule spheres, monocytes and dendritic cell precursors. More importantly, CLEC12A is not present on normal hematopoietic stem cells. Where reference is made herein to CLEC12A, it refers to human CLEC12A (SEQ ID NO: 1; Figure 19) unless specifically stated otherwise.

The term "CLEC12A" means all variants described herein (eg, spliced and mutated) and their isoforms that retain resolution of myeloid expression (both at surface expression and mRNA levels), including those of Bakker et al. Cancer Res 2004, 64, p8443-50 and as described in Marshall 2004-J Biol Chem 279(15), p14792-802B. While accession numbers are primarily provided as an additional method of identification, the actual protein sequence may vary, eg, due to mutations in the encoding gene, such as occurs in some cancers and the like.

The term "CD3" (cluster of differentiation 3) refers to a protein complex consisting of a CD3γ chain (SwissProt P09693), a CD3δ chain (SwissProt P04234), a CD3ε chain (SwissProt P07766) and a CD3ζ chain homodimer (SwissProt P20963). ). CD3ε is known by various aliases, some of which are: "CD3e molecule, epsilon (CD3-TCR complex)"; "CD3e antigen, epsilon polypeptide (TiT3 complex)"; T cell surface antigen T3/Leu-4ε chain; T3E; T cell antigen receptor complex, epsilon subunit of T3; CD3e antigen; CD3-ε3; IMD18; TCRE. The Ids of the CD3E gene are HGNC: 1674; EntrezGene: 916; Ensembl: ENSG00000198851; OMIM: 186830 and UniProtKB: P07766. These chains associate with the T cell receptor (TCR) and the zeta chain to form TCR complexes that generate activation messages in T lymphocytes upon mitogenic signaling. CD3 is expressed on T cells and NK T cells. Where CD3 is referred to herein, it refers to human CD3 (SEQ ID NOs: 2-5; Figure 20) unless specifically stated otherwise.

BCMA is also known as the tumor necrosis factor receptor superfamily, member 17 (TNFRSF17); TNFRSF13A2; B cell maturation antigen; BCM; B cell maturation factor; B cell maturation protein; CD269 or CD269 antigen. Ids: HGNC: 11913; EntrezGene: 608; Ensembl: ENSG00000048462; OMIM: 109545; UniProtKB: Q02223.

CD19 is also known as CD19 molecule; T cell surface antigen Leu-12; CD19 antigen; CvID3; differentiation antigen CD19; B4; B lymphocyte surface antigen B4; B lymphocyte antigen CD19. Ids: HGNC: 1633; Entrez Gene: 930; Ensembl: ENSG00000177455; OMIM: 107265; UniProtKB: P15391.

CD20 also known as transmembrane 4-domain, subfamily A, member 1 (MS4A1); MS4A2; CD20; S7; leukocyte surface antigen Leu-16; B lymphocyte antigen CD20; Bp35; B lymphocyte cell surface antigen B1; CD20 Antigen; CD20 receptor; CVID5; B lymphocyte surface antigen B1; B1; transmembrane 4-domain subfamily A member 1; LEU-16. Ids: HGNC: 7315; EntrezGene: 931; Ensembl: ENSG00000156738; OMIM: 112210; UniProtKB: P11836.

CD30 is also known as tumor necrosis factor receptor superfamily, member 8 (TNFRSF8); Ki-1 antigen; CD30; Ki-1; D1S166E; Family member 8; CD30L receptor; CD30 antigen. Ids: HGNC: 11923; Entrez Gene: 943; Ensembl: ENSG00000120949; OMIM: 153243; UniProtKB: P28908.

CD33 is also known as CD33 molecule; SIGLEC-3; CD33 antigen (Gp67); myeloid cell surface antigen CD33; sialic acid-binding Ig-like lectin 3; Siglec-3; SIGLEC3; CD33 antigen and gp67. Ids: HGNC: 1659; Entrez Gene: 945; Ensembl: ENSG00000105383; OMIM: 159590; 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-ribose hydrolase; NAD(+) nucleoside Enzyme; EC 3.2.2.5; Cyclic ADP-ribose hydrolase 1; CD38 antigen. Ids: HGNC: 1667; Entrez Gene: 952; Ensembl: ENSG00000004468; OMIM: 107270; UniProtKB: P28907.

CD44 is also known as CD44 molecule (Indian blood group); IN; MDU2; CD44 antigen (Homing function and Indian blood group system); MDU3; CDW44; MIC4; CSPG8; chondroitin sulfate proteoglycan 8; HCELL; hematopoietic cell E - and L-selectin ligand; MC56; extracellular matrix receptor III; Pgp1; heparan sulfate (Heparan Sulfate) proteoglycan; cell surface glycoprotein CD44; ); phagocyte glycoprotein 1; homing function and Indian blood group system; ECMR-III; CDw44; HUTCH-I; Homing/adhesion receptor; phagocyte glycoprotein I; Hermes antigen. Ids: HGNC: 1681; Entrez Gene: 960; Ensembl: ENSG00000026508; OMIM: 107269; UniProtKB: P16070.

CD123 is also known as cell division cycle 123; cell division cycle 123 homolog; C10orf7; cell division cycle 123 homolog; D 123; protein D123; HT-1080; CCEP123; PZ32; CEP89; cell division cycle 123 homolog (S. Cerevisiae); FLJ14640; chromosome 10 open reading frame 7. Ids: HGNC: 16827; EntrezGene: 8872; Ensembl: ENSG00000151465; OMIM: 615470; UniProtKB: 075794.

CD138 is also known as Syndecan 1 (SCD1); CD138; SDC; Heparan sulfate proteoglycan fibroblast growth factor receptor; Syndecan Proteoglycan1; Syndecan; SYND1; Syndecan-1; CD138 antigen. Ids: HGNC: 10658; EntrezGene: 6382; Ensembl: ENSG00000115884; OMIM: 186355; UniProtKB: P18827.

CEA is also known as carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5); meconium antigen 100; CD66e; carcinoembryonic antigen; CD66e antigen. Ids: HGNC: 1817; Entrez Gene: 1048; Ensembl: ENSG00000105388; OMIM: 114890; UniProtKB: P06731.

EGFR is also known as epidermal growth factor receptor; erythroblast leukemia virus (V-Erb-B) oncogene homolog (avian); ERBB1; PIG61; proto-oncogene C-ErbB-1; avian erythroblast leukemia virus (V-Erb-B) oncogene homolog; receptor tyrosine protein kinase ErbB-1; cytostatic protein 40; cell proliferation-inducing protein 61; HER1; mENA; EC 2.7.10.1; EC 2.7.10; Epidermal growth factor receptor (avian erythroblast leukemia virus (V-Erb-B) oncogene homolog). Ids: HGNC: 3236; Entrez Gene: 1956; Ensembl: ENSG00000146648; OMIM: 131550; 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 (DLL3) is also known as Delta-Like 3); Drosophila Delta homolog 3; Delta3; Delta (Drosophila) like 3; SCDO1. The Ids of DLL3 are: HGNC: 2909; Entrez Gene: 10683; Ensembl: ENSG00000090932; OMIM: 602768 and UniProtKB: Q9NYJ7.

LGR5 is a Leucine-Rich Repeat Containing G Protein-Coupled Receptor 5 (Leucine-Rich Repeat Containing G Protein-Coupled Receptor 5). Alternative names for this gene or protein are leucine-rich repeat-containing G protein-coupled receptor 5; leucine-rich repeat-containing G protein-coupled receptor 5; G protein-coupled receptor HG38; G protein G protein-coupled receptor 49; G protein-coupled receptor 67; GPR67; GPR49; Orphan GProtein-Coupled Receptor HG38; G protein-coupled receptor 49; GPR49; HG38 and FEX. The LGR5-binding protein or antibody of the invention binds human LGR5. Due to the similarity in sequence and tertiary structure between human and other mammalian xenologs, the LGR5 binding proteins or antibodies of the invention may also bind such xenologs, but not necessarily. The database accession numbers for the human LGR5 protein and the gene encoding it are (NC_000012.12; NT_029419.13; NC_018923.2; NP_001264155.1; NP_001264156.1; NP_003658.1).

MSLN or mesothelin Also known as Mesothelin; Pre-Pro-Megakaryocyte-Potentiating Factor; CAK1 Antigen; MPF; Soluble MPF Mesothelin-Associated Protein; Cell potentiation factor and SMRP. The Ids of MSLN are: HGNC: 7371; Entrez Gene: 10232; Ensembl: ENSG00000102854; OMIM: 601051; UniProtKB: Q13421.

Folate receptor 1 also known as FOLR1; folate receptor 1; ovarian tumor-associated antigen MOv18; adult folate binding protein; folate receptor, adult; KB cell FBP; FR-alpha; FOLR; FBP; folate binding protein; body 1. The Ids of FOLR1 are HGNC: 3791; Entrez Gene: 2348; Ensembl: ENSG00000110195; OMIM: 136430; UniProtKB: P15328.

Folate receptor 3 is also referred to as FOLR3; folate receptor 3(γ); FR-γ; folate receptor 3; γ-HFR; and FR-G. The Ids for FOLR3 are HGNC: 3795; Entrez Gene: 2352; Ensembl: ENSG00000110203; OMIM: 602469; and UniProtKB: P41439.

EPCAM is also known as Epithelial Cell Adhesion Molecule; EGP40; M4S1; ESA; MIC18; KS1/4; Tumor-Associated Calcium Signal Transducer 1; MK-1; TACSTD1; Human Epiglin-2 ; TROP1; membrane fraction, chromosome 4, surface marker (35kD glycoprotein); adenocarcinoma-associated antigen; EGP; cell surface glycoprotein Trop-1; Ep-CAM; Protein GA733-2; M1S2; EGP314; CD326 antigen; KSA; Epithelial cell surface antigen; D IAR5; Epithelin; HNPCC8; hEGP314; Ids: HGNC: 11529; Entrez Gene: 4072; Ensembl: ENSG00000119888; OMIM: 185535; UniProtKB: P16422.

HER2 also known as V-Erb-B2 avian erythroblast leukemia virus oncogene homolog 2; ERBB2; CD340; NGL; HER-2; HER-2/neu2; NEU2; TKR1; neuro/glioblastoma Derived oncogene homolog; C-Erb B2/Neu protein; metastatic lymph node gene 19 protein; herstatin; proto-oncogene C-ErbB-2; neuroblastoma/glioblastoma Tumor-derived oncogene homolog; proto-oncogene Neu; receptor tyrosine protein kinase ErbB-2; tyrosine kinase type cell surface receptor HER2; V-Erb-B2 erythroblast leukemia virus oncogene homolog Compound 2, neuro/glioblastoma-derived oncogene homolog; MLN 19; MLN19; p185erbB2; CD340 antigen; EC 2.7.10.1; EC 2.7.10; V-Erb-B2 avian erythroblast leukemia virus Oncogene homolog 2 (neural/glioblastoma-derived oncogene homolog). Ids:

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

HM1.24 is also known as BST2; Bone Marrow Stromal Cell Antigen 2; TETHERIN; BST-2; Bone Marrow Stromal Antigen 2; HM1.24 Antigen; Tetherin; CD317; CD317 Antigen; NPC-A-7. Ids: HGNC: 1119; EntrezGene: 684; Ensembl: ENSG00000130303; OMIM: 600534; UniProtKB: Q10589.

MCSP is also known as Sperm Mitochondria-Associated Cysteine-Rich Protein (SMCP); MCSP; MCS; Mitochondrial Capsule Selenoprotein; HSMCSGEN1; Body-associated cysteine-rich protein. Ids: HGNC: 6962; Entrez Gene: 4184; Ensembl: ENSG00000163206; OMIM: 601148; 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 allografting, autoimmune diseases and other disease states such as hepatitis. Binding of PD-L1 to PD-1 or B7.1 (CD80) transmits an inhibitory message which reduces the proliferation of PD-1 expressing T cells. It is generally believed that PD-1 can control the accumulation of foreign antigen-specific T cells through apoptosis. A variety of cancer cells express PD-L1, and its expression is thought to be responsible, at least in part, for slowing the immune response against cancer cells. PD-L1 is a member of the B7 family of proteins and is known by various other names such as CD274 molecule; CD274 antigen; B7 homolog 1; PDCD1 ligand 1; PDCD1LG1; PDCD1L1; B7H1; PDL1; planned cell death 1 ligand 1; planned death ligand 1; B7-H1; and B7-H. The external Ids of CD274 are HGNC: 17635; Entrez Gene: 29126; Ensembl: ENSG00000120217; OMIM: 605402; UniProtKB: Q9NZQ7.

PSMA also known as folate hydrolase (prostate-specific membrane antigen) 1; FOLH1; NAALAD1; FOLH; mGCP; glutamic acid carboxypeptidase II; N-acetylated-α-linked acid dipeptidase I; PSM; NAALADase I; PSMA; EC 3.4.17.21; Glutamate carboxylase II; GCP2; cytostatic gene 27 protein; NAALAdase; ; Membrane glutamic acid carboxypeptidase; N-acetylated α-linked acid dipeptidase 1; Pteroylpoly-γ-glutamic acid carboxypeptidase; Prostate specific membrane antigen variant F; FGCP; Folate hydrolase 1; GCPII; prostate specific membrane antigen. Ids: HGNC: 3788; Entrez Gene: 2346; Ensembl: ENSG00000086205; OMIM: 600934; UniProtKB: Q04609.

PSMA is not to be confused with the proteasome (Prosome, Macropain) subunit, alpha type, 1, which is also known by its alias as PSMA1.

Accession numbers are given primarily to provide an additional method of target identification, the actual sequence of the protein bound may vary, eg, due to mutations in the encoding gene, such as occurs in some cancers and the like. Antigen-binding addresses 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 a gene or protein is referred to herein, it refers to the native gene or protein as well as variant forms of the gene or protein, as can be detected in tumors, cancers and the like, preferably human tumors, cancers and the like Detectable in cancer.

Bispecific or multispecific antibodies of the invention preferably bind 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 human CLEC12A or a variant thereof. Preferred bispecific antibodies according to the invention bind human CD3 and human CLEC12A or a variant thereof. In a preferred embodiment, the multispecific antibody binds to CD3, PD-L1 and EGFR.

HGNC stands for HUGO Gene Nomenclature Committee. The number after the abbreviation is the accession number, and the information of the gene and the protein encoded by the gene can be retrieved from the HGNC database with the accession number. Entrez Gene provides accession number or gene ID, which can retrieve the information of the gene or the protein encoded by the gene from the NCBI (National Center for Biotechnology Information) database. Ensemble provides accession numbers with which information about the gene or the protein encoded by the gene can be obtained from the Ensemble database. Ensembl is a joint project between EMBL-EB and the Wellcome Trust Sanger Institute to develop a software system that automatically annotates and maintains selected eukaryotic genomes.

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

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

CDR1: SFGIS; CDR2: GFIPVLGTANYAQKFQG; CDR3: RGNWNPFDP

or

Includes the following amino acid sequences:

CDR1: SX ₁ TFTIS;

CDR2: GIIPX ₂ FGTITYAQKFQG;

CDR3: RGNWNPFDP;

in

X ₁ =K or R; X ₂ =L or I.

In a preferred embodiment, X ₁ =K; and X ₂ =L. In another preferred embodiment, X ₁ =R; and X ₂ =I.

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

CDR1: SKTLTIS; CDR2: GIIPIFGSITYAQKFQD; CDR3: RGNWNPFDP; or

Includes the following amino acid sequences:

CDR1: GSGIS; CDR2: GFIPFFGSANYAQKFRD; CDR3: RGNWNPX ₁₃ DP;

in

X ₁₃ =or L or F.

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

CDR1: RX3WIG; CDR2: IIYPGDSDTRYSPSFQG; CDR3: X ₄ IRYFX ₅ WSEDYHYYX ₆ DV;

in

_X3 =F or Y; _X4 =H or N _; X5=D or V _; and X6=L or M.

In preferred embodiments, X ₃ =F; X ₄ =H; X ₅ =D; and X ₆ =L; or X ₃ =Y; X ₄ =N; X ₅ =V; and X ₆ =M.

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

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

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

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

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

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

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

CDR1: DYTMH;

CDR2: DISSWX ₇ GX ₈ X ₉ X ₁₀ YADSVKG;

CDR3: DHX ₁₁ GYGDYEGGGFDX ₁₂ ;

in

X ₇ =S or G;

X ₈ =S or T;

X ₉ =I or T;

X ₁₀ =G or Y;

X ₁₁ =R or M;

X ₁₂ =H or Y,

Preferably X ₇ , X ₈ , X ₉ and X ₁₀ are S, S, I and G or G, S, I and Y or S, T, T and G, and preferably X ₁₁ and X ₁₂ are R and H , or R and Y, or M and Y, more preferably X ₇ , X ₈ , X ₉ , X ₁₀ , X ₁₁ and X ₁₂ are S, S, I, G, R and H or G, S, I, Y, R and Y are either S, T, T, G, M and Y, or in other words, preferably X ₇ , X ₈ , X ₉ and X ₁₀ are S, S, I and G, and X ₁₁ and X ₁₂ are R and H; or X ₇ , X ₈ , X ₉ and X ₁₀ are G, S, I and Y and X ₁₁ and X ₁₂ are R and Y; or X ₇ , X ₈ , X ₉ and X ₁₀ are S, T, T and G, and X ₁₁ and X ₁₂ are M and Y.

In a preferred embodiment, the light chain variable region comprises the IgVκ1-39*01 gene segment as depicted in Figure 11A, with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or amino acid sequences of combinations thereof. The amino acid sequence of IgV _K 1-39*01 is depicted in Figure 11A. IgVκ1-39 is the abbreviation for Immunoglobulin Variable κ1-39 Gene. This gene is also known as immunoglobulin kappa variable 1-39; IGKV139; IGKV1-39. The external Ids of this gene are HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. Preferred amino acid sequences of IgVκ1-39 are provided in Figure 11A. This row lists the sequence of V regions. The V zone can be combined with one of the five J zones. Figures 11B and 11D depict two preferred sequences for the IgVκ 1-39 sequence combined with a J region. The linked sequences are denoted as IGKV1-39/jk1 and IGKV1-39/jk5; alternative names are IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01 (named according to the IMGT database World Wide Web at imgt.org ).

IgVκ1-39*01 including the light chain variable region is preferred for the germline sequence. IGJκ1*01 or /IGJκ5*01 including the light chain variable region are preferred as germline sequences. In a preferred embodiment, the IGKV1-39/jk1 or IGKV1-39/jk5 light chain variable region is a germline sequence.

In a preferred embodiment, the light chain variable region includes germline series IgVκ1-39*01. In a preferred embodiment, the light chain variable region includes a κ light chain IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01. In a preferred embodiment, IgVκ1-39*01/IGJκ1*01. The light chain variable region preferably comprises a germline κ light chain IgVκ1-39*01/IGJκ1*01 or a germline κ light chain IgVκ1-39*01/IGJκ5*01, preferably a germline κ light chain IgVκ1-39*01/IGJκ1* 01.

Mature B cells that produce antibodies with a light chain typically produce a light chain that has undergone one or more mutations relative to the germline sequence, the normal sequence of the organism's non-lymphocytes. The process responsible for these mutations is often referred to as somatic (hyper)mutation. The resulting light chain is referred to as an affinity matured light chain. These light chains, when derived from the germline IgVκ1-39*01 sequence, are IgVκ1-39*01 derived light chains. In this specification, the term "IgVκ1-39*01" will include IgVκ1-39*01 derived light chains, mutations introduced by somatic hypermutation can also be introduced artificially in the laboratory. Other mutations or variants can also be introduced into the light chain in the laboratory without affecting the type, not necessarily quantitative, nature of the light chain. A light chain design has 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof, regardless of whether it comprises a sequence as depicted in Figure 11A, Figure 11 or Figure 11. How is an IgVκ1-39*01 light chain. In a preferred embodiment, the IgVκ1-39*01 light chain is a light chain comprising a sequence as depicted in Figure 11A, Figure 11B or Figure 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 a preferred embodiment, the IgVκ1-39*01 light chain is a light chain comprising a sequence as depicted in Figure 11A, Figure 11B or Figure 11C, which has 0-5, preferably 0-4, more Preferably 0-3 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof. In a preferred embodiment, the IgVκ1-39*01 light chain is a light chain comprising a sequence as depicted in Figure 11A, Figure 11B or Figure 11C, which has 0-2, more preferably 0-1, Most preferred are zero amino acid variations, insertions, deletions, substitutions, additions, or combinations thereof. In a preferred embodiment, the IgVκ1-39*01 light chain is a light chain comprising a sequence as depicted in Figure 11A or Figure 11B with the mentioned amino acid variations, insertions, deletions, substitutions, additions or the like The combination. In a preferred embodiment, the light chain includes the sequence of Figure 11B.

The light chains preferably include a common light chain variable region. The common light chain variable region preferably includes an IgVκ1-39 light chain variable region. The light chain variable region is preferably a germline IgVκ1-39*01 variable region. The light chain variable region preferably comprises a kappa light chain IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01. The light chain variable region preferably comprises a germline kappa light chain IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01. The light chain variable region preferably comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGSGSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVTITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTPEDTD FTLITQQ SSLQ SGVPS RFSGS GSGTPED FAT FGYY QGTRL EIK with 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 comprising the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTP, 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 of the light chain variable region, preferably not within the CDR1 or CDR2 region of the light chain variable region. In preferred embodiments, the light chain variable region does not include deletions, additions or variations with respect to the sequence shown. In this embodiment, the light chain variable region may have 0-5 amino acid substitutions for the amino acid sequence shown. Amino acid substitutions are preferably reserved amino acid substitutions. 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, ie 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 previously described is preferably a bispecific antibody. A "bispecific antibody" as described herein is an antibody wherein one domain of the antibody binds a first antigen and a second domain of the antibody binds 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 in which the VH can specifically recognize a first antigen and the VL paired with the VH of the immunoglobulin variable region can specifically recognize a second antigen. The resulting VH/VL pair will bind either Antigen 1 or Antigen 2. These so-called "two-in-one antibodies" are described, for example, in WO2008/027236, WO2010/108127 and Schaefer et al. (Cancer Cell 20, 472-486, October 2011). Bispecific antibodies such as the present invention are not limited to any particular bispecific format or method of production thereof.

The bispecific antibody preferably has a 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 an antigen on CD3. 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 a preferred embodiment, the bispecific antibody is a full-length antibody having a heavy/light (H/L) chain combination that binds CD3 and an H/L chain that binds another antigen, preferably a tumor antigen combination. 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 completely identical, that is, a similar or identical human light chain is a so-called "common light chain", which is a A light chain that can be combined with different heavy chains to form antibodies with functional antigen binding domains. In a preferred embodiment, 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 in the first and second H/L chain combinations are completely identical, that is, 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 that can be combined with different heavy chain variable regions to form an antibody with a functional antigen binding domain. The light chains comprising a common light chain variable region are preferably a common light chain.

The common light chain within the bispecific antibody is preferably an IgVκ1-39 light chain as indicated above.

The invention also provides optional bispecific antibody formats, such as Spiess, C. et al. B (Alternativemolecular formats and therapeutic applications for bispecificantibodies. Mol. Immunol. (2015) http:dx.doi.org/10.1016/j. such as described in molimm.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, monobody, VH and Fab fragment to provide a second binding activity.

In the aspect of the bispecific antibody of the present invention, the light chain in the CD3-binding H/L chain combination is preferably similar to the light chain in the H/V chain combination that binds an antigen other than CD3, preferably a tumor antigen. In a more preferred embodiment, the light chains in the two H/L chain combinations are completely identical, that is, the human light chain is a so-called "common light chain", which is a kind of light chain that can be combined with different heavy chains to Form the light chain of an antibody with a functional antigen-binding domain. Preferably, the common light chain has germline sequences. Preferred germline sequences are light chain variable regions that are commonly used in human repertoires and have good thermodynamic stability, yield and solubility. A preferred germline sequence is IgVκ1-39, preferably a rearranged germline human kappa light chain IgVκ1-39*01/IGJκ1*01 or a fragment or functional equivalent thereof (ie the same IgVκ1-39 gene segment but IGJκ different gene segments) (named according to the IMGT database World Wide Web at imgt.org).

The term "abnormal cell" as used herein includes tumor cells, more particularly blood-derived tumor cells, but also preleukemic cells such as those responsible for myelodysplastic syndrome (MDS) and leukemic cells such as acute myeloid leukemia (AML). ) tumor cells or chronic myeloid leukemia (CML) cells.

The term "immune effector cell" or "effector cell" as used herein refers to cells within the natural repertoire of cells within the mammalian immune system that can be activated to affect the viability of target cells. 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 Sexual pellets. Therefore, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells or neutrophils. Recruiting effector cells to abnormal cells means bringing immune effector cells into close proximity to abnormal target cells so that the effector cells can directly kill abnormal cells or indirectly initiate killing of abnormal cells.

As used herein, the terms "individual" and "patient" are used interchangeably and refer to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cows, horses, pigs, etc. (eg patients with cancer, eg human patients).

As used herein, the terms "treat," "treating," and "treatment" refer to performing any type of intervention or procedure on an individual or administering an active agent or combination of active agents to that individual , and for the purpose of reversing, alleviating, ameliorating, inhibiting or slowing down or preventing the progression, progression, severity or recurrence of a symptom, complication, condition or biochemical marker associated with the disease.

As used herein, "effective treatment" or "positive treatment response" refers to treatment that produces a beneficial effect, eg, alleviation of at least one symptom of a disease or disorder, such as cancer. A beneficial effect can be presented as an improvement from baseline, including improvement from 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 markers of cancer. Effective treatment can, 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 a formulation or combination of formulations that provides the desired biological, therapeutic and/or prophylactic result. The result may be a reduction, amelioration, mitigation, attenuation, delay and/or alleviation of one or more signs, symptoms or causes of a disease, or any other desired change in 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 can: (i) reduce the number of cancer cells; (ii) reduce the size of the tumor; (iii) to a certain extent inhibit, slow, slow and stop the infiltration of cancer cells into surrounding organs; ( iv) inhibiting tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying tumor development and/or recurrence; and/or (vii) alleviating to some extent one or more symptoms associated with cancer. In one example, a "therapeutic amount" is the amount of a CLEC12A/CD3 bispecific antibody that reduces cancer (eg, a reduction in the number of cancer cells) or slows cancer, eg, acute myeloid leukemia, myelodysplastic syndrome, or chronic myelogenous leukemia Progress.

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

There are 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 provide an antigen-binding protein that can bind to human CD3, preferably an antibody, comprising an antibody variable domain, the antibody variable domain comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is provided. chain variable region including amino acid sequence

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

Also 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 is chain variable region including amino acid sequence

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

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

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

The amino acid variation, insertion, deletion, substitution, addition or combination thereof is preferably not within the CDR3 region of the heavy chain variable 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 sequence shown. In a specific example, the heavy chain variable region may have 0-10, preferably 0-5, amino acid substitutions with respect to the amino acid sequence shown. In preferred embodiments, the heavy chain variable region includes 0-9, 0-8, 0-7, 0-6, 0-5 at positions other than such CDRs with respect to the indicated amino acid sequence , 0-4, preferably 0-3, preferably 0-2, preferably 0-1 and preferably 0 amino acid variations, insertions, deletions, substitutions, additions, or combinations thereof. Provided that the aligned sequences do not differ by more than 10 positions, preferably not by more than 5 positions, then a combination of insertions, additions, deletions or substitutions is the claimed combination. A gap in one of the aligned sequences will count as as many amino acids as skipped in the other sequence. An amino acid substitution, if any, is preferably a reserved amino acid substitution.

The present invention further provides an antigen-binding protein that binds human CD3, preferably an antibody, comprising an antibody variable domain, the 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 MF8057; MF8058 or MF8078 as depicted in Figure 13.

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

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

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

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

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNANTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDYWGQGTLVTVSS with 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)

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

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, among others, especially for this purpose herein It is mentioned and incorporated herein by reference. The heavy chain variable region of this heavy/light chain combination that will bind human EGFR or CLEC12A can have 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions, for the amino acid sequence shown or a combination thereof. In a preferred embodiment, the heavy chain variable region includes 0-9, 0-8, 0-7, 0-6, 0-5, 0-4 in terms of the shown amino acid sequence, preferably 0 -3, preferably 0-2, preferably 0-1 and preferably 0 amino acid variations, insertions, deletions, substitutions, additions, or a combination thereof. A combination of insertions, deletions, additions or substitutions is the claimed combination, provided that the aligned sequences do not differ by more than 5 positions. A gap in one of the aligned sequences will count as as many amino acids as skipped in the other sequence.

An amino acid variation, insertion, deletion, substitution, addition or combination thereof is preferably not made/present in the heavy and light chain binding interface.

Provided that if the amino acid in the interface of the H/L chain interaction is changed, it is preferred that the corresponding amino acid in the other chain is also changed to accommodate the change. The insertion or addition of amino acids preferably does not require insertion or addition of proline.

The addition of amino acids can in principle be considered the same as insertion. The addition of amino acids to one end of a polypeptide chain is sometimes not considered an insertion but rather a strict addition (extension). For the purposes of the present invention, both addition 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 of the heavy chain variable region, preferably not within the CDR1 or CDR2 region of the heavy chain variable region. In preferred embodiments, the heavy chain variable region does not include deletions, additions or variations with respect to the sequence shown. In this particular example, the heavy chain variable region may have 0-5 amino acid substitutions for the amino acid sequence shown. Amino acid substitutions are preferably reserved amino acid substitutions. The CD3 binding VH CDR1, CDR2 and CDR3 of the invention preferably comprise the combination of CD3 binding VH CDR1, CD2 and CDR3 depicted in Figure 13, preferably the VH of one of MF8057; MF8058; MF8078; MF8397; MF8508; MF9249 or MF9267 .

The constant regions of the antibodies of the invention, including bispecific or multispecific antibodies, are preferably human constant regions. 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 domains of the antibodies produced herein are derived from a library of human antibody variable domains. Therefore these variable domains are human. Unique CDR regions can be derived from humans, 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 exemplified in FIG. 12 without limitation.

Various methods of producing antibodies exist in the art. Antibodies are typically produced by cells expressing nucleic acid encoding the antibody. Suitable antibody producing cells are fusion tumor cells, Chinese Hamster Ovary (CHO) cells, NSO cells or PER-C6 cells. In a particularly preferred embodiment, the cells are CHO cells.

Various institutions 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, NSO cells or PER.C6 cells. These cells are also used for other purposes such as protein production. Cell lines developed for industrial scale production of proteins and antibodies are further referred to herein as industrial cell lines. In a preferred embodiment, the present invention provides an industrial cell line that produces the antibody of the present invention.

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

The present invention further provides a cell comprising the antibody of the present invention. Preferably the cell (typically an in vitro, isolated or recombinant cell) produces the antibody. In a preferred embodiment, the cells are fusion tumor cells, Chinese hamster ovary (CHO) cells, NSO cells or PER-C6 cells. In a particularly preferred embodiment the cells are CHO cells. Further provided is a cell culture comprising the cells of the present invention. Various institutions 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, NSO cells or PER.C6 cells. These cells are also used for other purposes such as protein production. Cell lines developed for industrial scale production of proteins and antibodies are further referred to herein as industrial cell lines. Thus, in a preferred embodiment, the present invention provides the use of developing a cell line for large-scale production of antibodies for producing the antibodies of the present invention. The present invention further provides a cell for producing 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 shown in Figure 13, a nucleic acid molecule encoding VH as shown by numeral 4327 or shown by numeral 8233, or a combination thereof.

The invention further provides a method for producing an antibody comprising culturing a 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 such as the present invention. The antibody is preferably purified from the culture medium. The antibody ratio is preferably affinity purified.

Cells of the invention are, for example, fusion tumor cell lines, CHO cells, 293F cells, NSO cells or another cell type known to be suitable for antibody production for clinical purposes. In a particularly preferred embodiment the cells are human cells. Preferred is a cell transformed with the E1 region of adenovirus or a functional equivalent thereof. A preferred example of such a cell line is the PER.C6 cell line or an effector thereof. In a particularly preferred embodiment the cells are CHO cells or variants thereof. Preferred is a variant that utilizes the glutamic acid synthase (GS) vector system for expression of the antibody.

The invention further provides a method for producing an antibody comprising culturing a 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 such as the present invention. The antibody is preferably purified from the culture medium. The antibody ratio is preferably affinity purified.

Bispecific antibodies are also typically produced by cells expressing the nucleic acid encoding the antibody. In this case the cells express different light and heavy chains that make up the bispecific antibody. To this end the cells express 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 with a heavy chain variable region and a second heavy chain with at least two heavy chain variable regions, such that these two heavy chain variable regions are expressed Heavy chain cells produce a monospecific antibody (two homodimers paired with a first heavy chain), a quadrovalent antibody (two homodimers paired with a second heavy chain), and trispecific antibodies Antibodies (heterodimers of first and second heavy chains). When cells express two or more light chains, the number of possible heavy/light chain combinations in the antibody produced increases. The production of the aforementioned "common light chain" is preferred in order to reduce the number of different antibody species (different heavy and light chain combinations).

An antibody-producing cell expressing a common light chain and equal amounts of both heavy chains typically produces 50% bispecific antibody and 25% each monospecific antibody (ie, having the exact same combination of heavy and light chains). Optionally, in the above example for a first heavy chain with one variable region and a second heavy chain with two variable regions, the two heavy chains would typically produce 50% trispecificity , 25% monospecific and 25% quadrospecific.

Several methods have been disclosed to facilitate the production of bispecific antibodies or conversely monospecific antibodies, which can further be used to facilitate the production of multispecific antibodies. It is preferred in the present invention that the cells favor the production of the bispecific antibody over the production of the corresponding monospecific antibody. This is usually accomplished by modifying the constant region of the heavy chain so that it favors heterodimerization (ie dimerization of the heavy chain with another heavy/light chain combination) over homodimerization. In a preferred embodiment the bispecific antibodies of the invention comprise two different immunoglobulin heavy chains with compatible heterodimeric domains. Various compatible heterodimeric domains have been described in the art. The compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. The art describes various methods by which heterodimerization of these heavy chains can be accomplished, including the use of "knob into hole" bispecific antibodies.

Compatible use is disclosed in US 13/866,747 (now issued as US 9,248,181), US 14/081,848 (now issued as US 9,358,286) and PCT/NL2013/050294 (published as WO2013/157954; incorporated herein by reference) The heteropolymeric two domains are provided for methods and building blocks for the production of bispecific antibodies. These components and methods may also be advantageously used in the present invention. In particular, preferred mutations that produce substantially only bispecific full-length IgG molecules are amino acid substitutions L351K and T366K (according to EU numbering) of the first CH3 domain and amino acids of the second domain ("KK-variant" heavy chain) Substitutes L351D and L368E ("DE-variant" heavy chains), 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 pair preferentially 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, homodimerization of DE-variant heavy chains (DEDE homodimers) or homodimerization of KK-variant heavy chains Mass dimerization (KKKK homodimer) hardly occurs. In one embodiment, a heavy chain/light chain combination that includes a variable domain that binds CD3 includes a KK variant of the heavy chain. This specific example includes a heavy chain/light chain combination that binds the variable domains of antigens other than CD3, including DE variants of the heavy chain. In a preferred specific example, the antigen other than CD3 is CLEC12A. In a preferred embodiment, the VH that binds to the variable domain of CLEC12A is MF4327 as depicted in Figure 13 .

Some antibodies are pre-modified in the CH2/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 mutant CH2 and/or lower hinge domains such that the interaction of the bispecific IgG antibodies with Fc-gamma receptors is reduced. 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, eg, to enhance Fc receptor interaction or to enhance C1q binding. With regard to specific examples for autoimmune indications, this modification may be preferred.

The invention further provides a method of treating an individual comprising administering an antigen binding protein, preferably an antibody, of the invention to the individual in need thereof. The present invention further provides an antigen binding protein of the present invention, preferably an antibody, for use in the treatment of an individual in need. The individual preferably has cells to be removed from the body. Such cells can be abnormal immune cells or cancer cells that are directed toward an autoimmune response, among others. Suitable variable domains for this purpose are those with a common light chain and the VHs of MF9249 and MF8397 and others. These have moderately low affinity and low cell killing activity but are functional under autoimmune reaction conditions. Suitable variable domains for this purpose are these and others with a common light chain and VH of MF9267, MF8057, MF8058, MF8078 and MF8508. These variable domains have suitable affinity and suitable cell killing activity for anticancer purposes.

In addition, an invention set forth herein includes an antigen binding protein or antibody having a high affinity variable domain with high cell killing activity that can be administered or expressed locally, including the binding domain MF8078. The present invention further provides a method of treating an individual comprising administering an antigen binding protein of the present invention, preferably an antibody, to the individual in need thereof via a localized means of administration as known to those skilled in the art , including, for example, an oncolytic virus for local treatment of melanoma or other cancers in isolated compartments such as the brain.

The present invention further provides an antigen binding protein of the present invention, preferably an antibody, for use in the treatment of an individual in need thereof, which preferably has moderate to high affinity and relatively high cytotoxicity such as those described herein. Suitable variable domains for this purpose are those with a common light chain and the VHs of MF8057, MF8058, MF9267, MF8508 and MF8078 and others.

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 include the following amino acid sequences:

CDR1: SFGIS

CDR2: GFIPVLGTANYAQKFQG

CDR3: RGNWNPFDP; or

Includes the following amino acid sequences:

CDR1: SX ₁ TFTIS;

CDR2: GIIPX ₂ FGTITYAQKFQG;

CDR3: RGNWNPFDP;

in

X ₁ =K or R;

X ₂ =L or I.

In a preferred embodiment, X ₁ =K; and X ₂ =L. In another preferred embodiment, X ₁ =R; and X ₂ =I.

The present invention further provides antigen binding proteins of the present invention, preferably antibodies, for use in the treatment of an individual in need thereof, which preferably have moderate to high affinity and relatively high cytotoxicity such as those described herein. Variable domains suitable for this purpose are those with a common light chain and the VH of MF8048, MF8056 and MF8101 and others.

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, which includes the following amino acid sequence:

CDR1: SKTLTIS;

CDR2: GIIPIFGSITYAQKFQD;

CDR3: RGNWNPFDP; or

Includes the following amino acid sequences:

CDR1: GSGIS;

CDR2: GFIPFFGSANYAQKFRD;

CDR3: RGNWNPX _13DP ;

in

X ₁₃ =or L or F.

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes an amino acid sequence

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

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 include the following amino acid sequences:

CDR1: RX ₃ WIG;

CDR2: IIYPGDSDTRYSPSFQG;

CDR3: X ₄ IRYFX ₅ WSEDYHYYX ₆ DV;

in

X ₃ =F or Y;

X ₄ =H or N;

X ₅ =D or V;

X ₆ =L or M.

In a specific example, X ₃ =F; X ₄ =H; X ₅ =D; and X ₆ =L. In another specific example, X ₃ =Y; X ₄ =N; X ₅ =V; and X ₆ =M.

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes an amino acid sequence

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

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 include the following amino acid sequences:

CDR1:SYALS;

CDR2:GISGSGRTTWYADSVKG;

CDR3: DGGYSYGPYWYFDL.

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 include the following amino acid sequences:

CDR1:SYALS;

CDR2: AISGSGRTTWYADSVKG;

CDR3: DGGYTYGPYWYFDL.

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes an amino acid sequence

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

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 include the following amino acid sequences:

CDR1: DYTMH;

CDR2: DISWSSGSIGYADSVKG;

CDR3: DHRGYGDYEGGGFDY.

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 include the following amino acid sequences:

CDR1: DYTMH;

CDR2: DISSWX ₇ GX ₈ X ₉ X ₁₀ YADSVKG;

CDR3: DHX ₁₁ GYGDYEGGGFDX ₁₂ ;

in

X ₇ =S or G;

X ₈ =S or T;

X ₉ =I or T;

X ₁₀ =G or Y;

X ₁₁ =R or M;

X ₁₂ =H or Y,

Preferably X ₇ , X ₈ , X ₉ and X ₁₀ are S, S, I and G or G, S, I and Y or S, T, T and G, and preferably X ₁₁ and X ₁₂ are R and H , or R and Y, or M and Y, more preferably X ₇ , X ₈ , X ₉ , X ₁₀ , X ₁₁ and X ₁₂ are S, S, I, G, R and H or G, S, I, Y, R and Y are either S, T, T, G, M and Y, or in other words, preferably X ₇ , X ₈ , X ₉ and X ₁₀ are S, S, I and G, and X ₁₁ and X ₁₂ are R and H; or X ₇ , X ₈ , X ₉ and X ₁₀ are G, S, I and Y and X ₁₁ and X ₁₂ are R and Y; or X ₇ , X ₈ , X ₉ and X ₁₀ are S, T, T and G, and X ₁₁ and X ₁₂ are M and Y.

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, preferably an antibody, which binds human CD3, comprising an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes an amino acid sequence

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

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

The antibody is preferably a human or humanized antibody. The antibody preferably comprises two different immunoglobulin heavy chains with compatible heterodimeric domains. The compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. The bispecific antibody is preferably an IgG antibody with mutant CH2 and/or lower hinge domains for tumor immunology applications such that the interaction of the bispecific or multispecific IgG antibody with Fc-gamma receptors is reduced. The 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, for autoimmune applications, the interaction of the bispecific or multispecific with Fc-gamma receptors is enhanced by modifying the CH2 and/or CH3 domains. For example, engineering (by introducing amino acid substitutions) the Fc region of a greater selective binding to an activated receptor can create the desired ability of an anticancer Mab or CD3 targeting binding arm to have greater mediator cytotoxic activity Antibodies for the treatment of autoimmune related diseases. For example, one reported technique for enhancing ADCC of antibodies is afucosylation. (See eg, Junttila, T.T., K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70(11):4481-4489). There is thus further provided a bispecific antibody according to the invention which is afucosylated. Alternatively, or additionally, various other strategies have been reported to be used to achieve ADCC enhancement, including, for example, glycoengineering (Kyowa Hakko/Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics). , all seek to improve Fc binding to low affinity activated FcγRIIIa, and/or to reduce binding to low affinity inhibitory FcγRIIb.

The antibodies preferably include 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 human CD3, wherein the variable domain Each includes a distinct heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds the variable domains of human CD3 includes a CDR1, CDR2, and CDR3, the CDR1, CDR2 and CDR3 includes the following amino acid sequence:

CDR1: SFGIS

CDR2: GFIPVLGTANYAQKFQG

CDR3: RGNWNPFDP; or

Includes the following amino acid sequences:

CDR1: SX ₁ TFTIS;

CDR2: GIIPX ₂ FGTITYAQKFQG;

CDR3: RGNWNPFDP;

in

X ₁ =K or R;

X ₂ =L or I.

In a preferred embodiment, X ₁ =K; and X ₂ =L. In another preferred embodiment, X ₁ =R; and X ₂ =I.

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 human CD3, wherein the variable domain Each includes a distinct heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds the variable domains of human CD3 includes a CDR1, CDR2, and CDR3, the CDR1, CDR2 and CDR3 includes the following amino acid sequence:

CDR1: SKTLTIS;

CDR2: GIIPIFGSITYAQKFQD;

CDR3: RGNWNPFDP; or

Includes the following amino acid sequences:

CDR1: GSGIS;

CDR2: GFIPFFGSANYAQKFRD;

CDR3: RGNWNPX _13DP ;

in

X ₁₃ =or 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 human CD3, wherein the variable domain Each includes a distinct heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds the variable domain of human CD3 includes the amino acid sequence

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

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 human CD3, wherein the variable domain Each includes a distinct heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds the variable domains of human CD3 includes a CDR1, CDR2, and CDR3, the CDR1, CDR2 and CDR3 includes the following amino acid sequence:

CDR1: RX ₃ WIG;

CDR2: IIYPGDSDTRYSPSFQG;

CDR3: X ₄ IRYFX ₅ WSEDYHYYX ₆ DV;

in

X ₃ =F or Y;

X ₄ =H or N;

X ₅ =D or V;

X ₆ =L or M.

In a specific example, X ₃ =F; X ₄ =H; X ₅ =D; and X ₆ =L. In another specific example, X ₃ =Y; X ₄ =N; X ₅ =V; and X ₆ =M.

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 human CD3, wherein the variable domain Each includes a distinct heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds the variable domain of human CD3 includes the amino acid sequence

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

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 human CD3, wherein the variable domain Each includes a distinct heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds the variable domains of human CD3 includes a CDR1, CDR2, and CDR3, the CDR1, CDR2 and CDR3 includes the following amino acid sequence:

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 to a tumor antigen and a variable domain that binds human CD3, which binds to human CD3 The variable domain includes an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 include the following amino acid sequences:

CDR1:SYALS;

CDR2: AISGSGRTTWYADSVKG;

CDR3: DGGYTYGPYWYFDL.

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 human CD3, wherein the variable domain Each includes a distinct heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds the variable domain of human CD3 includes the amino acid sequence

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

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 human CD3, wherein the variable domain Each includes a distinct heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds the variable domains of human CD3 includes a CDR1, CDR2, and CDR3, the CDR1, CDR2 and CDR3 includes the following amino acid sequence:

CDR1: DYTMH;

CDR2: DISWSSGSIGYADSVKG;

CDR3: DHRGYGDYEGGGFDY.

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 human CD3, which binds to human CD3 The variable domain includes an antibody variable domain, the antibody variable domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes a CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 include the following amino acid sequences:

CDR1: DYTMH;

CDR2: DISSWX ₇ GX ₈ X ₉ X ₁₀ YADSVKG;

CDR3: DHX ₁₁ GYGDYEGGGFDX ₁₂ ;

in

X ₇ =S or G;

X ₈ =S or T;

X ₉ =I or T;

X ₁₀ =G or Y;

X ₁₁ =R or M;

X ₁₂ =H or Y,

Preferably X ₇ , X ₈ , X ₉ and X ₁₀ are S, S, I and G or G, S, I and Y or S, T, T and G, and preferably X ₁₁ and X ₁₂ are R and H , or R and Y, or M and Y, more preferably X ₇ , X ₈ , X ₉ , X ₁₀ , X ₁₁ and X ₁₂ are S, S, I, G, R and H or G, S, I, Y, R and Y are either S, T, T, G, M and Y, or in other words, preferably X ₇ , X ₈ , X ₉ and X ₁₀ are S, S, I and G, and X ₁₁ and X ₁₂ are R and H; or X ₇ , X ₈ , X ₉ and X ₁₀ are G, S, I and Y and X ₁₁ and X ₁₂ are R and Y; or X ₇ , X ₈ , X ₉ and X ₁₀ are S, T, T and G, and X ₁₁ and X ₁₂ are M and Y.

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 human CD3, wherein the variable domain Each includes a distinct heavy chain variable region and a common light chain variable region, and wherein the heavy chain variable region that binds the variable domain of human CD3 includes the amino acid sequence

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

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

Amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNANTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAKDRHWHWWLDAFDYWGQGTLVTVSS

There are 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or combinations thereof at one or more positions other than the CDR.

In another specific example, the variable domain of the heavy chain of the variable domain that binds the tumor antigen preferably includes

Amino acid sequence of MF4327(CLEC12A)

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

The present invention further provides an antibody or derivative thereof of the present invention or a pharmaceutical composition of the present invention for use in the treatment of an individual in need thereof. With regard to treating an individual having 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 tumor antigens.

CD3/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 and endometrial cancers; prostate cancer, brain cancer and any other solid tumor.

Also provided are CD3/tumor antigen bispecific antibodies or derivatives thereof of the present invention or pharmaceutical compositions comprising these bispecific antibodies or derivatives thereof for use in the treatment of various bone marrow-derived leukemias and preleukemia diseases but in addition B cells lymphoma. Diseases that can be treated according to the present invention include myeloid leukemia or preleukemia diseases such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelogenous leukemia (CML), and Hodgkin's lymphoid tumor and most non-Hodgkin lymphomas. And B-ALL; T-ALL, adventitial cell lymphoma are also preferred therapeutic targets of the antibody of the present invention. Accordingly, the present invention provides a bispecific full-length IgG antibody according to the present invention for use in the treatment of myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), multiple myeloma (MM) or preferably acute myeloid Preparations for Leukemia (AML). Also provided is the use of a bispecific IgG antibody according to the invention for the manufacture of a medicament for the treatment or prevention of MDS, CML, MM or preferably AML.

The amount to be administered to a patient as an antibody of the invention typically falls within the 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 obtain the desired therapeutic effect, the greater the therapeutic range will typically be. Thus, it is preferred that the antibodies of the present invention exert sufficient therapeutic effects at low doses.

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 a major negative determinant of outcome in AML and long-term (five-year) survival in severe elderly AML patients is approximately 10%. Disease progression was observed within 3 years in almost all patients who had been in remission after induction chemotherapy. Current post-remission treatments have shown limited, if any, value in older patients with AML. Thus, the burden of residual resistant leukemia remains high, and the subpopulation of surviving drug-resistant leukemia cells rapidly relapses. These chemotherapy-unresponsive AML tumor cells require novel drugs with completely different modes of action in order to induce and support complete remission. Although complete remission (CR) can be achieved in more than 50% of elderly AML patients and approximately 80% of younger patients with many intensified chemotherapy combinations, progression of response and survival remains a significant research challenge. In a recently published network meta-analysis of 65 randomized clinical trials (15.110 patients) in older patients with AML, most modified study-induced regimens had similar or even worse efficacy analysis compared to Traditionally use daunorubicin and cytarabine for 3+7 induction. This standard of AML treatment is associated with high morbidity and even mortality. The majority of patients with CR recurrence are due to residual leukemia stem cells after chemotherapy. Further dose escalation was limited by unacceptable toxicity. An urgent need for new therapeutic modalities, preferably less toxic, has thus emerged, especially in elderly patients with AML.

Treatment of AML that does not respond to chemotherapy can be accomplished by using bispecific antibodies to relocate T cells from the patient's own immune system to AML tumor cells and subsequently activate tumor-specific T cells. This method is also referred to as the "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, the CD3xCLEC12A bispecific IgG antibody effectively resets T cells to AML tumor cells, thereby inducing AML tumor lysis.

Example

cell line

BxPC3 human pancreatic cancer cell line.

HCT-116 human colon cancer cell line.

小鼠immunization with CD3

mouse

小鼠。有关于特异性重链可变区，或者具有此处所公开的序列的三价多聚体，其可借由具有本领域中的通常技艺的人士所知道的方式来生成。To generate human antibodies that bind CD3, the human common light chain and a human heavy chain (HC) minilocus (comprising a selected set of human V gene segments, all human Ds, and all human Js) Instead, transgenic mice (see WO2009/157771, incorporated herein by reference) were immunized with TCR/CD3 containing lipoparticles (Intergral Molecular). These mice are called

mice. There are specific heavy chain variable regions, or trivalent multimers having the sequences disclosed herein, which can be produced by means known to those of ordinary skill in the art.

小鼠以含有Hek293T-衍生的人类5D5M TCR/CD3的脂质球，继而为人类T细胞被免疫，以产生一种抗-TCR/CDR3免疫反应及产生抗-TCR/CD3抗体群组。

Mice were immunized with lipid globules containing Hek293T-derived human 5D5M TCR/CD3 followed by human T cells to generate an anti-TCR/CDR3 immune response and to generate a population of anti-TCR/CD3 antibodies.

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

The lipid globules used for immunization in this study contained the 5D5M TCRαβ combination. A vector containing the 5D5M TCRαβ combination was synthesized, transfected and used to generate lipid globules containing this TCR/CD3 combination by transient transfection into HEK293T cells (Intergral Molecular).

5D5M TCRα

5D5M TCRβ

小鼠使用于使用TCR/CD3脂质球及初级人类T细胞的免疫作用。

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

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

For all immunized mice, collect and store lymphatic material for antibody discovery when:

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

The titers for human TCR/CD3 were <1/300 and >1/100 and did not increase during the final booster immunization.

Priming immunisation using lipid globules

小鼠对于TCR/CD3的体液性免疫反应，使用含有人类5D5M TCRαβ组合的脂质球用于免疫。脂质球和Gerbu佐剂一起使用于第一次和第二次注射。To activate (prime)

Humoral immune response to TCR/CD3 in mice using lipid globules containing a combination of human 5D5M TCRαβ for immunization. Lipid globules were used with Gerbu adjuvant for the first and second injections.

Booster immunization using polyline T cells

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

Screening of sera from immunized mice by ELISA

Interim serum IgG titers were screened by ELISA using TCR/CD3 containing lipid globules and null lipid globules. Serum IgG titers were determined using anti-mouse IgG staining, as this staining proved to be the most sensitive.

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

Inguinal lymph nodes from successfully immunized mice were used to construct "immune" phage antibody libraries. Trizol LS was used to extract RNA from lymphoid tissue and 1 μg of total RNA was used in an RT reaction with IgG-CH1 specific primers. The resulting cDNA was then used to amplify a polyclonal pool of VH-encoding cDNA using in-house developed VH-specific primers, essentially as described in Marks et al. (J Mol Biol. 1991 Dec 5; 222(3) : 581-97) described in text B. The resulting PCR product was then cloned into a phagemid vector for visualization of Fab fragments on phage, as described in Haard et al. (J Biol Chem. 1999 Jun 25; 274(26): 18218-30) B described, with the exception that the light chain is the same for each antibody and encoded by the vector. After ligation, such phagemids were used to transform E. coli TG1 bacteria and the transformed bacteria were plated on LB-agar plates containing ampicillin and glucose. All phage libraries contained >10e6 transformants and had an insert frequency of >80%. Bacteria were harvested after overnight growth and used to prepare phage according to established procedures (de Haard et al., J Biol Chem. 1999 Jun 25;274(26):18218-30).

Phages carrying Fab fragments that specifically bind human CD3 were selected.

Phage libraries were rescued according to standard procedures (J Mol Biol. 1991 Dec 5; 222(3): 581-97; J Biol Chem. 1999 Jun 25; 274(26): 18218-30) and in one or more Rounds of immune phage antibody library selection to pick phage. In round 1, recombinant CD3 protein was coated onto wells of maxisorp ^™ ELISA plates or NUNC immunotubes, while in round 2, recombinant CD3 protein or cells overexpressing human CD3 protein were used. Maxisorp ^™ ELISA plates or immunotubes were blocked with 4% ELK. Phage antibody pools were also blocked with 4% ELK and also blocked with excess human IgG to deplete Fc region binders prior to addition of the phage pools to the coated antigen.

Incubation of the phage pool with the coated protein was performed at room temperature for 2 hrs under shaking conditions. The plate or tube was then washed with 0.05% Tween-20 in PBS, followed by 5 to 10 washes with PBS. Bound phage was eluted using 50 mM glycine (pH 2.2) and added to E. coli TG-1 and incubated at 37°C for phage infection.

Subsequently, infected bacteria were plated onto agar plates containing ampicillin and glucose and incubated overnight at 37°C. After the 1st round of selection, colonies were scraped from the plate and pooled and then rescued and amplified to make an enriched 1st round of phage pools for synthetic repertoires. For immune pools, single colonies were screened for target binding after the first round of phage selection.

Antibody colonization and production

The bispecific antibodies used herein generally differ from each other only by the specific amino acid sequence of the heavy chain variable region of one or two variable domains. Antibody production is used to express heavy and light chains by breeding the heavy chain variable regions into expression vectors. Methods for producing bispecific antibodies are known in the art.

Briefly, DNA encoding the heavy chain variable region of the CD3-targeted variable domain was cloned into the MV1624 vector (see Figure 10A), which encodes KK residues (L351K, T366K) in the CH3 region for generation IgG heavy chain heterodimers (WO2013/157954 and WO2013/157953). The Fc constant region contains mutations within CH2 to silence Fc effector function. The DNA encoding the heavy chain variable region replaces the 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 the MV1625 vector (Fig. 10B), which expresses the second heavy chain of the antibody portion of the bispecific antibody basis with L351D-L368E Mutations were made within the CH3 region (WO2013/157954 and WO2013/157953). The DNA encoding the heavy chain variable region replaces the stuffer region within the construct. The variable region is preceded by an encoded HC signal peptide (not shown). Both constructs also contained an expression cassette for expression of the IGKV1-39/jk1 light chain. Simultaneous representation of this 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, 293-F cell stock was split in 293-F medium at a ratio of 1:1 and at 155 rpm orbital shaking speed at 37°C and 8% Incubate overnight under _CO2 . Cells were diluted to a density of ^5x105 cells/mL the day before transfection. 4 mL of suspension cells were seeded into 24 deep well culture plates, covered with a gas permeable seal and incubated overnight at 37°C and 8% CO ₂ with a rotary shaking speed of 285 rpm. On the day of transfection, 4.8 mL of 293-F medium was mixed with 240 μg of linear polyethylenimine (PEI) (MW 25,000). For each IgG to be produced, 200 μL of the 293F medium-PEI mixture was supplemented with 8 μL of DNA (4 μL of DNA encoding each heavy chain for the heterodimer). The mixture was incubated at room temperature for 20 minutes before being gently added to the cells. One day after transfection, penicillin-streptomycin (Pen Strep) diluted in 500 [mu]L of 293F medium was added to each well. Such plates were incubated at 37° C. and 8% CO ₂ with a rotary shaking speed of 285 rpm until harvesting 7 days after transfection. The plates were centrifuged at 500 g for 5 min and the IgGs-containing supernatant was filtered using 10-12 μm melt blown polypropylene filter plates and stored at -20°C prior to purification.

Antibody purification from culture supernatant

Antibody-containing medium was harvested and centrifuged to remove cellular debris. Protein A Sepharose beads were then added to the medium. The medium and Protein A Sepharose beads were incubated with the antibody to allow binding.

After incubation the beads were passed through a vacuum filter to detach the beads from the medium and washed. Such antibodies are eluted from the beads by incubation with lysis buffer.

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

buffer exchange

To desalt the purified antibody, a filter plate or filter column is used to centrifuge the antibody fraction. Centrifuge the filter plate or column to reduce the volume of the antibody fraction. Subsequently, PBS or the desired buffer is added to the portion to replace the buffer with a low salt buffer. Optionally, this centrifugation step followed by the buffer addition step is repeated to further desalt the antibody storage buffer.

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

The specific CD3 x tumor antigen bispecific IgG combination induced tumor antigen specific T cell activation and tumor antigen positive tumor cell lysis was tested in a cytotoxicity assay. Effector cells are healthy donor-derived resting T cells and target cells are BxPC3 cells or HTC-116 cells.

Resting T cells were isolated from whole blood from healthy donors according to standard techniques using Ficoll and the EasySep Human T Cell Isolation Kit, analyzed by anti-CD3 antibody using flow cytometry T cells were checked for >95% purity and subsequently cryopreserved. For cytotoxicity assays, cryopreserved T cells were thawed and used if their viability was >90% when thawed by standard trypan blue staining. Cytotoxicity Assay Briefly, thawed resting T cells and BxPC3 or HCT116 target cells were co-cultured at a 5:1 E:T ratio for 48 hours. Antibodies were tested at a dilution range. A CD3 monospecific antibody and an EGFR monospecific antibody, and an irrelevant IgG1 isotype control mAb were included in the assay as controls (eg, an antibody that binds CD3 and another antigen such as tetanus toxoid (TT)) . T cell activation was quantified using flow cytometry; CD8 T cells were gated according to CD8 expression and their activation status was subsequently analyzed by measuring CD69 expression on T cells. Target cell lysis was determined by measuring the fraction of viable cells as assessed by ATP levels as assessed by CellTiterGlo (Promega). ATP levels, measured by luminescence on an Envision microplate reader, result in relative light unit (RLU) values, which are analyzed using GraphPad Prism.

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

% Kill = (100-(RLU sample/RLU no IgG) x 100).

In this assay, the bispecific antibody has two binding domains. One targets EGFR and the other targets CD3. Both binding domains have the same (common) light chain variable region (VL) and different heavy chain variable regions (VH). The EGFR-targeted binding domain has a VH with the amino acid sequence of MF8233. The CD3-targeted binding domain has a VH with the amino acid sequence of one of the designated MFs for CD3. This bispecific antibody contains mutations in CH2 to silence Fc effector function.

The antibody MF8233xMF8397 induced upregulation of CD69 (Fig 4-6) and CD25 (Fig 4-6) on CD4 and CD8 T cells, which were determined after 48 hours of co-culture at a 5:1 E±T ratio. Lysis of the T cell vehicle 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, binding to membrane expressed CD3 on HPB-ALL cells (DSMZ, ACC 483) can be assessed by mobile cytometry (according to the FACS procedure as previously described in WO2014/051433). In one embodiment, binding of a candidate EGFR/CD3 bispecific antibody to CD3 on HPB ALL cells was demonstrated 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 CD3δ/ε or CD3γ/ε. Binding of this candidate bispecific IgG1 to EGFR can be determined using BxPC3 and HCT-116 and CHO cells transfected with EGFR expressing constructs; a CD3 monospecific antibody and an EGFR monospecific antibody, and unrelated An IgGl isotype control mAb was included in the assay as a control (eg, an antibody that binds CD3 and another antigen such as tetanus toxoid (TT)).

Generation of additional clones from supergroups 1, 3 and 4

From immune phage library screening (as described in the section "Selecting phages carrying Fab fragments that specifically bind human CD3"), additional clones carrying Fab fragments that specifically bind human CD3 were characterized. Additional clones were identified from supergroup 1, including MF8048, MF8101 and MF8056. Additional clones were identified from supergroup 3 and supergroup 4, including MF8562 in supergroup 3 and MF8998 in supergroup 4.

小鼠呈现的VH基因库(pool)被执行。为了这个目的，从不同的小鼠获得的序列资料集与属于超级群4MF的MF序列作比较。此导致识别出属于超级群4序列变异体殖株MF10401及MF10428。不同序列的HCDR1及HCDR2内发现到数种不同的突变。Additional neocolons were identified from supergroup 4 using Next Generation Sequencer (NGS) analysis. NGS was used to generate anti-CD3 cohorts from

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

The VH sequences of all additional clones from supergroups 1, 3 and 4 were cloned into the MV1624 (DM-KK) vector and presented in a CD3xEGFR bispecific format for further characterization, as described in "Antibody clones and Production" section.

Characterization of additional clones from supergroups 1 and 4

Assays(eBiosciences^TM)、遵照标准制造商的指示来判定自以HCT-116细胞进行的EGFRxCD3细胞毒性分析衍生之上清液内的IFN-γ及TNF-α细胞介素生产(图14F-G)。Additional clones from supergroup 1 were characterized for their functional activity in a bispecific format. The EGFR binding arm of this bispecific CD3xEGFR antibody has the amino acid sequence encoded by MF8233. As a control, these CD3 clones were also tested with another antigen (eg, tetanus toxoid) of the amino acid sequence encoded by MF1337. Reference MFs (MF8057 and MF8058) from Supergroup 1 are included to be based on, as above: "Antibody Tumor Antigen Specific T Cell Activation and Lysis of BxPC3 Cells or HTC-116 Cells" and "CD3 Bispecific Antibody Characterization" The affinities of the sequence variants were directly compared with those of the MF clones from supergroup 1 that had been characterized as described in the "L" section. Binding affinity of HPB-ALL cells expressing human CD3-TCR complexes using mobile cytometry (FIG. 14A and FIG. 18) and tumor antigen-positive tumor cells with cytotoxicity assay (FIG. 14B-E) (HCT-116) T cell activation and lysis. No target cell lysis was observed with the bispecific antibody with the MF1337 control arm. Target cell lysis was observed in a dose-dependent manner for the different CD3 clones tested. Low target cell lysis was observed with MF8048. Activation markers CD69 and CD25 expression levels on CD4 and CD8 T cells were measured with FACS staining to assess T cell activation. Dose-dependent T cell activation was observed in all clones, and no T cell activation was observed in the negative control. Finally, use after 48hrs

Assays (eBiosciences ^™ ), following standard manufacturer's instructions to determine IFN-gamma and TNF-alpha interleukin production in supernatants derived from EGFRxCD3 cytotoxicity assays with HCT-116 cells (Figure 14F-G) .

分析来测量IL-6、IFN-γ及TNF-α的细胞介素电平。For characterization of additional clones belonging to supergroup 4, binding affinity to HPB-ALL cells was determined by FACS (Figure 15). PG1337, a monovalent antibody with two identical MF1337 arms specific for tetanus toxoid, was used as a negative control. For cytotoxicity assays, HCT-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 MF10428. A CD3xTAA bispecific antibody known to be highly active was included as a positive control. Target cell lysis was quantified using cell viability measurements. Using the supernatant from the cytotoxicity assay,

Assays to measure IL-6, IFN-γ and TNF-α interferon levels.

The three strains of supergroup 4 tested were thus found to exhibit different binding but similar lytic activity. Although the lytic activity of these clones was similar, reduced interleukin production was observed.

Table 1

MF combination AUC MF8233xMF8998 13630 MF8233xMF10428 2700 MF8233xMF10401 9646 MF1337xMF1337 255

Table 1 : AUC values for binding of the indicated CD3 clones referred to in Figure 15A.

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 Fake 11 - CD3xTAA: +ctrl TAA 369 4.5

Table 2: AUC and EC50 values for target cell lysis of the indicated CD3 clones mentioned in Figure 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: AUC and EC50 values of the indicated interferon amounts and CD3 clones mentioned in Figure 15A.

All additionally identified MFs were observed to be functional as assessed by cytotoxicity assays. Next, a graph was plotted between dissolution on the Y-axis and binding affinity on the X-axis (FIG. 16) to understand the relationship between dissolution and affinity within and across supergroups. Overall, a diverse cohort of anti-CD3 Fabs was generated which consists of a diverse supergroup and covers a range of affinities. Interestingly, supergroups 1 and 4 resulted in clones exhibiting similar activity but different CD3 binding (FIG. 16B). Comparing supergroups 1 and 3 revealed clones that exhibited 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: AUC and EC50 values for target cell lysis of the indicated CD3 clones mentioned in Figures 16B and 16C.

CD3 antigen characterization

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

table 5

Table 5: AUC and EC50 values of the indicated bispecific antibodies mentioned in Figure 17 in target cell lysis. Negative controls showed no significant measurable activity.

binding affinity

Additional CD3 clones were analyzed by FACS for binding affinity to HPB-ALL cells expressing human CD3 as described in the "Characterization of CD3 Bispecific Antibodies" section. The affinity of MF6955 and MF6964 for CD3 was measured by surface plasmon resonance (SPR) technique using BIAcore ^™ T100. Anti-human IgG mouse monoclonal antibody (Becton and Dickinson, cat. Nr. 555784) was coupled to the CM5 sensor chip surface using free amine chemistry (NHS/EDC). The CD3xTAA bispecific antibody is then captured on the sensor surface. A range of concentrations of recombinantly purified antigenic human CD3δε-Fc protein was then moved across 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. Figure 18 illustrates the range of binding affinity for the CD3 panel generated.

Claims (33)

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

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.

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

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.

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

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.

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

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.

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.

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