JP7090780B2 - Antibodies that bind to CD3 - Google Patents

Description

The present invention generally relates to antibodies that bind to CD3, including, for example, multispecific antibodies for activating T cells. In addition, the invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells containing such polynucleotides. The present invention relates to a method for producing the antibody and a method for using the antibody for the treatment of a disease.

CD3 (differentiation antigen group 3) is a protein complex consisting of four subunits, a CD3γ chain, a CD3δ chain, and two CD3ε chains. CD3 associates with T cell receptors and ζ chains to generate activation signals in T lymphocytes.

CD3 has been extensively investigated as a drug target. Monoclonal antibodies that target CD3 have been used as immunosuppressive therapies in the treatment of autoimmune diseases such as type I diabetes or transplant rejection. The CD3 antibody muromonab-CD3 (OKT3) was the first monoclonal antibody to date approved for clinical use in humans in 1985.

More recent applications of CD3 antibodies are in the form of bispecific antibodies that bind CD3 on the one hand and tumor cell antigens on the other. Simultaneous binding of such antibodies to both of its targets enhances the transient interaction between target cells and T cells, resulting in activation of any cytotoxic T cell followed by lysis of the target cell. cause.

For therapeutic purposes, an important requirement for an antibody to be achieved is to be sufficiently stable both in vitro (for drug storage) and in vivo (after administration to a patient).

Modifications such as asparagine deamidation are common degradations of recombinant antibodies and can affect both in vitro stability and in vivo biological function.

Given the incredible therapeutic potential of antibodies, especially bispecific antibodies for activating T cells, there is a need for CD3 antibodies with optimized properties.

INDUSTRIAL APPLICABILITY The present invention provides an antibody that binds to CD3 and is resistant to degradation by, for example, asparagine deamidation, and thus comprises a particularly stable multispecific (eg, bispecific) antibody required for therapeutic purposes. do. The provided (multispecific) antibody further combines good efficacy and productivity with low toxicity and favorable pharmacokinetic properties.

As shown herein, an antibody comprising a multispecific antibody that binds to CD3, provided by the present invention, is determined by surface plasmon resonance (SPR) at pH 6, -80 ° C for 2 weeks. Compared to the subsequent binding activity, the binding activity to CD3 is maintained by more than about 90% after 2 weeks at pH 7.4, 37 ° C.

In one aspect, the invention is an antibody that binds to CD3, wherein the antibody comprises the heavy chain complementarity determining regions (HCDR) 1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 5. A th chain comprising a heavy chain variable region (VH) and a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and a light chain variable region (VL) comprising LCDR3 of SEQ ID NO: 10. An antibody, which comprises one antigen binding domain, is provided. In one embodiment, VH comprises at least about 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 7, and / or VL is sequence. It contains at least about 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence to the amino acid sequence of number 11.

In a further aspect, the invention provides an antibody that binds to CD3 and comprises a first antigen binding domain comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11.

In one aspect, the first antigen binding domain is a Fab molecule.

In one aspect, the antibody comprises an Fc domain consisting of first and second subunits.

In one aspect, the antibody comprises a second antigen binding domain and optionally a third antigen binding domain that binds to the second antigen.

In one aspect, the second antigen-binding domain and / or, if present, the third antigen-binding domain is a Fab molecule.

In one aspect, the first antigen binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain or the constant domains CL and CH1, in particular the variable domains VL and VH, are replaced with each other.

In one aspect, the second antigen-binding domain and, if present, the third antigen-binding domain is a conventional Fab molecule.

In one embodiment, the second antigen-binding domain and, if present, the third antigen-binding domain is a Fab molecule, in which the amino acid at position 124 is independently lysine (K), arginine in the constant domain CL. It has been replaced by (R) or histidine (H) (numbering by Kabat) and the amino acid at position 123 has been independently replaced by lysine (K), arginine (R) or histidine (H) (Kabat). In the constant domain CH1, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering by Kabat EU index), and the amino acid at position 213 is independent. And have been replaced with glutamate (E) or aspartic acid (D) (numbering by Kabat EU index).

In one aspect, the first and second antigen binding domains are optionally fused to each other via a peptide linker.

In one embodiment, the first and second antigen-binding domains are Fab molecules, respectively, and (i) the second antigen-binding domain is the C-terminus of the Fab heavy chain, and the Fab heavy chain of the first antigen-binding domain. Either it is fused to the N-terminus of the Fab heavy chain, or (ii) the first antigen-binding domain is fused to the C-terminus of the Fab heavy chain and to the N-terminus of the Fab heavy chain of the second antigen-binding domain. On the other hand.

In one aspect, the first antigen-binding domain, the second antigen-binding domain and, if present, the third antigen-binding domain are Fab molecules, respectively, and the antibody consists of first and second subunits. Containing the Fc domain, (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and the first antigen-binding domain is Fab. At the C-terminus of the heavy chain, fused to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding domain is at the C-terminus of the Fab heavy chain, the second antigen-binding domain. The Fab heavy chain is fused to the N-terminus of the Fab heavy chain, and the second antigen-binding domain is either fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain. And, if present, a third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

In one aspect, the Fc domain is an IgG, particularly the Fc domain of IgG ₁ . In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc domain comprises modifications that facilitate association of the first and second subunits of the Fc domain. In one aspect, the Fc domain comprises one or more amino acid substitutions that reduce binding to the Fc receptor and / or reduce effector function.

In one aspect, the second antigen is a target cell antigen, particularly a tumor cell antigen.

In one aspect, the second antigen is TYRP-1. In one aspect, the second antigen-binding domain and, if present, the third antigen-binding domain are VH comprising HCDR1 of SEQ ID NO: 15, HCDR2 of SEQ ID NO: 16, and HCDR3 of SEQ ID NO: 17 and SEQ ID NO: 19. LCDR1, LCDR2 of SEQ ID NO: 20, and VL including LCDR3 of SEQ ID NO: 21. In one aspect, the second antigen-binding domain and, if present, the third antigen-binding domain are at least about 95%, 96%, 97%, 98%, 99% or of the amino acid sequence of SEQ ID NO: 18. A VH containing 100% identical amino acid sequence and / or a VL containing at least about 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 22. include.

In one aspect, the second antigen is EGFRvIII. In one aspect, the second antigen-binding domain and, if present, the third antigen-binding domain are VH comprising HCDR1 of SEQ ID NO: 85, HCDR2 of SEQ ID NO: 86, and HCDR3 of SEQ ID NO: 87, and SEQ ID NO: 89. LCDR1, LCDR2 of SEQ ID NO: 90, and VL including LCDR3 of SEQ ID NO: 91. In one aspect, the second antigen-binding domain and, if present, the third antigen-binding domain are at least about 95%, 96%, 97%, 98%, 99% or of the amino acid sequence of SEQ ID NO: 88. A VH containing 100% identical amino acid sequence and / or a VL containing at least about 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 92. include.

According to a further aspect of the invention, an isolated polynucleotide encoding an antibody of the invention and a host cell containing the isolated polynucleotide of the invention are provided.

In another embodiment, it binds to CD3, comprising: (a) culturing the host cells of the invention under conditions suitable for antibody expression, and optionally (b) recovering the antibody. A method for producing an antibody is provided. The invention also includes antibodies that bind to CD3 produced by the methods of the invention.

The present invention further provides a pharmaceutical composition comprising the antibody of the invention and a pharmaceutically acceptable carrier.

Methods of using the antibodies and pharmaceutical compositions of the present invention are also included in the present invention. In one aspect, the invention provides an antibody or pharmaceutical composition according to the invention for use as a pharmaceutical. In one aspect, an antibody or pharmaceutical composition according to the invention is provided for use in the treatment of a disease. In a specific embodiment, the disease is cancer.

Also provided are the use of the antibodies or pharmaceutical compositions of the invention in the manufacture of pharmaceuticals, the use of the antibodies or pharmaceutical compositions of the invention in the manufacture of pharmaceuticals for the treatment of diseases, in particular cancer. The present invention also provides a method of treating a disease in an individual, comprising administering to the individual an effective amount of an antibody or pharmaceutical composition according to the invention.

It is an exemplary structure of the (multispecific) antibody of the present invention. (FIGS. 1A, 1D) FIG. 3 is a diagram of a "1 + 1 CrossMab" molecule. (FIGS. 1B, 1E) FIG. 1 is a diagram of a "2 + 1 IgG Crossfab" molecule in which the order of the Crossfab component and the Fab component is different ("inverted"). (FIG. 1C, FIG. 1F) FIG. 6 is a diagram of a "2 + 1 IgG Crossfab" molecule. (FIG. 1G, FIG. 1K) FIG. 1 is a diagram of a "1 + 1 IgG Crossfab" molecule in which the order of the Crossfab component and the Fab component is different (“inverted”). (FIG. 1H, FIG. 1L) FIG. 1 is a diagram of a "1 + 1 IgG Crossfab" molecule. (FIG. 1I, FIG. 1M) FIG. 6 is a diagram of a "2 + 1 IgG Crossfab" molecule with two CrossFabs. (FIG. 1J, FIG. 1N) FIG. 6 is a diagram of a "2 + 1 IgG Crossfab" molecule having two CrossFabs with different order of Crossfab and Fab components ("inverted"). (FIG. 1O, FIG. 1S) FIG. 3 is a diagram of a "Fab-Crossfab" molecule. (FIG. 1P, FIG. 1T) FIG. 3 is a diagram of a "Crossfab-Fab" molecule. (FIG. 1Q, FIG. 1U) FIG. 6 is a diagram of a "(Fab) ₂ -Crossfab" molecule. (FIG. 1R, FIG. 1V) FIG. 6 is a diagram of a "Crossfab- (Fab) ₂ " molecule. (FIG. 1W, FIG. 1Y) FIG. 1 is a diagram of a "Fab- (Crossfab) ₂ " molecule. (FIG. 1X, FIG. 1Z) "(Crossfab) ₂ -Fab" molecule diagram. Black dot: Any modification in the Fc domain that promotes heterodimerization. ++,-: Amino acids of opposite charge introduced arbitrarily in the CH1 and CL domains. The Crossfab molecule is shown to include the exchange of VH and VL regions, but in particular, in embodiments where no charge modification has been introduced into the CH1 and CL domains, the CH1 domain is substituted. It may include exchange with the CL domain. Recombinant CD3 of the original CD3 binder, CD3 _orig and optimized CD3 binder, CD3 _opt , measured by SPR at 40 ° C, pH 6 after 14 days, or at 37 ° C, pH 7.4, 14 days under non-stress conditions. Relative binding activity to (IgG format). Binding of the original CD3 binder, CD3 _orig and optimized CD3 binder, CD3 _opt to Jurkat NFAT cells as measured by flow cytometry (IgG format). Antibody binding to Jurkat NFAT cells was detected using a fluorescently labeled anti-human Fc-specific secondary antibody. FIG. 3 is a schematic representation of the CD3 activation assay used in Example 3. Jurkat NFAT cell activation with the original CD3 binder, CD3 _orig and optimized CD3 binder, CD3 _opt (IgG format). Jurkat NFAT reporter cells were co-incubated with anti-PGLALA-expressing CHO (CHO-PGLALA) cells in the presence of CD3 _orig IgG PGLALA or CD3 _opt IgG PGLALA, or CD3 _opt IgG wt as a negative control. CD3 activation was quantified by measuring luminescence after 24 hours. FIG. 3 is a schematic diagram of a T cell bispecific antibody (TCB) molecule prepared in an example. All TCB antibodies tested were "2 + 1 IgG CrossFab," inverted "" with charge modification (VH / VL exchange in CD3 binder, charge modification in target antigen binder, EE = 147E, 213E; RK = 123R, 124K). Was produced as. TYRP1 TCB containing the original CD3 binder, CD3 _orig or optimized CD3 binder, CD3 _opt , measured by SPR at 40 ° C, pH 6 after 14 days, or at 37 ° C, pH 7.4, 14 days under non-stress conditions. Relative binding activity to recombinant CD3. The original CD3 binder, CD3 _orig or optimized CD3 binder, CD3 _opt , or corresponding, measured by SPR at 40 ° C, pH 6 after 14 days, or at 37 ° C, pH 7.4, 14 days after non-stress conditions. TYRP1 TCB-containing relative binding activity to recombinant TYRP1. Binding of TYRP1 TCBs containing the original CD3 binder, CD3 _orig or optimized CD3 binder, CD3 _opt , as measured by flow cytometry, to Jurkat NFAT cells. TCB binding to Jurkat NFAT cells was detected using a fluorescently labeled anti-human Fc-specific secondary antibody. Jurkat NFAT cell activation by TYRP1 TCB containing the original CD3 binder or the optimized CD3 binder. Jurkat NFAT cell reporter cells were co-incubated with the melanoma cell line M150543 in the presence of TYRP1 TCB CD3 _orig or TYRP1 TCB CD3 _opt . CD3 activation in the presence of TCB was quantified by measuring luminescence after 24 hours. Tumor cell killing and T cell activation by TYRP1 TCB containing the original CD3 binder or the optimized CD3 binder. Killing of melanoma cell line M150543 during treatment with TYRP1 TCB CD3 _orig and TYRP1 TCB CD3 _opt by PBMCs from three different healthy donors (FIGS. 11A-11F: donor 1, 11G-11L: donors). 2. FIGS. 11M-11R: Donor 3) were determined by LDH release after 24 hours (FIGS. 11A, 11G, 11M) and 48 hours (FIGS. 11B, 11H, 11N). At the same time, CD8 T cells (FIG. 11E, FIG. 11F, FIG. 11K, FIG. 11L, FIG. 11Q, FIG. 11R) and CD4 T cells (FIG. 11C, FIG. 11D, FIG. 11I, FIG. 11J, FIG. 11O, FIG. 11P) in PBMC. The CD25 upward control (FIG. 11C, FIG. 11E, FIG. 11I, FIG. 11K, FIG. 11O, FIG. 11Q) and the CD69 upward control (FIG. 11D, FIG. 11F, FIG. 11J, FIG. 11L, FIG. 11P, FIG. 11R) are described above. After 48 hours, it was measured by flow cytometry as a marker for T cell activation. It is a specific binding of EGFRvIII IgG PGLALA. Specific binding of the EGFRvIII IgG PGLALA antibody to EGFRvIII, which is not cross-reactive to EGFRwt, flows for CHO-EGFRvIII (FIG. 12A), EGFRvIII positive DK-MG (FIG. 12B), and EGFRwt-expressing MKN-45 (FIG. 12C). Tested by cytometry. Cetuximab was included as a positive control for EGFR wt expression. CAR J activation with EGFRvIII IgG PGLALA. Jurkat NFAT reporter cells expressing anti-PGLALA CAR were co-incubated with EGFRvIII expressing DK-MG cells and EGFRvIII IgG PGLALA antibody or DP47 IgG PGLALA as negative controls. Activation of Jurkat NFAT cells was quantified by measuring luminescence after 22 hours. EGFRvIII IgG PGLALA and corresponding TCB binding to EGFRvIII. Specific binding of EGFRvIII binders as IgG PGLALA and EGFRvIII binders converted to TCB to CHO-EGFRvIII cells (FIG. 14A) and MKN-45 cells (FIG. 14B) was measured by flow cytometry. Jurkat NFAT activation by EGFRvIII TCB. Jurkat NFAT activation was determined as a marker for CD3 involvement with EGFRvIII TCB in the presence of EGFRvIII positive DK-MG cells. DP47 TCB was included as a negative control. Tumor cytolysis by EGFRvIII TCB. Induction of specific tumor cytolysis by EGFRvIII TCB was performed with PBMC immediately after isolation and either EGFRvIII-positive DK-MG cells (FIGS. 16A, 16B) or EGFRwt-positive MKN-45 cells (FIGS. 16C, 16D). Determined in co-culture for 24 hours (FIGS. 16A, 16C) or 48 hours (FIGS. 16B, 16D). T cell activation by EGFRvIII TCB. Induction of T cell activation by EGFRvIII TCB is performed with PBMC immediately after isolation and EGFRvIII-positive DK-MG cells (FIGS. 17A, 17B, 17E, 17F) or EGFRwt-positive MKN-45 cells (FIGS. 17C, 17D). , FIG. 17G, FIG. 17H), the activation marker CD25 (FIGS. 17A, 17C, FIG. 17A) on CD4 T cells (FIGS. 17A-17D) or CD8 T cells (FIGS. 17E-17H). 17E, FIG. 17G) or CD69 (FIGS. 17B, 17D, 17F, 17H). Cytokine release by EGFRvIII TCB. Induction of the release of INFγ (FIGS. 18A, 18D), TNFα (FIGS. 18B, 18E), and Granzyme B (FIG. 18C, 18F) by EGFRvIII TCB was performed with PBMC immediately after isolation and EGFRvIII-positive DK-MG cells. It was determined in co-culture with either (FIGS. 18A-18C) or EGFR wt-positive MKN-45 cells (FIGS. 18D-18F). Affinity maturation is a specific binding of mature EGFRvIII IgG PGLALA. The specific binding of the affinity matured EGFRvIII antibody to EGFRvIII was compared to the parent EGFRvIII binder on U87MG-EGFRvIII cells (FIG. 19A) and the EGFRwt positive cell line MKN-45 (FIG. 19B). Jurkat NFAT activation by EGFRvIII TCB. Jurkat NFAT activation was determined as a marker for CD3 involvement with EGFRvIII TCB in the presence of EGFRvIII positive DK-MG cells (FIG. 20A), U87MG-EGFRvIII cells (FIG. 20B), and MKN-45 (FIG. 20C). .. DP47 TCB was included as a negative control. An EGFRvIII TCB containing the original CD3 binder, CD3 _orig or optimized CD3 binder, CD3 _opt , measured by SPR at 40 ° C, pH 6 after 14 days, or at 37 ° C, pH 7.4, 14 days under non-stress conditions. Relative binding activity to recombinant CD3. An EGFRvIII TCB containing the original CD3 binder, CD3 _orig or optimized CD3 binder, CD3 _opt , measured by SPR at 40 ° C, pH 6 after 14 days, or at 37 ° C, pH 7.4, 14 days under non-stress conditions. Relative binding activity to recombinant CD3. Binding of EGFRvIII TCBs containing the original CD3 binder, CD3 _orig or optimized CD3 binder, CD3 _opt , as measured by flow cytometry, to Jurkat NFAT cells. TCB binding to Jurkat NFAT cells was detected using a fluorescently labeled anti-human Fc-specific secondary antibody. Binding of EGFRvIII TCB containing P063.056 EGFRvIII binder or P056.021 EGFRvIII binder to U87MG-EGFRvIII cells as measured by flow cytometry. TCB binding to U87MG-EGFRvIII cells was detected using a fluorescently labeled anti-human Fc-specific secondary antibody. Tumor cytolysis and T cell activation by EGFRvIII TCB. Induction of specific tumor cytolysis (FIGS. 25A, 25B) and T cell activation (FIG. 25C, FIG. 25D) by EGFRvIII TCB was performed in co-culture with PBMC and U87MG-EGFRvIII cells immediately after isolation for 24 hours (FIG. 25C, FIG. 25D). 25A, 25C) or 48 hours (25B, 25D). DP47 TCB was included as a negative control. Jurkat NFAT activation comparing the 2 + 1 and 1 + 1 formats of EGFRvIII TCB. Jurkat NFAT activation was determined as a marker for CD3 involvement with EGFRvIII TCB in 2 + 1 inverted format and 1 + 1 head-to-tail format in the presence of EGFRvIII positive U87MG-EGFRvIII cells. Tumor cytolysis and T cell activation comparing the 2 + 1 and 1 + 1 formats of EGFRvIII TCB. Induction of specific tumor cell lysis (FIGS. 27A, 27B) and T cell activation (FIGS. 27C, 27D) by EGFRvIII TCB in 2 + 1 inverted format and 1 + 1 head-to-tail format was performed immediately after isolation of PBMC and U87MG. -Determined at 24 hours (FIG. 27A, FIG. 27C) or 48 hours (FIG. 27B, FIG. 27D) in co-culture with EGFRvIII cells. T cell activation and proliferation by EGFRvIII TCB. Induction of T cell proliferation (FIG. 28A, FIG. 28C) and T cell activation of CD4 T cells (FIGS. 28A, 28B) and CD8 T cells (FIGS. 28A, 28D) by EGFRvIII TCB is healthy with U87MG-EGFRvIII. Determined in co-culture with PBMCs isolated from donors. Tumor cell lysis, T cell activation, and cytokine release by EGFRvIII TCB. Tumor cytolysis (FIGS. 29A, 29B) by EGFRvIII TCB, T cell activation (FIGS. 29C, 29D), and induction of release of IFNγ and TNFα (FIG. 29E, 29F) are U87MG-EGFRvIII cells with PBMC. Was determined in co-culture of. Tumor cell lysis was measured 24 and 48 hours after treatment, and T cell activation and cytokine release were measured after 48 hours. Tumor cell lysis, T cell activation, and cytokine release by TYRP-1 TCB. Tumor cytolysis by TYRP-1 TCB (FIGS. 30A, 30B), T cell activation (FIGS. 30C, 30D), and induction of release of IFNγ and TNFα (FIGS. 30E, 30F) are derived from patients with PBMCs. It was determined in co-culture of the melanoma cell line M150543. Tumor cell lysis was measured 24 and 48 hours after treatment, and T cell activation and cytokine release were measured after 48 hours. The in vivo efficacy of TYRP-1 TCB. IGR-1 human melanoma cell lines were subcutaneously injected into humanized NSG mice to study tumor growth suppression in a melanoma subcutaneous xenograft model. Significant tumor growth suppression (TGI) was observed in the TYRP-1 TCB group compared to the vehicle group (68% TGI, p = 0.0058 ^* ). The in vivo efficacy of EGFRvIII TCB. A U87-huEGFRvIII human glioblastoma cell line was subcutaneously injected into humanized NSG mice to study tumor growth suppression in a glioblastoma subcutaneous xenograft model. Significant tumor control was observed in the EGFRvIII TCB group and all mice achieved complete remission.

I. Definition

The terms are used herein as they are commonly used in the art, unless defined below to have other meanings.

As used herein, the terms "first," "second," and "third" with respect to antigen-binding domains and the like are used when two or more parts of each type are present. Used to simplify the distinction. The use of these terms is not intended to give a particular order or orientation of the antibody unless otherwise explicitly indicated.

The terms "anti-CD3 antibody" and "antibody that binds to CD3" are capable of binding to CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in the targeting of CD3. Refers to an antibody that is. In one aspect, the degree of binding of the anti-CD3 antibody to an unrelated non-CD3 protein is less than about 10% of the binding of the antibody to CD3, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, the antibody that binds to CD3 has an equilibrium dissociation constant ( _KD ) of ≦ 1 μM, ≦ 500 nM, ≦ 200 nM, or ≦ 100 nM. For example, if the antibody has a _KD of 1 μM or less as measured by SPR, the antibody is referred to as “specifically binding” to CD3. In certain embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3s from different species.

The term "antibody" is used in the broadest sense herein and is a monoclonal antibody, polyclonal antibody, multispecific antibody (eg, bispecific antibody), and as long as they exhibit the desired antigen-binding activity. Includes, but is not limited to, various antibody structures including, but not limited to, antibody fragments.

"Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F (ab') ₂ , diabody, linear antibody, single-chain antibody molecules (eg, scFv and scFab), single domain antibodies, And multispecific antibodies formed from antibody fragments, but not limited to these. For a review of specific antibody fragments, see Holliger and Hoodson, Nature Biotechnology 23: 1126-1136 (2005).

The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the native antibody structure.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous set of antibodies. That is, a possible variant, wherein the individual antibodies included in the assembly bind to the same and / or the same epitope, but include, for example, naturally occurring mutations or mutations that occur during the manufacture of monoclonal antibody formulations. Excludes antibodies. Such variants are generally present in small quantities. In contrast to polyclonal antibody formulations that contain different antibodies that typically direct to different determinants (epitope), each monoclonal antibody in the monoclonal antibody formulation is relative to a single determinant on one antigen. Oriented. Therefore, the modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially uniform set of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibody is a hybridoma method, a recombinant DNA method, a phage display method, and a method of utilizing a transgenic animal containing all or a part of a human immunoglobulin loci, for example, the monoclonal antibody described herein. It may be made by a variety of techniques including, but not limited to, such methods and other exemplary methods for making.

An "isolated antibody" is an antibody isolated from a component of its natural environment. In some embodiments, the antibody is, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC, affinity chromatography). If determined by the imaging, size exclusion chromatography) method, it is purified to a purity greater than 95% or greater than 99%. As a review of the test method for antibody purification, for example, Flatman et al. , J. Chromatogr. See B 848: 79-87 (2007). In some embodiments, the antibody provided by the present invention is an isolated antibody.

The term "chimeric" antibody refers to an antibody in which part of a heavy chain and / or light chain is derived from a particular source or species and the rest of the heavy chain and / or light chain is derived from a different source or species.

A "humanized" antibody refers to a chimeric antibody that comprises an amino acid residue from a non-human CDR and an amino acid residue from a human FR. In certain embodiments, the humanized antibody is at least one of almost all or almost all CDRs corresponding to the variable domain of the non-human antibody, and almost all or almost all FRs corresponding to the variable domain of the human antibody. Usually contains two variable domains. Such variable domains are referred to herein as "humanized variable regions". The humanized antibody may optionally include at least a portion of the antibody constant region derived from the human antibody. In some embodiments, some FR residues in a humanized antibody are, for example, from a non-human antibody (eg, an antibody from which a CDR residue is derived) in order to restore or improve antibody specificity or affinity. Replaced by the corresponding residue of. An "humanized form" of an antibody, eg, a non-human antibody, refers to an antibody that has undergone humanization.

A "human antibody" possesses an amino acid sequence corresponding to an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. be. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues. In certain embodiments, the human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain embodiments, the human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from the human antibody library are also considered to be human antibodies or human antibody fragments of the invention.

The term "antigen binding domain" refers to a portion of an antibody that binds to some or all of an antigen and contains regions complementary to some or all of the antigen. The antigen binding domain may be conferred, for example, by one or more antibody variable domains (also referred to as antibody variable regions). In a preferred embodiment, the antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or antibody light chain involved in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of the native antibody (VH and VL, respectively) generally have similar structures, with each domain complementing the four conserved framework regions (FRs). Includes determining regions (CDRs). For example, Kindt et al. , Kuby Immunology, 6th ed. , W. H. Freeman & Co. , Page 91 (2007). A single VH domain or VL domain may be sufficient to provide antigen binding specificity. Further, the antibody that binds to a specific antigen may use the VH domain or VL domain of the antibody that binds to the antigen, and screen and isolate a library of complementary VL domains or VH domains, respectively. For example, Portolano et al. , J. Immunol. 150: 880-887 (1993); Clarkson et al. , Nature 352: 624-628 (1991). As used herein in combination with variable region sequences, "Kabat numbering" is referred to as Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Refers to the numbering system described by Public Health Service, National Institutes of Health, Bethesda, MD (1991).

As used herein, the amino acid positions of all constant regions and domains of heavy and light chains are described in Kabat, et al. , Sequences of Proteins of Immunological Interest, 5th ed. , Public Health Service, National Institutes of Health, Bethesda, MD (1991), numbered according to the Kabat numbering system, referred to herein as "Kabat numbering" or "Kabat numbering". Specifically, Kabat numbering system (Kabat, et al., Sequences of Protects of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, page 19), page 64, Bethesda, Bethesda. , Kappa and lambda isotype light chain constant domain CL, and Kabat EU index numbering system (see pages 661-723) used for heavy chain constant domains (CH1, hinge, CH2 and CH3), in this case. Is further clarified by referring to "Kabat EU index numbering" or "Kabat EU index numbering".

As used herein, the term "hypervariable region" or "HVR" is a region of an antibody variable domain that is hypervariable within a sequence and determines antigen binding specificity, eg, "complementarity determination". It means each of the "regions"("CDRs"). Generally, the antibody comprises 6 CDRs, 3 in VH (HCDR1, HCDR2, HCDR3) and 3 in VL (LCDR1, LCDR2, LCDR3). The exemplary CDRs herein refer to.
(A) Amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3). Variable loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));
(B) Amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3). CDR (Kabat et al., Publics of Proteins of Immunological Interest, 5th Ed. Antigen contact (MacCallum et al. J. Mol. Biol. 262: 732) occurring at 55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3). -745 (1996)).

Unless otherwise instructed, CDRs are determined according to Kabat et al., Above. Those skilled in the art will appreciate that the notation of a CDR can be determined according to the Chothia, McCallum, or any other scientifically accredited naming system.

"Framework" or "FR" refers to variable domain residues other than complementarity determining regions (CDRs). The variable domain FR generally consists of four FR domains, FR1, FR2, FR3 and FR4. Therefore, the HVR and FR sequences generally appear in VH (or VL) in the following order: That is, FR1-HCDR1 (LCDR1) -FR2-HCDR2 (LCDR2) -FR3-HCDR3 (LCDR3) -FR4.

Unless otherwise indicated herein, CDR residues and other residues (eg, FR residues) in the variable domain are described in Kabat et al. Numbered herein according to (see above).

The "acceptor human framework" for the purposes of this specification is a light chain variable domain (VL) framework or weight derived from the human immunoglobulin framework or human consensus framework as defined below. It is a framework containing the amino acid sequence of the chain variable domain (VH) framework. The human immunoglobulin framework or the human consensus framework "derived" acceptor human framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. In general, subgroups of sequences include Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. It is a subgroup as described in 1-3.

The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, an IgG class immunoglobulin is a heterotetramer glycoprotein of about 150,000 daltons and is composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also known as a variable heavy chain domain or heavy chain variable region, followed by three constant domains (CH1, CH2 and) also called heavy chain constant regions. It has CH3) and. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also referred to as a variable light chain domain or light chain variable region, followed by a constant light chain (CL), also referred to as a light chain constant region. Has a domain. The heavy chain of immunoglobulin may be assigned to one of five types called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which may be assigned. For example, further subtypes such as γ ₁ (IgG ₁ ), γ ₂ (IgG ₂ ), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ) and α ₂ (IgA ₂ ). May be done. The light chain of an immunoglobulin may be assigned to one of two types called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. The immunoglobulin is substantially composed of two Fab molecules and an Fc domain that are connected via the immunoglobulin hinge region.

An antibody or immunoglobulin "class" refers to the type of constant domain or constant region possessed by a heavy chain of antibody or immunoglobulin. There are five major classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM, some of which are subclasses (isotypes) such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . Heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The "Fab molecule" is a protein consisting of a VH domain and a CH1 domain of an immunoglobulin heavy chain ("Fab heavy chain") and a VL domain and a CL domain of an immunoglobulin light chain ("Fab light chain"). Point to.

By "crossover" Fab molecule (also referred to as "Crossfab") is meant a Fab molecule in which the variable and constant domains of the Fab heavy and light chains have been exchanged (ie, replaced with each other). That is, the crossover Fab molecule is a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, from the N-terminal to the C-terminal), and a heavy chain variable domain VH and a light chain constant. Contains a peptide chain composed of the domain CL (VH-CL, from the N-terminus to the C-terminus). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and Fab heavy chain have been exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the (crossover) Fab molecule. It is called the "heavy chain" of. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light chain and Fab heavy chain have been exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Is called.

In contrast, a "conventional" Fab molecule means a Fab molecule in its natural format, i.e., a heavy chain composed of a heavy chain variable domain and a constant domain (VH-CH1, from the N-terminus). Includes (towards the C-terminus) and a light chain (VL-CL, from the N-terminus to the C-terminus) composed of a light chain variable domain and a constant domain.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variable Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational cleavage of one or more, in particular one or two amino acids, from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full heavy chain may contain the full heavy chain or may contain a cleaved variant of the full heavy chain. good. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered by the Kabat EU index). Therefore, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447) may or may not be present. Amino acid sequences of heavy chains containing the Fc region (or subunits of the Fc domain as defined herein) are herein free of the C-terminal glycine-lysine dipeptide, unless otherwise indicated. Shown. In one aspect, the heavy chain comprising the Fc region (subunit) specified herein, contained in the antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered by Kabat EU index). .. In one aspect, the heavy chain comprising the Fc region (subunit) specified herein, contained in the antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbered by Kabat EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is described in Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Follows an EU numbering system (also referred to as EU index) as described in Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, the "subunit" of an Fc domain comprises one of the two polypeptides forming the Fc domain of the dimer, i.e., the C-terminal constant region of the immunoglobulin heavy chain. It means a polypeptide capable of stable self-association. For example, subunits of the IgG Fc domain include IgG CH2 and IgG CH3 constant domains.

"Fused" means that the components (eg, Fab molecule and Fc domain subunit) are linked either directly by peptide bond or via one or more peptide linkers. That is.

The term "multispecificity" means that an antibody can specifically bind to at least two distinct antigenic determinants. The multispecific antibody can be, for example, a bispecific antibody. Typically, bispecific antibodies contain two antigen binding sites, each specific for a different antigenic determinant. In certain embodiments, a multispecific (eg, bispecific) antibody can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed in two distinct cells.

The term "valence" as used herein refers to the presence of a particular number of antigen binding sites within an antigen binding molecule. In this case, the term "monovalent binding to antigen" refers to the presence of one (and no more than one) antigen-binding site specific to the antigen within the antigen-binding molecule.

"Antigen binding site" refers to the site of an antigen binding molecule that interacts with an antigen, i.e., one or more amino acid residues. For example, the antigen binding site of an antibody comprises amino acid residues from complementarity determining regions (CDRs). Native immunoglobulin molecules typically contain two antigen binding sites and Fab molecules typically have one antigen binding site.

As used herein, the term "antigen determinant" or "antigen" refers to a site on a polypeptide polymer to which an antigen-binding domain binds (eg, an amino acid) that forms an antigen-binding domain-antigen complex. Refers to an antigenic composition composed of discontinuous amino acids in the continuous extension part or different regions of. Useful antigenic determinants are released in serum, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other affected cells, on the surface of immune cells, and / Or can be found in the extracellular matrix (ECM). In a preferred embodiment, the antigen is a human protein.

"CD3" is any vertebrate, including mammals such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys), and rodents (eg, mice and rats), unless otherwise indicated. Refers to any native CD3 of origin. The term includes "full length" unprocessed CD3, and any form of CD3 resulting from processing in cells. The term also includes naturally occurring variants of CD3, such as splice variants or allelic variants. In one aspect, CD3 is the epsilon subunit (CD3ε) of human CD3, in particular human CD3. The amino acid sequence of human CD3ε is shown in SEQ ID NO: 112 (without signal peptide). See also UniProt (www.uniprot.org) deposit number P07766 (version 189) or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. In another aspect, the CD3 is a cynomolgus monkey (Macafascicularis) CD3, in particular a cynomolgus monkey CD3ε. The amino acid sequence of cynomolgus monkey CD3ε is shown in SEQ ID NO: 113 (without signal peptide). See also NCBI GenBank number BAB71849.1. In certain embodiments, the antibodies of the invention bind to CD3 antigens from different species, particularly to the epitopes of CD3 stored in human CD3 and cynomolgus monkey CD3. In a preferred embodiment, the antibody binds to human CD3.

As used herein, a "target cell antigen" is presented on the surface of a target cell, eg, a cancer cell or (in the case of a "tumor cell antigen") a cell within a tumor, such as a tumor stromal cell. Means an antigenic determinant. Preferably, the target cell antigen is other than CD3, which is not CD3 and / or is expressed on a different cell. In a preferred embodiment, the target cell antigen is TYRP-1, especially human TYRP-1. In another aspect, the target cell antigen is EGFRvIII, in particular human EGFRvIII.

"TYRP1" or "TYRP-1" is an abbreviation for tyrosine-related protein 1, which is an enzyme involved in melanin synthesis. The mature form of TYRP1, initially also referred to as gp75, is a 75 kDa transmembrane glycoprotein. The sequence of human TYRP1 is shown in SEQ ID NO: 114 (without signal peptide). See also UniProt entry number P17643 (version 185). As used herein, "TYRP1" refers to mammals such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys), and rodents (eg, mice and rats), unless otherwise indicated. Refers to any native TYRP1 from any vertebrate source, including animals. The term includes "full length" unprocessed TYRP1 and any form of TYRP1 resulting from processing in cells. The term also includes naturally occurring variants of TYRP1, such as splice variants or allelic variants. In one aspect, the TYRP1 is a human TYRP1.

"EGFRvIII" is an abbreviation for Epidermal Growth Factor Receptor Variant III, which results in the deletion of 267 amino acids with a glycine substitution at the junction, in-frame of Exxon 2-7. A variant of EGFR formed by deletion. The sequence of human EGFRvIII is shown in SEQ ID NO: 115 (without signal peptide). The sequence of wild-type human EGFR is shown in SEQ ID NO: 116 (without signal peptide). See also UniProt entry number P00533 (version 258). As used herein, "EGFRvIII" refers to mammals such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys), and rodents (eg, mice and rats), unless otherwise indicated. Refers to any native EGFRvIII from any vertebrate source, including animals. The term includes any form of "full length" unprocessed EGFRvIII (but not wild-type EGFR), and EGFRvIII resulting from processing in cells (eg, EGFRvIII without a signal peptide). In one aspect, EGFRvIII is human EGFRvIII.

"Affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise stated, "binding affinity" as used herein refers to a specific binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of the molecule X for its partner Y can generally be expressed by the dissociation constant ( _KD ). Affinity can be measured by well-established methods known in the art, including those described herein. A preferred method for measuring affinity is surface plasmon resonance (SPR).

An "affinity maturation" antibody is an antibody against an antigen that has one or more changes in one or more complementarity determining regions (CDRs) as compared to a parent antibody that has no changes. Refers to an antibody that improves affinity.

"Reduced binding", eg, reduced binding to Fc receptors, refers, for example, to reduced affinity for each interaction, as measured by SPR. For clarity, the term also includes that the affinity drops to zero (or falls below the detection limit of the analytical method), i.e., the interaction is completely lost. Conversely, "increased binding" refers to an increase in binding affinity for individual interactions.

As used herein, "T cell activation" is selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers, T lymphocytes. In particular, refers to the cellular response of one or more cytotoxic T lymphocytes. Assays suitable for determining T cell activation are known in the art and are described herein.

"Modifications that promote association of the first and second subunits of the Fc domain" reduce or prevent association with the same polypeptide as the peptide containing the Fc domain subunit to form a homodimer. , Manipulation of the peptide backbone or post-translational modification of the Fc domain subunit. Modifications that promote association, when used herein, are preferably separate for each of the two Fc domain subunits (ie, the first and second subunits of the Fc domain) for which it is desirable to associate. The modifications are complementary to each other so as to facilitate the association of the two Fc domain subunits. For example, the association-promoting modifications may alter the structure or charge of one or both of the Fc domain subunits to achieve the desired steric or electrostatic association, respectively. Thus, (hetero) dimerization occurs between the polypeptide containing the first Fc domain subunit and the polypeptide containing the second Fc domain subunit, and further components fused to the respective subunits (heterogeneous). For example, they do not have to be the same in the sense that they are not the same (antigen binding domain). In some embodiments, modifications that facilitate association of the first and second subunits of the Fc domain include amino acid mutations within the Fc domain, specifically amino acid substitutions. In a preferred embodiment, modifications that facilitate association of the first and second subunits of the Fc domain include distinct amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

The term "effector function" refers to the biological activity resulting from the Fc region of an antibody and depends on the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagia (ADCP), cytokine secretion, immune complex-mediated antigen by antigen-presenting cells Uptake, down-regulation of cell surface receptors (eg, B cell receptors), and B cell activation.

An "activated Fc receptor" is an Fc receptor that induces a signaling event that stimulates cells containing the receptor to exert effector function after association with the Fc domain of the antibody. Human activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (CD89).

Antibody-dependent cellular cytotoxicity (ADCC) is an immune mechanism that causes the lysis of antibody-coated target cells by immune effector cells. The target cell is a cell to which an antibody containing an Fc region or a derivative thereof specifically binds to the Fc region via a protein moiety that is generally N-terminal. As used herein, the term "reduction of ADCC" is defined above, a reduction in the number of target cells lysing at a given antibody concentration in the medium surrounding the target cells at a given time. Increased antibody concentration in the medium surrounding the target cells required to achieve lysis of a given number of target cells by the mechanism of ADCC and / or at a given time. Defined as. The reduction in ADCC is for ADCC mediated by the same antibody that is produced by the same type of host cell but not engineered using the same standard production, purification, formulation and storage methods (known to those of skill in the art). For example, an amino acid substitution that lowers ADCC, which is a decrease in ADCC mediated by an antibody containing that Fc domain, is for ADCC mediated by the same antibody that does not contain this amino acid substitution in the Fc domain. Assays suitable for measuring ADCC are well known in the art (see, eg, PCT Application International Publication No. 2006/082515 or 2012/130831).

As used herein, the term "manipulate, manipulate, manipulate" is naturally occurring or after any manipulation or translation of the peptide backbone of recombinant polypeptides or fragments thereof. Considered to include modifications. Manipulation involves modification of amino acid sequences, modification of glycosylation patterns, or modification of side chain groups of individual amino acids, and a combination of these approaches.

The term "amino acid mutation", as used herein, is intended to include substitutions, deletions, insertions, and modifications of amino acids. Any combination of substitutions, deletions, insertions and modifications will reach the final construct as long as the final construct has the desired characteristics, eg, reduced binding to the Fc receptor, or increased association with another peptide. Can be done like this. Deletions and insertions of amino acid sequences include deletions and insertions of amino acid at the amino and / or carboxy terminus. A preferred amino acid mutation is an amino acid substitution. For example, non-conservative amino acid substitutions (ie, replacing one amino acid with another amino acid with different structural and / or chemical properties) are particularly preferred for the purpose of altering the binding characteristics of the Fc region. Amino acid substitutions may be with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of 20 standard amino acids (eg 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Replacement can be mentioned. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods include mutagenesis of specific sites, PCR, gene synthesis and the like. It is envisioned that methods other than genetic engineering that alter the side chain groups of amino acids, such as chemical modifications, may also be useful. Various names may be used herein to indicate the same amino acid mutation. For example, the replacement of proline for glycine at position ₃₂₉ of the Fc domain can be indicated as 329G, G329, G329, P329G or Pro329Gly.

"Amino acid sequence identity rate (%)" to the reference polypeptide sequence is part of the sequence identity after aligning the sequences and introducing gaps if necessary to achieve maximum sequence identity rate. Is defined as the proportion of amino acid residues in the candidate sequence that is identical to the amino acid residues in the reference polypeptide sequence, without considering any conservative substitutions. Alignments for determining the amino acid sequence identity rate can be made in various manners within the skill of the art, eg, publicly available computer software such as BLAST, BLAST-2, Clustal W. It can be achieved using the Megalign (DNASTAR) software or the FASTA program package. One of skill in the art can determine the appropriate parameters for aligning the sequences, including any algorithm required to achieve maximum alignment over the overall length of the sequences being compared. Alternatively, the identity ratio value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is described by Genentech, Inc. The source code is U.S.A. S. It is filed in the user documentation of Copyright Office (Washington DC, 20559) and is described in U.S.A. S. Copyright Registration No. It is registered under TXU51087 and is described in International Publication No. 2001/007611.

However, for purposes herein, the% amino acid sequence identity value is generated using the FASTA package version 36.3.8c ggseerch program or subsequently using the BLOSUM50 comparison matrix. The FASTA program package is available from W.A. R. Pearson and D. J. Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988): 2444-2448), W. et al. R. Pearson (“Effective protein section comparison” Meth. Enzymol. 266 (1996): 227-258), and Pearson et. al. Created by (Genomics 46 (1997) 24-36) and published and available from www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/fasta be. Instead, use a public server accessible at fasta.bioch.virginia.edu/fasta_www2/index.cgi with the ggseearch (global protein: protein) program and default options (BLOSUM50; open: -10; Using ext: -2; Ktup = 2), it is possible to reliably perform global alignment rather than local alignment and compare sequences. The amino acid identity rate is given in the output alignment header.

The term "polynucleotide" or "nucleic acid molecule" includes any compound and / or substance, including polymers of nucleotides. Each nucleotide is composed of a base, specifically a purine base or a pyrimidine base (that is, cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), sugar. (Ie, deoxyribose or ribose), and a phosphate group. In many cases, a nucleic acid molecule is described by a base sequence, where the base represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented from 5'to 3'. As used herein, the term nucleic acid molecule refers to deoxyribonucleic acid (DNA), such as complementary DNA (DNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), DNA or synthetic forms of RNA. And include mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes sense and antisense strands, as well as both single-stranded and double-stranded forms. In addition, the nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides, including derivatized sugars or phosphate skeletal bonds or chemically modified residues, include modified nucleotide bases. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and / or in vivo, for example in a host or patient. Such a DNA vector (eg, cDNA) or RNA vector (eg, mRNA) may be unmodified or may be modified. For example, the mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that the mRNA can be injected into the subject to produce an antibody in vivo (. See, for example, Stadler et al. (2017) Nature Medicine 23: 815-817 or European Patent No. 2101823 (B1)).

An "isolated" nucleic acid molecule is one in which the nucleic acid molecule is separated from its constituents of its natural environment. The isolated nucleic acid molecule includes a nucleic acid molecule in which a nucleic acid molecule normally contained in a cell containing the nucleic acid molecule is present outside the chromosome or at a chromosomal position different from the original chromosomal position thereof.

"An isolated polynucleotide (or nucleic acid) encoding an antibody" refers to one or more polynucleotide molecules encoding the heavy and light chains (or fragments thereof) of an antibody, a single vector or a separate vector. Such a polynucleotide molecule (s) include such a polynucleotide molecule (s) located at one or more positions in a host cell.

As used herein, the term "vector" refers to a nucleic acid molecule to which it can propagate another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure and a vector integrated into the genome of the host cell into which the vector has been introduced. Certain vectors can direct the expression of nucleic acids to which they are functionally linked. Such vectors are referred to herein as "expression vectors".

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which are the primary transformed cells and the progeny derived from the primary transformed cells, regardless of the number of passages. including. The progeny may not have the exact same nucleic acid content as the parent cell, but may contain mutations. Progeny of variants having the same function or biological activity as screened or selected for the originally transformed cells are included in the invention. The host cell is any type of cell line that can be used to produce the antibodies of the invention. Host cells include cultured cells, eg, cultured mammalian cells, eg, HEK cells, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma, to name just a few. Cells, PER cells, PER. Examples include C6 cells or hybridoma cells, yeast cells, insect cells and plant cells, but also include cells contained in transgenic animals, transgenic plants or cultured plants or animal tissues. In one aspect, the host cell of the invention is a eukaryotic cell, particularly a mammalian cell. In one aspect, the host cell is not a cell in the human body.

The term "pharmaceutical composition" or "pharmaceutical formulation" is a form in which the biological activity of the active ingredient contained in the preparation is effective and is acceptable to the subject to whom the composition will be administered. Refers to a preparation that does not contain any additional ingredients that are too toxic.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" (and its grammatical variants, eg, "treat" or "treating"), is in the individual being treated. Refers to clinical intervention in an attempt to change the original course of the disease, which can be done prophylactically or during the course of clinical pathology. The desired effects of treatment include preventing the onset or recurrence of the disease, reducing symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing disease progression. These include letting, remission or alleviation of the condition, and recovery or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of the disease or to slow the progression of the disease.

The "individual" or "subject" is a mammal. Mammals include domestic animals (eg, cows, sheep, cats, dogs and horses), primates (eg, non-human primates such as humans and monkeys), rabbits, rodents (eg, mice and rats). However, but not limited to these. In certain embodiments, the individual or subject is a human.

An "effective amount" of a drug, eg, a pharmaceutical composition, refers to the dosage required to achieve the desired therapeutic or prophylactic outcome and the amount effective for the required time period.

The term "package package insert" is a commercial product of a therapeutic product that contains information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings regarding the use of such therapeutic product. Used to refer to the instructions normally included in the package.

II. Compositions and methods

The present invention provides antibodies that bind to CD3, including multispecific antibodies that bind to CD3 and a second antigen. Antibodies exhibit excellent stability, for example, in combination with other properties that are beneficial for therapeutic applications related to efficacy and safety, pharmacokinetics, and reproducibility. The antibody of the present invention is useful for treating diseases such as cancer, for example.

A. Anti-CD3 antibody

In one aspect, the invention provides an antibody that binds to CD3. In one aspect, an isolated antibody that binds to CD3 is provided. In one aspect, the invention provides an antibody that specifically binds to CD3. In certain embodiments, the anti-CD3 antibody is at pH 7.4, 37 ° C, as determined by surface plasmon resonance (SPR), compared to binding activity at pH 6, -80 ° C after 2 weeks. After 2 weeks, the binding activity to CD3 is maintained over about 90%.

In one aspect, the invention is an antibody that binds to CD3, wherein the antibody comprises the heavy chain complementarity determining regions (HCDR) 1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 5. A th chain comprising a heavy chain variable region (VH) and a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and a light chain variable region (VL) comprising LCDR3 of SEQ ID NO: 10. An antibody, which comprises one antigen binding domain, is provided.

In one aspect, the antibody is a humanized antibody. In one aspect, the antigen binding domain is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, VH and / or VL are humanized variable regions.

In one aspect, VH and / or VL include an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

In one aspect, the VH comprises one or more heavy chain framework sequences (ie, FR1, FR2, FR3 and / or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO: 7. In one aspect, VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In one aspect, VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. In one aspect, VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity is substituted (eg, conservative substitution), inserted, or compared to the reference sequence. An antibody that contains a deletion but contains that sequence retains the ability to bind to CD3. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 7. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the CDR (ie, in FR). In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 7. Optionally, VH comprises the amino acid sequence of SEQ ID NO: 7, including post-translational modifications of that sequence.

In one aspect, the VL comprises one or more light chain framework sequences (ie, FR1, FR2, FR3 and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO: 11. In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In one aspect, the VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 11. In one aspect, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity is substituted (eg, conservative substitution), inserted, or compared to the reference sequence. An antibody that contains a deletion but contains that sequence retains the ability to bind to CD3. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 11. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the CDR (ie, in FR). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 11. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 11 and comprises a post-translational modification of that sequence.

In one aspect, VH comprises at least about 95%, 96%, 97%, 98%, or 99% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 7, and VL to the amino acid sequence of SEQ ID NO: 11. It contains at least about 95%, 96%, 97%, 98%, or 99% identical amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 7, and VL comprises the amino acid sequence of SEQ ID NO: 11.

In a further aspect, the invention comprises an antibody that binds to CD3 and comprises a first antigen binding domain comprising a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 11. offer.

In a further aspect, the invention provides an antibody that binds to CD3 and comprises a first antigen binding domain comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11.

In another aspect, the invention is the first comprising an antibody that binds to CD3, a VH comprising a heavy chain CDR sequence of VH of SEQ ID NO: 7 and a VL comprising a light chain CDR sequence of VL of SEQ ID NO: 11. An antibody, which comprises an antigen binding domain, is provided.

In a further aspect, the first antigen binding domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of VH of SEQ ID NO: 7 and the LCDR1, LCDR2 and LCDR3 amino acid sequences of VL of SEQ ID NO: 11.

In one aspect, the VH has at least 95%, 96%, 97%, 98% or 99% sequence identity to the heavy chain CDR sequence of VH of SEQ ID NO: 7 and the framework sequence of VH of SEQ ID NO: 7. Includes a framework with. In one aspect, the VH comprises a heavy chain CDR sequence of VH of SEQ ID NO: 7 and a framework having at least 95% sequence identity to the framework sequence of VH of SEQ ID NO: 7. In one aspect, the VH comprises a heavy chain CDR sequence of VH of SEQ ID NO: 7 and a framework having at least 98% sequence identity to the framework sequence of VH of SEQ ID NO: 7.

In one aspect, the VL has at least 95%, 96%, 97%, 98% or 99% sequence identity to the light chain CDR sequence of VL of SEQ ID NO: 11 and the framework sequence of VL of SEQ ID NO: 11. Includes a framework with. In one aspect, the VL comprises a light chain CDR sequence of VL of SEQ ID NO: 11 and a framework having at least 95% sequence identity to the framework sequence of VL of SEQ ID NO: 11. In one aspect, the VL comprises a light chain CDR sequence of VL of SEQ ID NO: 11 and a framework having at least 98% sequence identity to the framework sequence of VL of SEQ ID NO: 11.

In one aspect, the invention comprises an antibody that binds to CD3 and comprises a first antigen binding domain comprising the VH sequence of any of the embodiments provided above and the VL sequence of any of the embodiments provided above. , Antibodies.

In one aspect, the antibody comprises a human constant region. In one aspect, the antibody is an immunoglobulin molecule comprising a human constant region, particularly an IgG class immunoglobulin molecule comprising the human CH1, CH2, CH3 and / or CL domains. Exemplary sequences of human constant domains are given in SEQ ID NOs: 120 and 121 (human kappa and lambda CL domains, respectively), and SEQ ID NO: 122 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one aspect, the antibody is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 120 or SEQ ID NO: 121, particularly the amino acid sequence of SEQ ID NO: 120. Contains the light chain constant region containing the sequence. In one aspect, the antibody comprises a heavy chain constant region comprising at least about 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 122. In particular, the heavy chain constant region may contain amino acid mutations in the Fc domain as described herein.

In one aspect, the first antigen binding domain comprises a human constant region. In one aspect, the first antigen binding moiety is a Fab molecule containing a human constant region, in particular the human CH1 and / or CL domain. In one aspect, the first antigen binding domain is at least about 95%, 96%, 97%, 98%, 99% or with respect to the amino acid sequence of SEQ ID NO: 120 or SEQ ID NO: 121, particularly the amino acid sequence of SEQ ID NO: 120. Contains a light chain constant region containing 100% identical amino acid sequences. In particular, the light chain constant region may contain the amino acid mutations described herein in a "charge-modified" state and / or, in the case of a crossover Fab molecule, one or more (particularly 2). It may contain a deletion or substitution of the N-terminal amino acid. In one aspect, the first antigen binding domain is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 122. Includes a heavy chain constant region containing. In particular, the heavy chain constant region (specifically the CH1 domain) may contain the amino acid mutations described herein in a "charge-modified" state.

In one aspect, the antibody is a monoclonal antibody.

In one aspect, the antibody is an IgG, in particular an IgG ₁ antibody. In one aspect, the antibody is a full length antibody.

In another aspect, the antibody is an antibody fragment selected from the group of Fv molecule, scFv molecule, Fab molecule, F (ab') ₂ molecule, in particular Fab molecule. In another aspect, the antibody fragment is a diabody, triabody, or tetrabody.

In one aspect, the first antigen binding domain is a Fab molecule. In a preferred embodiment, the first antigen binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of the Fab light chain and Fab heavy chain are replaced with each other ( That is, the first antigen-binding domain is a crossover Fab molecule).

In a further aspect, the antibody according to any of the above embodiments is described in Section II. A. 1. 1. ~ 8. Any of the features described in may be incorporated alone or in combination.

In a preferred embodiment, the antibody comprises an Fc domain, in particular an IgG Fc domain, more preferably an IgG1 Fc domain. In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc domain is an IgG ₁ Fc domain. The Fc domain consists of the first and second subunits and may incorporate any of the features described below in connection with the Fc domain variant, alone or in combination (section). II.A.8.).

In another preferred embodiment, the antibody comprises a second antigen binding domain and optionally a third antigen binding domain that binds to the second antigen (ie, the antibody is further described herein below. It is a multispecific antibody to be used (Section II.A.7.).

1. 1. Antibody fragment

In certain embodiments, the antibodies provided herein are antibody fragments.

In one aspect, the antibody fragment is a Fab molecule, a Fab'molecule, a Fab'-SH molecule, or _two F (ab') molecules, particularly the Fab molecule described herein. The "Fab'molecule" differs from the Fab molecule only in the addition of a residue at the carboxy terminus of the CH1 domain containing one or more cysteines from the antibody hinge region. Fab'-SH is a Fab'molecule in which a cysteine residue (s) in the constant domain retains a free thiol group. Pepsin treatment gives two F (ab') molecules with _two antigen binding sites (two Fab molecules) and a portion of the Fc region.

In another aspect, the antibody fragment is a diabody, triabody, or tetrabody. A "diabody" is an antibody fragment having two antigen binding sites that can be divalent or bispecific. For example, European Patent No. 404,097, International Publication No. 1993/01161, Hudson et al. , Nat. Med. 9: 129-134 (2003), and Hollinger et al. , Proc. Natl. Acad. Sci. See USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al. , Nat. Med. It is also described in 9: 129-134 (2003).

In a further aspect, the antibody fragment is a single-stranded Fab molecule. The "single-chain Fab molecule" or "scFab" refers to an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL). , And the linker, the antibody domain and the linker are in the following order: a) VH-CH1-linker-VL-CL, b) VL-CL-in the direction from the N-terminus to the C-terminus. It has one of a linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide of at least 30 amino acids, preferably 32-50 amino acids. The single-stranded Fab molecule is stabilized by a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-stranded Fab molecules are further subjected to the formation of interchain disulfide bonds by insertion of cysteine residues (eg, variable heavy chain position 44 and variable light chain position 100 according to Kabat numbering). It will be stabilized.

In another aspect, the antibody fragment is a single chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the heavy chain (VH) and light chain (VL) variable domains of an antibody linked by a linker. In particular, the linker is a short polypeptide of 10-25 amino acids, usually rich in glycine for flexibility and rich in serine or threonine for solubility, and the N-terminus of VH. Can be connected to the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody despite removal of constant regions and introduction of linkers. As a review of scFv fragments, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. , Springer-Verlag, New York, pp. Please refer to International Publication No. 93/16185 and US Pat. Nos. 5,571,894 and 5,587,458.

In another aspect, the antibody fragment is a single domain antibody. A "single domain antibody" is an antibody fragment comprising all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Pat. No. 6,248,516 (B1)).

Antibody fragments are made by a variety of techniques, including, but not limited to, digestion of intact antibodies by proteolysis and recombinant production by recombinant host cells (eg, E. coli), as described herein. May be good.

2. 2. Humanized antibody

In certain embodiments, the antibody provided herein is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while preserving the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody contains one or more variable domains in which the CDR (or part thereof) is derived from a non-human antibody and the FR (or part thereof) is derived from a human antibody sequence. The humanized antibody optionally also comprises at least a portion of the human constant region. In some embodiments, some FR residues in a humanized antibody are, for example, from a non-human antibody (eg, an antibody from which a CDR residue is derived) in order to restore or improve antibody specificity or affinity. Replaced by the corresponding residue of.

For information on humanized antibodies and methods for producing them, see Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008) and further described below: Richmann et al. , Nature 332: 323-329 (1988); Queen et al. , Proc. Nat'l Acad. Sci. USA 86: 12002-10033 (1989); US Pat. Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al. .. , Methods 36: 25-34 (2005) (description of sex-determining region (SDR) grafts); Padlan, Mol. Immunol. 28: 489-498 (1991) (description for resurfacing); Dollar'Acqua et al. , Methods 36: 43-60 (2005) (description for FR shuffle); and Osbourn et al. , Methods 36: 61-68 (2005) and Klimka et al. , Br. J. Cancer, 83: 252-260 (2000) (FR Shuffle, "Description of Guided Choice Approach".

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (eg, Sims et al. J. Immunol. 151: 2296). (1993)); Framework regions derived from human antibody consensus sequences of specific subtypes of heavy or light chain variable regions (eg, Carter et al. Proc. Natl. Acad. Sci. USA, 89). : 4285 (1992); and Presta et al. J. Immunol., 151: 2623 (1993)); Human maturation (somatic cell mutation) framework region or human germline framework region (eg, Almagro). And Francson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from FR library screening (eg, Baca et al., J. Biol. Chem. 272: 10678-). 10684 (1997) and Rosok et al., J Biol. Chem. 271: 22611-22618 (1996)).

3. 3. Glycosylation variant

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites to an antibody can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

If the antibody contains an Fc region, the oligosaccharide attached to the antibody may be changed. Native antibodies produced by mammalian cells typically contain branched bifurcated oligosaccharides that are generally bound to Asn297 in the CH2 domain of the Fc region by N-binding. For example, Wright et al. See TIBTECH 15: 26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to the "stem" GlcNAc of the bifurcated oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention may be made to create antibody variants with certain improved properties.

In one aspect, a non-fucosylated oligosaccharide, i.e., an antibody variant having an oligosaccharide structure lacking fucose binding (direct or indirect) to the Fc region is provided. Such non-fucosylated oligosaccharides (also referred to as "afcosylated" oligosaccharides) are, in particular, N-linked oligosaccharides lacking the fucose residue to which the first GlcNAc is attached to the stem of the bifurcated oligosaccharide structure. In one aspect, an antibody variant is provided in which the proportion of non-fucosylated oligosaccharides in the Fc region is increased as compared to the native antibody or the parent antibody. For example, the proportion of non-fucosylated oligosaccharides is at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (ie, in the absence of fucosylated oligosaccharides). May be good. The proportion of non-fucosylated oligosaccharides is, for example, the sum of all oligosaccharides bound to Asn297 (eg, complex, hybrid, and) as measured by MALDI-TOF mass spectrometry as described in WO 2006/082515. The (average) amount of oligosaccharides lacking fucose residues with respect to the high mannose structure). Asn297 refers to an asparagine residue located at a position within the Fc region of about 297 (EU numbering of Fc region residues). However, Asn297 may also be located upstream or downstream of about ± 3 amino acids from position 297, i.e., between positions 294-300, due to minor sequence mutations in the antibody. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region can have improved FcγRIIIa receptor binding and / or improved effector function, especially improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003/0151782; 2004/093621.

As an example of a cell line capable of producing an antibody with reduced fucosylation, Lec13CHO cells deleted by protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US patent application published. 2003/0157108; and WO 2004/056312, especially Example 11), and knockout cell lines, eg, FUT8 knockout CHO cells of the α-1,6-fucosyltransferase gene (eg, Yamane-Ohnuki et). Al. Biotech. Bioeng. 87: 614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); and International Publication No. 2003/085107. Or cells with reduced or extinguished activity of GDP-fucose synthesis or transporter proteins (see, eg, US Patent Application Publication Nos. 2004259150, 2005031613, 2004432140, 2004110282). I want to be).

In a further aspect, the antibody variant is provided, for example, with a bisect oligosaccharide in which a bifurcated oligosaccharide bound to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described, for example, in Umana et al. , Nat Biotechnol 17, 176-180 (1999); Ferrara et al. , Biotechn Bioeng 93,851-861 (2006); International Publication No. 99/54342; No. 2004/06540, No. 2003/011878.

Also provided are antibody variants having at least one galactose residue within the oligosaccharide bound to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087, 1998/58964, and 1999/22764.

4. Cysteine Manipulative Antibody Variant

In certain embodiments, it may be desirable to make a cysteine engineered antibody, eg, THIOMAB ™, in which one or more residues of the antibody are replaced with cysteine residues. In a preferred embodiment, the substituted residue occurs at the accessible site of the antibody. By substituting these residues with cysteine, reactive thiol groups are thereby positioned at the accessible sites of the antibody, which can be used to antibody to other moieties such as drug moieties or linker-drug moieties. Can be complexed to make an immune complex as further described herein. Cysteine-operated antibodies are, for example, US Pat. Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or International Publication No. 2016040856. Can be made as described in.

5. Antibody derivative

In certain embodiments, the antibodies provided herein may be further modified to include additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3. , 6-Trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycols, polypropylene glycol homopolymers, polypolylone oxide / ethylene oxide copolymers, poly Examples include, but are not limited to, oxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymer may have any molecular weight, may be branched, or may not be branched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, the polymers can be the same molecule or different molecules. In general, the number and / or type of polymer used for derivatization includes the specific properties or functions of the antibody to be improved, whether the antibody derivative is used in a therapy under specified conditions, and the like. It may be determined based on considerations not limited to these.

6. Immune complex

The invention also comprises cytotoxic agents, chemotherapeutic agents or chemotherapeutic agents, growth inhibitors, toxins (eg, protein toxins, bacterial fungi, plant, or enzyme-active toxins of animal origin, or fragments thereof), or radioactive. Provided are an immune complex comprising an anti-CD3 antibody herein combined with one or more therapeutic agents such as isotopes.

In one aspect, the immune complex is an antibody drug conjugate (ADC) in which the antibody is complexed into one or more of the therapeutic agents described above. Antibodies are typically linked to one or more therapeutic agents using a linker. A review of ADC techniques, including examples of therapeutic agents and drugs and linkers, is described in Pharmacol Review 68: 3-19 (2016).

In another aspect, the immune complex comprises an enzyme-active toxin or an antibody of the invention complexed to a fragment thereof, a diphtheria A chain, a non-binding active fragment of a diphtheria toxin, an exotoxin A chain (Rypus bacillus). From), ricin A chain, abrin A chain, modesin A chain, α-salcin, cinnamon abragiri protein, dianthin protein, yoshuyamagobo protein (PAPI, PAPII, and PAP-S), turleishi inhibitor, curcin , Crotin, savonsaw inhibitor, geronin, mitgerin, restrictocin, phenomycin, enomycin, and tricotesene, but not limited to these.

In another aspect, the immune complex comprises an antibody described herein of the invention, which is complexed with a radioactive atom to form a radioactive complex. Various radioisotopes are available for the production of radioactive complexes. Examples include radioactive isotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and Lu. When a radiocomplex is used for detection, of a radioatom for scintillation studies, such as Tc ^99m or I ¹²³ , or magnetic resonance imaging (also known as magnetic resonance imaging, MRI). May include spin labels such as I ¹²³ , I ¹³¹ , In ¹¹¹ , F ¹⁹ , C ¹³ , N ¹⁵ , O ¹⁷ , gadolinium, manganese, or iron.

Complexes of antibodies and cytotoxic agents include N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), 4- (N-maleimidemethyl) cyclohexane-1-carboxylate succinimidyl (SMCC), iminothiolan (IT). ), Bifunctional derivatives of imide esters (such as dimethyl HCl for adipimide acid), active esters (such as dysuccinimidyl sverate), aldehydes (such as glutaaldehyde), bis-azido compounds (bis (p-azidobenzoyl) hexanediamine). , Etc.), bis-diazonium derivatives (bis- (p-diazonium benzoyl) -ethylenediamine, etc.), diisocyanates (eg, toluene2,6-diisocyanate, etc.), and bis-active fluorine compounds (1,5-difluoro-2,4). -Can be made using a variety of bifunctional protein binding agents such as (dinitrobenzene, etc.). For example, the ricin immunotoxin is described in Vitetta et al. , Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocianatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See International Publication No. 94/11026. The linker may be a "cleavable linker" that facilitates the release of cytotoxic drugs within the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photosensitive linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al, Cancer Res. 52: 127-131 (1992); US Pat. No. 5,208,020). ) May be used.

The immunoconjugates or ADCs herein are BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS. , Sulf-MBS, Sulf-SIAB, Sulf-SMCC, and Sulf-SMBP, as well as commercially available SVSBs (eg, from Pierce Biotechnology, Inc., Rockford, IL., USA) ((4-vinyl). Sulfones) succinimidyl benzoate), but not limited to, expressly contemplate such complexes prepared with cross-linking agent reagents.

7. Multispecific antibody

In certain embodiments, the antibodies provided herein are multispecific antibodies, in particular bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different antigenic determinants (eg, two different proteins or two different epitopes in the same protein). In certain embodiments, the multispecific antibody has three or more binding specificities. In certain embodiments, one of the binding specificities is for CD3 and the other is for any other antigen. In certain embodiments, the multispecific antibody may bind to two (or more) different epitopes of CD3. Multispecific (eg, bispecific) antibodies can also be used to localize cytotoxic agents or cells to cells expressing CD3. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for producing multispecific antibodies include recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and ". The "konb-in-hole" operation (see, eg, US Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270: 26 (1997)) However, but not limited to these. Multispecific antibodies also manipulate the electrostatic steering effect to make antibody Fc heterodimer molecules (see, eg, WO 2009/089004); of two or more antibodies or fragments. Cross-linking (see, eg, US Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); production of bispecific antibodies using leucine zippers (eg, Kosterny et.). al., J. Immunol., 148 (5): 1547-1553 (1992) and WO 2011/034605); General light chains to avoid light chain mismatching problems. Use of technology (see, eg, WO 98/50431); use of "diabody" technology for making bispecific antibody fragments (eg, Hollinger et al., Proc. Natl. Accad). Sci. USA 90: 6444-6448 (1993); and the use of single-stranded Fv (sFv) dimers (eg, Gruber et al., J. Immunol., 152: 5368 (1994)). See); and, for example, Tutt et al. J. Immunol. It can be made by the preparation of the trispecific antibody described in 147: 60 (1991).

For example, an engineered antibody having three or more antigen binding sites, including an "Octopus antibody", or DVD-Ig is also included herein (eg, WO 2001/77342 and WO 2008 /. See 024715). Other examples of multispecific antibodies with three or more antigen binding sites are WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792. And can be found in International Publication No. 2013/026831. The multispecific antibody or antigen-binding fragment thereof also comprises a "double-acting FAb" or "DAF" containing an antigen-binding site that binds to CD3, as well as to another different antigen or to two different epitopes of CD3 (. See, for example, US Patent Application Publication No. 2008/0069820 and International Publication No. 2015/095539).

Multispecific antibodies are asymmetrical forms with domain crossovers in one or more binding arms of the same antigen specificity, i.e., the VH / VL domain (eg, WO 2009/080252 and WO 2015 / See 150447), CH1 / CL domain (see, eg, International Publication No. 2009/080253) or complete Fab arm (eg, International Publication No. 2009/082511, International Publication No. 2016/016299). See also Schaefer et al, PNAS, 108 (2011) 1187-1191 and Klein at al., MAbs 8 (2016) 1010-20) (so-called "CrossMab"). It can also be provided by "technology". Asymmetric Fab arms can also be manipulated by introducing charged or uncharged amino acid mutations into the domain interface to orient the correct Fab pairing. See, for example, International Publication No. 2016/172485.

Various additional molecular formats of multispecific antibodies are known in the art and are included herein (see, eg, Spirits et al., Mol Immunol 67 (2015) 95-106).

Certain types of multispecific antibodies, also included herein, include surface antigens of target cells, such as tumor cells, and activated invariant components of the T cell receptor (TCR) complex, such as T cells. A bispecific antibody designed to simultaneously bind to CD3 for retargeting and killing target cells. Thus, in a preferred embodiment, the antibodies provided herein are multispecific antibodies, particularly bispecific, in which one of the binding specificities is to CD3 and the other is to the target cell antigen. It is a sex antibody.

An example of a bispecific antibody format that may be useful for this purpose is the so-called "BiTE" (bispecific T cell engager) molecule, in which two scFv molecules are fused by a flexible linker. (For example, International Publication No. 2004/106381, International Publication No. 2005/061547, International Publication No. 2007/042261 and International Publication No. 2008/119567, Nagarsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011). (See); Diabody (Holliger et al., Prot Eng 9,299-305 (1996)) and derivatives thereof, such as Tandem Diabody (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41). -56 (1999)); "DART" (bi-affinity retargeting) molecule based on the Diabody format, but characterized by C-terminal disulfide bridges for further stabilization (Johnson et al., J Mol Biol. 399,436-449 (2010)), as well as all hybrid mouse / rat IgG molecules, the so-called triomab (as outlined in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). However, it is not limited to these. Specific T cell bispecific antibody formats included herein are WO 2013/026833, WO 2013/026839, WO 2016/20309; Bacac et al. , Oncoimmunology 5 (8) (2016) e1203498.

Preferred embodiments of the multispecific antibody of the present invention are described below.

In one aspect, the invention comprises a first antigen-binding domain that binds to CD3 as described herein, and a second and optionally a third antigen-binding domain that binds to a second antigen. Provided are antibodies that bind to CD3, including.

According to a preferred embodiment of the invention, the antigen-binding domain contained in the antibody is a Fab molecule (ie, an antigen-binding domain composed of heavy and light chains, including a variable domain and a constant domain, respectively). In one embodiment, the first, second and / or third antigen binding domain, if present, is a Fab molecule. In one embodiment, the Fab molecule is of human origin. In a preferred embodiment, the Fab molecule has been humanized. In yet another embodiment, the Fab molecule comprises a human heavy chain and light chain constant domain.

Preferably, at least one of the antigen binding domains is a crossover Fab molecule. Such modifications reduce the mispairing of heavy and light chains of different Fab molecules, thereby improving the yield and purity of the (multispecific) antibodies of the invention in recombinant production. In the preferred crossover Fab molecule useful for the (multispecific) antibody of the invention, the variable domains of the Fab light chain and Fab heavy chain (VL and VH, respectively) are exchanged. However, even with this domain exchange, the preparation of (multispecific) antibodies results in certain by-products due to the so-called Bens-Jones-type interactions between mispaired heavy and light chains. May include (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To further reduce the mismatching of heavy and light chains of different Fab molecules, thereby increasing the purity and yield of the desired (multispecific) antibody, the first antigen is a charged amino acid with opposite charges. It can be introduced at specific amino acid positions in the CH1 and CL domains of either the Fab molecule that binds to (CD3) or the Fab molecule that binds to a second antigen (eg, target cell antigen, eg TYRP-1 or EGFRvIII). Yes, and further described herein. The charge modification is a conventional Fab molecule contained in a (multispecific) antibody (eg, shown in FIGS. 1A-C, GJ), or a VH / VL crossover contained in a (multispecific) antibody. It is done in one of the Fab molecules (eg, shown in FIGS. 1D-F, K-N) (but not both). In a preferred embodiment, the charge modification is carried out on a conventional Fab molecule contained in the (multispecific) antibody (in a preferred embodiment, it binds to a second antigen, such as a target cell antigen, such as TYRP-1 or EGFRvIII).

In a preferred embodiment according to the invention, the (multispecific) antibody can simultaneously bind to the first antigen (ie, CD3) and the second antigen (eg, target antigen, eg, TYRP-1 or EGFRvIII). .. In one embodiment, the (multispecific) antibody can crosslink T cells and target cells by simultaneously binding to CD3 and target cell antigens. In an even more preferred embodiment, such co-binding results in lysis of target cells, particularly target cell antigens (eg, TYRP-1 or EGFRvIII) -expressing tumor cells. In one embodiment, such simultaneous binding results in activation of T cells. In another embodiment, such co-binding is T lymphocytes selected from the group of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers, especially cytotoxic. It produces a cellular response of T lymphocytes. In one embodiment, binding of the (multispecific) antibody to CD3 without simultaneous binding to the target cell antigen does not result in T cell activation.

In one embodiment, the (multispecific) antibody is capable of redirecting the cytotoxic activity of T cells to target cells. In a preferred embodiment, this redirection is independent of MHC-mediated peptide antigen presentation and / or T cell specificity by the target cell.

Preferably, the T cells according to any aspect of the invention are cytotoxic T cells. In some embodiments, the T cell is a CD4 ⁺ or CD8 ⁺ T cell, in particular a CD8 ⁺ T cell.

a) First antigen-binding domain

The (multispecific) antibody of the present invention comprises at least one antigen binding domain (first antigen binding domain) that binds to CD3. In a preferred embodiment, the CD3 is a human CD3 (SEQ ID NO: 112) or a cynomolgus monkey CD3 (SEQ ID NO: 113), particularly human CD3. In one embodiment, the first antigen binding domain is cross-reactive (ie, specifically binds to) human and cynomolgus monkey CD3. In some embodiments, CD3 is the epsilon subunit of CD3 (CD3 epsilon).

In a preferred embodiment, the (multispecific) antibody comprises no more than one antigen binding domain that binds to CD3. In one embodiment, the (multispecific) antibody provides monovalent binding to CD3.

In one embodiment, the antigen binding domain that binds to CD3 is an antibody fragment selected from the group of Fv molecule, scFv molecule, Fab molecule and F (ab') ₂ molecule. In a preferred embodiment, the antigen binding domain that binds to CD3 is a Fab molecule.

In a preferred embodiment, the antigen-binding domain that binds to CD3 is the crossover Fab molecule described herein, that is, the variable domains VH and VL of the Fab heavy and light chains or the constant domains CH1 and CL are exchanged with each other. It is a Fab molecule that is / is being replaced. In such an embodiment, the antigen binding domain that binds to a second antigen (eg, target cell antigen, eg, TYRP-1 or EGFRvIII) is preferably a conventional Fab molecule. (Multispecific) In certain embodiments, the antigen-binding domain that binds to CD3 is preferably a crossover Fab molecule, and in certain embodiments, the antigen-binding domain that binds to a second antigen contained in the antibody is more than one, particularly a Fab molecule. The antigen-binding domain that binds to the antigen of 2 is a conventional Fab molecule.

In an alternative embodiment, the antigen binding domain that binds to CD3 is a conventional Fab molecule. In such an embodiment, the antigen-binding domain that binds to a second antigen (eg, target cell antigen, eg, TYRP-1 or EGFRvIII) is the crossover Fab molecule described herein, i.e., a Fab heavy chain. And the light chain variable domains VH and VL or constant domains CH1 and CL are exchanged / replaced Fab molecules with each other. (Multi-specificity) In certain embodiments, the antigen-binding domain that binds to a second antigen is preferably a crossover Fab molecule, and in certain embodiments, the antigen-binding domain that binds to CD3 contained in the antibody is more than one, particularly a Fab molecule. The antigen-binding domain that binds to CD3 is a conventional Fab molecule.

In a preferred embodiment, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain or the constant domains CL and CH1, in particular the variable domains VL and VH, are replaced with each other (i.e. , According to such an embodiment, the first antigen binding domain is a crossover Fab molecule in which the variable or constant domains of the Fab light chain and Fab heavy chain have been exchanged). In one such embodiment, the second (and, if present, third) antigen-binding domain is a conventional Fab molecule.

In one embodiment, no more than one antigen binding domain that binds to CD3 is present in the (multispecific) antibody (ie, this antibody provides monovalent binding to CD3).

b) Second (and third) antigen-binding domain

In certain embodiments, the (multispecific) antibody of the invention comprises at least one antigen binding domain that binds to a second antigen, particularly a Fab molecule. The second antigen is preferably not CD3, i.e. different from CD3. In one embodiment, the second antigen is an antigen expressed in cells different from CD3 (eg, expressed in cells other than T cells). In one aspect, the second antigen is a target cell antigen, particularly a tumor cell antigen. In a specific embodiment, the second antigen is TYRP-1. In another specific embodiment, the second antigen is EGFRvIII. The second antigen binding domain can direct the (multispecific) antibody to the target site, eg, to a particular type of tumor cell expressing the second antigen.

In one embodiment, the antigen binding domain that binds to the second antigen is an antibody fragment selected from the group of Fv molecule, scFv molecule, Fab molecule and F (ab') ₂ molecule. In a preferred embodiment, the antigen binding domain that binds to the second antigen is the Fab molecule.

In certain embodiments, the (multispecific) antibody comprises two antigen binding domains that bind to a second antigen, particularly the Fab molecule. In such a preferred embodiment, each of these antigen binding domains binds to the same antigenic determinant. In an even more preferred embodiment, all of these antigen binding domains are identical, i.e., have the same molecular format (eg, conventional or crossover Fab molecule) and are described herein (if present). The CH1 and CL domains contain amino acid sequences containing the same amino acid substitutions. In one embodiment, the (multispecific) antibody comprises no more than two antigen binding domains that bind to the second antigen, particularly the Fab molecule.

In a preferred embodiment, the antigen binding domain that binds to the second antigen is a conventional Fab molecule. In such an embodiment, the antigen-binding domain that binds to CD3 is the crossover Fab molecule described herein, that is, the variable domains VH and VL of the Fab heavy and light chains or the constant domains CH1 and CL are exchanged with each other. Fab molecules that have been / replaced.

In an alternative embodiment, the antigen-binding domain that binds to the second antigen is the crossover Fab molecule described herein, i.e., variable domains VH and VL or constant domains CH1 and CL of Fab heavy and light chains. Are Fab molecules that have been exchanged / replaced with each other. In such an embodiment, the antigen binding domain that binds to CD3 is a conventional Fab molecule.

In one embodiment, the second (and, if present, third) antigen-binding domain comprises a human constant region. In one embodiment, the second (and, if present, third) antigen-binding domain is a Fab molecule comprising a human constant region, particularly the human CH1 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs: 120 and 121 (human kappa and lambda CL domains, respectively), and SEQ ID NO: 122 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one embodiment, the second (and third, if any) antigen-binding domain is at least about 95%, 96%, 97% to the amino acid sequence of SEQ ID NO: 120 or SEQ ID NO: 121, particularly the amino acid sequence of SEQ ID NO: 120. , 98%, 99% or 100% contains a light chain constant region containing the same amino acid sequence. In particular, the light chain constant region may contain the amino acid mutations described herein in a "charge-modified" state and / or, in the case of a crossover Fab molecule, one or more (particularly 2). It may contain a deletion or substitution of the N-terminal amino acid. In some embodiments, the second (and third, if any) antigen-binding domain is at least about 95%, 96%, 97%, 98%, to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 122. Contains heavy chain constant regions containing 99% or 100% identical amino acid sequences. In particular, the heavy chain constant region (specifically the CH1 domain) may contain the amino acid mutations described herein in a "charge-modified" state.

TYRP-1

In a preferred embodiment, the second antigen is TYRP-1, in particular human TYRP-1 (SEQ ID NO: 114).

In one embodiment, the second (and, if present, third) antigen-binding domain comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 15, HCDR2 of SEQ ID NO: 16 and HCDR3 of SEQ ID NO: 17. It comprises a chain variable region (VH) and a light chain variable region (VL) comprising the light chain complementarity determining regions (LCDRs) 1 of SEQ ID NO: 19, LCDR2 of SEQ ID NO: 20 and LCDR3 of SEQ ID NO: 21.

In one embodiment, the second (and, if present, third) antigen-binding domain is (and derives from) a humanized antibody. In one embodiment, the second (and, if present, third) antigen-binding domain is a humanized antigen-binding domain (ie, the antigen-binding domain of a humanized antibody). In one embodiment, the VH and / or VL of the second (and, if present, third) antigen-binding domain is a humanized variable region.

In one embodiment, the VH and / or VL of the second (and third, if any) antigen-binding domain comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

In one embodiment, the VH of the second (and third, if any) antigen-binding domain is the heavy chain framework sequence of one or more heavy chain framework sequences of SEQ ID NO: 18 (ie, FR1, FR2, FR3 and / or FR4 sequences). )including. In one embodiment, VH comprises at least about 95%, about 96%, about 97%, about 98% or about 99% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, VH sequences having at least about 95%, about 96%, about 97%, about 98% or about 99% identity are substituted (eg, conservative substitutions), as compared to the reference sequence. An antibody containing an insertion or deletion, but containing that sequence, retains the ability to bind TYRP-1. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 18. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the CDR (ie, in FR). In one embodiment, VH comprises the amino acid sequence of SEQ ID NO: 18. Optionally, VH comprises the amino acid sequence of SEQ ID NO: 18, including post-translational modifications of that sequence.

In one embodiment, the VL of the second (and third, if any) antigen-binding domain is one or more light chain framework sequences of SEQ ID NO: 22 (ie, FR1, FR2, FR3 and / or FR4 sequences). )including. In one embodiment, the VL comprises an amino acid sequence of at least about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of SEQ ID NO: 22. In one embodiment, the VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 22. In one embodiment, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 22. In certain embodiments, VL sequences having at least about 95%, about 96%, about 97%, about 98% or about 99% identity are substituted (eg, conservative substitutions), as compared to the reference sequence. An antibody containing an insertion or deletion, but containing that sequence, retains the ability to bind TYRP-1. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 22. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the CDR (ie, in FR). In one embodiment, VL comprises the amino acid sequence of SEQ ID NO: 22. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 22 and comprises a post-translational modification of that sequence.

In one embodiment, the VH of the second (and third, if any) antigen-binding domain is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18. The VL of the second (and third, if any) antigen-binding domain comprising the sequence is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 22. Contains an array. In one embodiment, VH comprises the amino acid sequence of SEQ ID NO: 18 and VL comprises the amino acid sequence of SEQ ID NO: 22.

In a further embodiment, the second (and, if present, third) antigen binding domain comprises a VH comprising the sequence of SEQ ID NO: 18 and a VL comprising the sequence of SEQ ID NO: 22.

In a further embodiment, the second (and, if present, third) antigen binding domain comprises the VH sequence of SEQ ID NO: 18 and the VL sequence of SEQ ID NO: 22.

In another embodiment, the second (and third, if any) antigen binding domain comprises a VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 18 and a VL comprising the light chain CDR sequence of VL of SEQ ID NO: 22. include.

In a further embodiment, the second (and, if present, third) antigen-binding domain is the amino acid sequence of HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 18, and the amino acids of LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 22. Contains an array.

In one embodiment, the VH of the second (and third, if any) antigen-binding domain is at least 95%, 96, to the heavy chain CDR sequence of VH of SEQ ID NO: 18 and the framework sequence of VH of SEQ ID NO: 18. Includes a framework with%, 97%, 98% or 99% sequence identity. In one embodiment, the VH comprises a heavy chain CDR sequence of VH of SEQ ID NO: 18 and a framework having at least 95% sequence identity to the framework sequence of VH of SEQ ID NO: 18. In another embodiment, the VH comprises a heavy chain CDR sequence of VH of SEQ ID NO: 18, and a framework having at least 98% sequence identity to the framework sequence of VH of SEQ ID NO: 18.

In one embodiment, the VL of the second (and third, if any) antigen-binding domain is at least 95%, 96, to the light chain CDR sequence of VL of SEQ ID NO: 22 and the framework sequence of VL of SEQ ID NO: 22. Includes a framework with%, 97%, 98% or 99% sequence identity. In one embodiment, the VL comprises a framework having at least 95% sequence identity to the light chain CDR sequence of VL of SEQ ID NO: 22 and the framework sequence of VL of SEQ ID NO: 22. In another embodiment, the VL comprises a framework having at least 98% sequence identity to the light chain CDR sequence of VL of SEQ ID NO: 22 and the framework sequence of VL of SEQ ID NO: 22.

EGFRvIII

In a preferred embodiment, the second antigen is EGFRvIII, in particular human EGFRvIII (SEQ ID NO: 115).

In one embodiment, the second (and, if present, third) antigen-binding domain comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 85, HCDR2 of SEQ ID NO: 86 and HCDR3 of SEQ ID NO: 87. It comprises a chain variable region (VH) and a light chain variable region (VL) comprising the light chain complementarity determining regions (LCDRs) 1 of SEQ ID NO: 89, LCDR2 of SEQ ID NO: 90 and LCDR3 of SEQ ID NO: 91.

In one embodiment, the second (and, if present, third) antigen-binding domain is (and derives from) a humanized antibody. In one embodiment, the second (and, if present, third) antigen-binding domain is a humanized antigen-binding domain (ie, the antigen-binding domain of a humanized antibody). In one embodiment, the VH and / or VL of the second (and, if present, third) antigen-binding domain is a humanized variable region.

In one embodiment, the VH and / or VL of the second (and third, if any) antigen-binding domain comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

In one embodiment, the VH of the second (and, if present, third) antigen-binding domain is one or more heavy chain framework sequences of SEQ ID NO: 88 (ie, FR1, FR2, FR3 and / or FR4 sequences). )including. In one embodiment, VH comprises an amino acid sequence of at least about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of SEQ ID NO: 88. In one embodiment, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 88. In one embodiment, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 88. In certain embodiments, VH sequences having at least about 95%, about 96%, about 97%, about 98% or about 99% identity are substituted (eg, conservative substitutions), as compared to the reference sequence. Antibodies containing the sequence, including insertions or deletions, retain the ability to bind EGFRvIII. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 88. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the CDR (ie, in FR). In one embodiment, VH comprises the amino acid sequence of SEQ ID NO: 88. Optionally, VH comprises the amino acid sequence of SEQ ID NO: 88, including post-translational modifications of that sequence.

In one embodiment, the VL of the second (and third, if any) antigen binding domain is the one or more light chain framework sequences of SEQ ID NO: 92 (ie, FR1, FR2, FR3 and / or FR4 sequences). )including. In one embodiment, the VL comprises an amino acid sequence of at least about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of SEQ ID NO: 92. In one embodiment, the VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 92. In one embodiment, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 92. In certain embodiments, VL sequences having at least about 95%, about 96%, about 97%, about 98% or about 99% identity are substituted (eg, conservative substitutions), as compared to the reference sequence. Antibodies containing the sequence, including insertions or deletions, retain the ability to bind EGFRvIII. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 92. In certain embodiments, substitutions, insertions, or deletions occur in the outer region of the CDR (ie, in FR). In one embodiment, VL comprises the amino acid sequence of SEQ ID NO: 92. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 92, including post-translational modifications of that sequence.

In one embodiment, the VH of the second (and third, if any) antigen-binding domain is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88. The VL of the second (and third, if any) antigen-binding domain comprising the sequence is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 92. Contains an array. In one embodiment, VH comprises the amino acid sequence of SEQ ID NO: 88 and VL comprises the amino acid sequence of SEQ ID NO: 92.

In a further embodiment, the second (and, if present, third) antigen binding domain comprises a VH comprising the sequence of SEQ ID NO: 88 and a VL comprising the sequence of SEQ ID NO: 92.

In a further embodiment, the second (and, if present, third) antigen binding domain comprises the VH sequence of SEQ ID NO: 88 and the VL sequence of SEQ ID NO: 92.

In another embodiment, the second (and third, if any) antigen binding domain comprises a VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 88 and a VL comprising the light chain CDR sequence of VL of SEQ ID NO: 92. include.

In a further embodiment, the second (and, if present, third) antigen-binding domain is the amino acid sequence of HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 88, and the amino acids of LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 92. Contains an array.

In one embodiment, the VH of the second (and third, if any) antigen-binding domain is at least 95%, 96 to the heavy chain CDR sequence of VH of SEQ ID NO: 88 and the framework sequence of VH of SEQ ID NO: 88. Includes a framework with%, 97%, 98% or 99% sequence identity. In one embodiment, the VH comprises a heavy chain CDR sequence of VH of SEQ ID NO: 88 and a framework having at least 95% sequence identity to the framework sequence of VH of SEQ ID NO: 88. In another embodiment, the VH comprises a heavy chain CDR sequence of VH of SEQ ID NO: 88 and a framework having at least 98% sequence identity to the framework sequence of VH of SEQ ID NO: 88.

In one embodiment, the VL of the second (and third, if any) antigen-binding domain is at least 95%, 96 to the light chain CDR sequence of VL of SEQ ID NO: 92 and the framework sequence of VL of SEQ ID NO: 92. Includes a framework with%, 97%, 98% or 99% sequence identity. In one embodiment, the VL comprises a framework having at least 95% sequence identity to the light chain CDR sequence of VL of SEQ ID NO: 92 and the framework sequence of VL of SEQ ID NO: 92. In another embodiment, the VL comprises a framework having at least 98% sequence identity to the light chain CDR sequence of VL of SEQ ID NO: 92 and the framework sequence of VL of SEQ ID NO: 92.

In an alternative embodiment, the second (and if present, third) antigen binding domain is provided above in this section with respect to the VH sequence of any of the embodiments provided above in this section, and with respect to EGFRvIII. Contains the VL sequence of any of the embodiments described, but comprising SEQ ID NOs: 85 (HCDR1), 86 (HCDR2), 87 (HCDR3), 88 (VH), 89 (LCDR1), 90 (LCDR2), 91 (LCDR3). And instead of 92 (VL), it is based on the following sequence (in order of column).

In one embodiment, the second (and, if present, third) antigen binding domain contains the VH sequence of any of the embodiments provided above in this section and the VL sequence of any of the embodiments provided above this section. include.

Anti-TYRP-1 and anti-EGFRvIII antibodies

The present invention also comprises a VH sequence of any of the embodiments provided above in this section with respect to TYRP-1, and a VL sequence of any of the embodiments provided above of this section with respect to TYRP-1. Antibodies that bind to -1 (eg, the heavy chain variable region (VH) containing the heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO: 15, HCDR2 of SEQ ID NO: 16 and HCDR3 of SEQ ID NO: 17, and the heavy chain variable region (VH) of SEQ ID NO: 19). Contains an antibody that binds to TYRP-1, or the sequence of SEQ ID NO: 18, comprising a light chain complementarity determination region (LCDR) 1, a light chain variable region (VL) comprising LCDR2 of SEQ ID NO: 20 and LCDR3 of SEQ ID NO: 21. An antibody that binds to TYRP-1) comprising VH and VL comprising the sequence of SEQ ID NO: 22 is also provided.

The invention also comprises an antibody that binds to EGFRvIII, comprising the VH sequence of any of the embodiments provided above in this section with respect to EGFRvIII, and the VL sequence of any of the embodiments provided above of this section with respect to EGFRvIII. (For example, a heavy chain variable region (VH) containing the heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO: 85, HCDR2 of SEQ ID NO: 86 and HCDR3 of SEQ ID NO: 87, and a light chain complementarity determination region of SEQ ID NO: 89. (LCDR) 1, an antibody that binds to EGFRvIII, comprising a light chain variable region (VL) comprising LCDR2 of SEQ ID NO: 90 and LCDR3 of SEQ ID NO: 91, or a sequence of VH and SEQ ID NO: 92 comprising the sequence of SEQ ID NO: 88. Also provided are antibodies that bind to TYRP-1, including VL.

In a further embodiment, the antibody that binds to TYRP-1 or EGFRvIII according to any of the above embodiments can incorporate any of the features described for the antibody that binds to CD3, either alone or in combination (anti-CD3). Antibodies, eg (unless the binding sequence is explicitly specified).

c) Charge modification

The (multispecific) antibody of the present invention exchanges VH / VL in the binding arm of one (or more in the case of a molecule containing more than two antigen-binding Fab molecules) to the Fab molecule contained therein. Includes amino acid substitutions that are particularly efficient in reducing light chain mispairs (Bens-Jones type by-products) with incompatible heavy chains that can result from the production of Fab-based multispecific antibodies with. (See also PCT Application International Publication No. 2015/150447, which is incorporated herein by reference in its entirety). Ratio of desired (multispecific) antibody compared to undesired by-products, in particular Bence Jones-type by-products resulting in multispecific antibodies having VH / VL domain exchange in one of the binding arms. Can be improved by introducing charged amino acids having opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

Thus, the first and second (and, if present, third) antigen-binding domains of the (multispecific) antibody are both Fab molecules and one of the antigen-binding domains (particularly the first antigen binding). In some embodiments, the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other in the domain).
i) In the constant domain CL of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is replaced with a positively charged amino acid (numbered by Kabat), second (and present). If so, in the constant domain CH1 of the antigen-binding domain of the third), the amino acid at position 147 or the amino acid at position 213 is replaced with a negatively charged amino acid (numbering by Kabat EU index), or ii) first. In the constant domain CL of the antigen-binding domain, the amino acid at position 124 is replaced with a positively charged amino acid (numbering by Kabat), and the amino acid or position at position 147 in the constant domain CH1 of the first antigen-binding domain. The 213 amino acids have been replaced with negatively charged amino acids (numbering by Kabat EU index).

The (multispecific) antibody does not contain both the modifications described in i) and ii). The constant domains CL and CH1 of the antigen-binding domain with VH / VL exchange are not replaced (ie, remain unexchanged) with each other.

In a more specific embodiment
i) In the constant domain CL of the second (and third, if any) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H). In the constant domain CH1 of the cage (numbering by Kabat), the second (and third, if any) antigen-binding domain, the amino acid at position 147 or 213 is due to glutamate (E) or aspartic acid (D). Independently substituted (numbered by Kabat EU index), or ii) in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is lysine (K), arginine (R) or histidine (H). (Numbering by Kabat), in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or amino acid at position 213 is independent of glutamic acid (E) or aspartic acid (D). (Numbering by Kabat EU index).

In one such embodiment, in the constant domain CL of the second (and third, if any) antigen-binding domain, the amino acid at position 124 is by lysine (K), arginine (R) or histidine (H). In the constant domain CH1 of the second (and third, if any) antigen-binding domain, which is independently substituted (numbered by Kabat), the amino acid at position 147 or 213 is glutamic acid (E) or It is independently substituted with aspartic acid (D) (numbering by Kabat EU index).

In a further embodiment, in the constant domain CL of the second (and third, if any) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H). In the constant domain CH1 of the second (and third, if any) antigen-binding domain, the amino acid at position 147 is independent of glutamic acid (E) or aspartic acid (D). Substituted (numbering by Kabat EU index).

In a preferred embodiment, in the constant domain CL of the second (and third, if any) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H). (Numbering by Kabat), the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering by Kabat), second (and if present). , 3) In the constant domain CH1 of the antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering by Kabat EU index), and the amino acid at position 213. Is independently substituted with glutamate (E) or aspartic acid (D) (numbering by Kabat EU index).

In a more preferred embodiment, in the constant domain CL of the second (and third, if any) antigen-binding domain, the amino acid at position 124 is replaced by lysine (K) (numbered by Kabat) and the amino acid at position 123. Is substituted with lysine (K) (numbered by Kabat) and the amino acid at position 147 is substituted with glutamic acid (E) in the constant domain CH1 of the second (and, if present, third) antigen-binding domain. The cage (numbering by Kabat EU index), the amino acid at position 213 is replaced with glutamic acid (E) (numbering by Kabat EU index).

In an even more preferred embodiment, in the constant domain CL of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is replaced by lysine (K) (numbered by Kabat) at position 123. The amino acid has been replaced by arginine (R) (numbered by Kabat) and the amino acid at position 147 has been replaced by glutamic acid (E) in the constant domain CH1 of the second (and third, if any) antigen-binding domain. (Numbering by Kabat EU index), the amino acid at position 213 is replaced with glutamic acid (E) (numbering by Kabat EU index).

In a preferred embodiment, if the amino acid substitution according to the above embodiment is performed on the constant domain CL and constant domain CH1 of the second (and if present, third) antigen-binding domain, the second (and if present, third). The constant domain CL of the antigen-binding domain of) is a kappa isotype.

Alternatively, the amino acid substitution according to the above embodiment replaces the constant domain CL and constant domain CH1 of the second (and, if present) antigen-binding domain with the constant domain CL and constant domain of the first antigen-binding domain. It is done in CH1. In a preferred embodiment, the constant domain CL of the first antigen binding domain is a kappa isotype.

Therefore, in one embodiment, in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering by Kabat). ), In the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering by Kabat EU index). ..

In a further embodiment, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering by Kabat). In the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering by Kabat EU index).

In yet another embodiment, in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering by Kabat). ), The amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering by Kabat) and at position 147 in the constant domain CH1 of the first antigen binding domain. Amino acids are independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D). Substituted (numbering by Kabat EU index).

In one embodiment, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is replaced by lysine (K) (numbering by Kabat) and the amino acid at position 123 is replaced by lysine (K). (Numbering by Kabat), in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 is replaced by glutamic acid (E) (numbering by Kabat EU index), and the amino acid at position 213 is due to glutamic acid (E). Substituted (numbering by Kabat EU index).

In another embodiment, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is replaced with lysine (K) (numbering by Kabat) and the amino acid at position 123 is replaced with arginine (R). In the cage (numbering by Kabat), the amino acid at position 147 is replaced with glutamic acid (E) in the constant domain CH1 of the first antigen-binding domain (numbering by Kabat EU index), and the amino acid at position 213 is glutamic acid (E). Replaced by (numbering by Kabat EU index).

In a preferred embodiment, the (multispecific) antibody of the invention is:
(A) A first antigen-binding domain that binds to CD3, in which the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, and SEQ ID NO: 2 Heavy chain complementarity determining regions (HCDR) 1, heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 3 and HCDR3 of SEQ ID NO: 5, and light chain complementarity determining regions (LCDR) 1 of SEQ ID NO: 8, sequence. A first antigen binding domain, which is a Fab molecule, comprising a light chain variable region (VL) comprising LCDR2 of No. 9 and LCDR3 of SEQ ID NO: 10.
(B) A second and optional third antigen-binding domain that binds to the second antigen, at position 124 in the constant domain CL of the second (and, if present, third) antigen-binding domain. Amino acids are independently substituted with lysine (K), arginine (R) or histidine (H) (preferably independently with lysine (K) or arginine (R)) (numbering by Kabat). , The amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (preferably independently with lysine (K) or arginine (R)) (Kabat). In the constant domain CH1 of the second (and third, if any) antigen-binding domain, the amino acid at position 147 is independently substituted with glutamate (E) or aspartic acid (D) (numbered by). Kabat EU index numbering), amino acids at position 213 are independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), second antigen binding domain and third antigen. Includes a combined domain.

d) Multispecific antibody format

The (multispecific) antibody according to the present invention can have various structures. An exemplary structure is depicted in FIG.

In a preferred embodiment, the antigen binding domain contained in the (multispecific) antibody is a Fab molecule. In such an embodiment, the first, second, third and the like antigen-binding domains may be referred to herein as first, second, third and the like Fab molecules, respectively.

In one embodiment, the first and second antigen binding domains of the (multispecific) antibody are optionally fused to each other via a peptide linker. In a preferred embodiment, the first and second antigen-binding domains are Fab molecules, respectively. In one such embodiment, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain. In another such embodiment, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain. (I) The first antigen-binding domain is fused at the C-terminal of the Fab heavy chain to the N-terminal of the Fab heavy chain of the second antigen-binding domain, or (ii) the second antigen-binding domain is the Fab heavy chain. In the embodiment in which the C-terminal of the first antigen-binding domain is fused to the N-terminal of the Fab heavy chain of the first antigen-binding domain, the Fab light chain of the first antigen-binding domain and the Fab light chain of the second antigen-binding domain are additionally fused. May optionally fuse to each other via a peptide linker.

A (multispecific) antibody having a single antigen binding domain (eg, Fab molecule) capable of specifically binding to a second antigen, eg, a target cell antigen, eg, TYRP-1 or EGFRvIII (eg, Figure. 1A, D, G, H, K, L) are particularly useful when internal translocation of the second antigen is predicted after binding of the high affinity antigen binding domain. In such cases, the presence of more than one antigen-binding domain specific for the second antigen may enhance the internal translocation of the second antigen, thereby reducing the availability of the second antigen.

However, in other cases, a second antigen, eg, two or more antigen binding domains (eg, Fab molecules) specific for a target cell antigen, may be optimized or targeted, for example, to a target site. Having (multispecific) antibodies contained to allow cross-linking of cellular antigens (see, eg, those shown in FIGS. 1B, 1C, 1E, 1F, 1I, 1J, 1M or 1N). Is advantageous.

Therefore, in a preferred embodiment, the (multispecific) antibody of the invention comprises a third antigen binding domain.

In one embodiment, the third antigen binding domain binds to a second antigen, such as a target cell antigen, such as TYRP-1 or EGFRvIII. In one embodiment, the third antigen binding domain is a Fab molecule.

In one embodiment, the third antigen binding domain is identical to the second antigen binding domain.

In some embodiments, the third and second antigen-binding domains are Fab molecules, respectively, and the third antigen-binding domain is identical to the second antigen-binding domain. Thus, in these embodiments, the second and third antigen binding domains contain the same heavy and light chain amino acid sequences and have the same domain arrangement (ie, conventional or crossover). Further, in these embodiments, the third antigen binding domain, if present, comprises the same amino acid substitutions as the second antigen binding domain. For example, the amino acid substitution described herein as "charge modification" is performed in the constant domain CL and constant domain CH1, respectively, of the second antigen binding domain and the third antigen binding domain. Alternatively, the amino acid substitution can be made in the constant domain CL and constant domain CH1 of the first antigen binding domain (in a preferred embodiment, which is also a Fab molecule), but the second antigen binding domain and the third antigen. It is not performed in the constant domain CL and the constant domain CH1 of the binding domain.

Similar to the second antigen binding domain, the third antigen binding domain is preferably a conventional Fab molecule. However, embodiments are also considered in which the second and third antigen binding domains are crossover Fab molecules (and the first antigen binding domain is a conventional Fab molecule). Therefore, in a preferred embodiment, the second and third antigen-binding domains are conventional Fab molecules, respectively, and the first antigen-binding domain is the crossover Fab molecule described herein, i.e., a Fab heavy chain. And the light chain variable domains VH and VL or constant domains CL and CH1 are exchanged / replaced with each other Fab molecules. In another embodiment, the second and third antigen-binding domains are crossover Fab molecules, respectively, and the first antigen-binding domain is a conventional Fab molecule.

When a third antigen binding domain is present, in a preferred embodiment, the first antigen binding domain binds to CD3 and the second and third antigen binding domains are the second antigen, particularly the target cell antigen, eg, TYRP. -1 or binds to EGFRvIII.

In a preferred embodiment, the (multispecific) antibody of the invention comprises an Fc domain consisting of first and second subunits. The first and second subunits of the Fc domain are capable of stable association.

The (multispecific) antibodies of the invention can have different structures, i.e., the first, second (and optionally, third) antigen binding domains fuse with each other and in different ways to the Fc domain. You may be doing it. The components may be fused to each other directly or preferably via one or more suitable peptide linkers. If the fusion of the Fab molecule is to the N-terminus of the subunit of the Fc domain, it is typically via the immunoglobulin hinge region.

In some embodiments, the first and second antigen binding domains are Fab molecules, respectively, and the first antigen binding domain is the C-terminus of the Fab heavy chain and the first or second subunit of the Fc domain. It is fused to the N-terminal. In such an embodiment, the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain or the N-terminus of the other subunit of the Fc domain. May be good. In a preferred embodiment, the second antigen-binding domain is a conventional Fab molecule and the first antigen-binding domain is the crossover Fab molecule described herein, i.e., a variable of Fab heavy chain and light chain. Fab molecules in which the domains VH and VL or the constant domains CL and CH1 are exchanged / replaced with each other. In other such embodiments, the second antigen-binding domain is a crossover Fab molecule and the first antigen-binding domain is a conventional Fab molecule.

In one embodiment, the first and second antigen-binding domains are Fab molecules, respectively, and the first antigen-binding domain is the C-terminus of the Fab heavy chain and the N-terminus of the first or second subunit of the Fc domain. The second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain. In a specific embodiment, the (multispecific) antibody is substantially from the first and second Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers. The second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminal of the Fab heavy chain, and the first Fab molecule is Fc at the C-terminal of the Fab heavy chain. It is fused to the N-terminus of the first or second subunit of the domain. Such structures are schematically depicted in FIGS. 1G and 1K (the first antigen binding domain in these examples is the VH / VL crossover Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be additionally fused to each other.

In another embodiment, the first and second antigen-binding domains are Fab molecules, respectively, and the first and second antigen-binding domains are the N-terminus of the Fc domain at the C-terminus of the Fab heavy chain, respectively. It is fused to the terminal. In a specific embodiment, the (multispecific) antibody is substantially from the first and second Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers. The first and second Fab molecules are each fused to the N-terminus of one of the subunits of the Fc domain at the C-terminus of the Fab heavy chain. Such a structure is schematically depicted in FIGS. 1A and 1D (in these examples, the first antigen binding domain is a VH / VL crossover Fab molecule and the second antigen binding domain is conventional. Fab molecule). The first and second Fab molecules may be fused directly to the Fc domain or via a peptide linker. In a preferred embodiment, the first and second Fab molecules are fused to the Fc domain, respectively, via the immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG ₁ hinge region, in particular the Fc domain is an IgG ₁ Fc domain.

In some embodiments, the first and second antigen binding domains are Fab molecules, respectively, and the second antigen binding domain is the C-terminus of the Fab heavy chain and the first or second subunit of the Fc domain. It is fused to the N-terminal. In such an embodiment, the first antigen binding domain is the C-terminus of the Fab heavy chain and the N-terminus of the Fab heavy chain of the first antigen binding domain or the other subunit of the Fc domain (as described above). It may be fused to the N-terminus of the unit. In a preferred embodiment, the second antigen-binding domain is a conventional Fab molecule and the first antigen-binding domain is a crossover Fab molecule described herein, i.e., a Fab heavy chain and a light chain. Fab molecules in which the variable domains VH and VL or the constant domains CL and CH1 are exchanged / replaced with each other. In other such embodiments, the second antigen-binding domain is a crossover Fab molecule and the first antigen-binding domain is a conventional Fab molecule.

In one embodiment, the first and second antigen-binding domains are Fab molecules, respectively, and the second antigen-binding domain is the C-terminus of the Fab heavy chain and the N-terminus of the first or second subunit of the Fc domain. The first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. In a specific embodiment, the (multispecific) antibody is substantially from the first and second Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers. The first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminal of the Fab heavy chain, and the second Fab molecule is Fc at the C-terminal of the Fab heavy chain. It is fused to the N-terminus of the first or second subunit of the domain. Such a structure is schematically depicted in FIGS. 1H and 1L (in these examples, the first antigen binding domain is a VH / VL crossover Fab molecule and the second antigen binding domain is conventional. Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be additionally fused to each other.

In some embodiments, the third antigen binding domain, in particular the third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such a preferred embodiment, the second and third antigen-binding domains are conventional Fab molecules, respectively, and the first antigen-binding domain is the crossover Fab molecule described herein, i.e., Fab weight. Fab molecules in which the variable domains VH and VL of the chains and light chains or the constant domains CL and CH1 are exchanged / replaced with each other. In other such embodiments, the second and third antigen-binding domains are crossover Fab molecules, respectively, and the first antigen-binding domain is a conventional Fab molecule.

In a preferred embodiment, the first and third antigen-binding domains are fused to the N-terminus of one of the Fc domain subunits at the C-terminus of the Fab heavy chain, respectively, and the second antigen-binding domain is: It is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain. In a specific embodiment, the (multispecific) antibody is an Fc domain consisting of first, second and third Fab molecules, first and second subunits, and optionally one or more peptide linkers. The second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the first Fab molecule is the C-terminus of the Fab heavy chain. At the terminus, it is fused to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Such structures are shown in FIGS. 1B and 1E (in these examples, the first antigen binding domain is a VH / VL crossover Fab molecule and the second and third antigen binding domains are conventional Fab molecules). , And FIGS. 1J and 1N (in these examples, the first antigen binding domain is a conventional Fab molecule and the second and third antigen binding domains are VH / VL crossover Fab molecules). It is depicted. The first and third Fab molecules may be fused directly to the Fc domain or via a peptide linker. In a preferred embodiment, the first and third Fab molecules are fused to the Fc domain, respectively, via the immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG ₁ hinge region, in particular the Fc domain is an IgG ₁ Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be additionally fused to each other.

In another such embodiment, the second and third antigen-binding domains are fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, respectively. , The C-terminus of the Fab heavy chain is fused to the N-terminus of the Fab heavy chain of the second antigen binding domain. In a specific embodiment, the (multispecific) antibody is an Fc domain consisting of first, second and third Fab molecules, first and second subunits, and optionally one or more peptide linkers. The first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the second Fab molecule is the C-terminus of the Fab heavy chain. At the terminus, it is fused to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Such structures are shown in FIGS. 1C and 1F (in these examples, the first antigen binding domain is a VH / VL crossover Fab molecule and the second and third antigen binding domains are conventional Fab molecules). , And FIGS. 1I and 1M (in these examples, the first antigen binding domain is a conventional Fab molecule and the second and third antigen binding domains are VH / VL crossover Fab molecules). It is depicted. The second and third Fab molecules may be fused directly to the Fc domain or via a peptide linker. In a preferred embodiment, the second and third Fab molecules are fused to the Fc domain, respectively, via the immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG ₁ hinge region, in particular the Fc domain is an IgG ₁ Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be additionally fused to each other.

Two Fab molecules, the hinge region, in the structure of the antibody in which the Fab molecule is fused to the N-terminus of each subunit of the Fc domain at the C-terminus of the Fab heavy chain via the immunoglobulin hinge region (multispecific). And Fc domains substantially form immunoglobulin molecules. In a preferred embodiment, the immunoglobulin molecule is an IgG class immunoglobulin. In an even more preferred form, the immunoglobulin is an IgG ₁ subclass immunoglobulin. _In another embodiment, the immunoglobulin is an IgG4 subclass immunoglobulin. In a more preferred embodiment, the immunoglobulin is a human immunoglobulin. In another embodiment, the immunoglobulin is a chimeric or humanized immunoglobulin. In one embodiment, the immunoglobulin comprises a human constant region, particularly a human Fc region.

In some of the (multispecific) antibodies of the invention, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are optionally fused to each other via a peptide linker. Depending on the structure of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused to the N-terminus of the Fab light chain of the second Fab molecule at the C-terminus, or the second. The Fab light chain of the Fab molecule of 2 may be fused to the N-terminal of the Fab light chain of the first Fab molecule at the C-terminal. Fusion of the Fab light chains of the first and second Fab molecules further reduces the mismatching of incompatible Fab heavy and light chains and is required for the expression of some (multispecific) antibodies of the invention. It also reduces the number of plasmids.

The antigen-binding domain may be fused to the Fc domain or directly or via a peptide linker containing one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G ₄ S) _n , (SG ₄ ) _n , (G ₄ S) _n or G ₄ (SG ₄ ) _n peptide linkers. "N" is generally an integer of 1 to 10, typically 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment 5-100, and in a further aspect 10-50 amino acids. In one embodiment, the peptide linker is (GxS) _n or ( _GxS ) _n Gm, G = glycine, S = serine and (x = 3, n = 3, 4, 5 or 6, m = 0, 1, 2 or 3) or (x = 4, n = 2, 3, 4 or 5, m = 0, 1, 2 or 3), and in one embodiment, x = 4 and n = 2 or 3. In a further embodiment, x = 4 and n = 2. In one embodiment, the peptide linker is ( _{G4S) 2 .} A peptide linker particularly suitable for fusing the Fab light chains of the first and second Fab molecules to each other is ( _{G4S) 2 .} Exemplary peptide linkers suitable for connecting the Fab heavy chains of the first and second Fab fragments include SEQ ID NOs: (D)-( _{G4 S) 2 (} SEQ ID NOs: 118 and 119). Another suitable such linker comprises sequence ( _{G4S) 4 .} In addition, the linker may include (part of) an immunoglobulin hinge region. In particular, if the Fab molecule is fused to the N-terminus of the Fc domain subunit, it may be fused with or without an additional peptide linker via the immunoglobulin hinge region or part thereof.

In certain embodiments, the (multispecific) antibody of the invention shares the Fab light chain variable region of the first Fab molecule with the Fab heavy chain constant region of the first Fab molecule (the carboxy-terminal peptide binding is shared with the Fab heavy chain constant region of the first Fab molecule. That is, the first Fab molecule contains the crossover Fab heavy chain, and the heavy chain variable region is replaced with the light chain variable region), and the Fab heavy chain constant region of the first Fab molecule has a carboxy-terminal peptide bond. The (VL ₍₁₎ -CH1 ₍₁ ) -CH2-CH3 (-CH4)) polypeptide shared with the Fc domain subunit, as well as the Fab heavy chain of the second Fab molecule, carboxy-terminal peptide binding to the Fc domain subunit. Contains (VH ₍₂₎ -CH1 ₍₂ ) -CH2-CH3 (-CH4)) polypeptides shared with. In some embodiments, the (multispecific) antibody is a polypeptide (VH) in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule. ₍₁₎ -CL ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included. In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

In certain embodiments, the (multispecific) antibodies of the invention share the Fab heavy chain variable region of the first Fab molecule with the Fab light chain constant region of the first Fab molecule (the carboxy-terminated peptide binding). That is, the first Fab molecule contains the crossover Fab heavy chain, and the heavy chain constant region is replaced with the light chain constant region), and the Fab light chain constant region of the first Fab molecule has a carboxy-terminal peptide bond. The (VH ₍₁₎ -CL ₍₁ ) -CH2-CH3 (-CH4)) polypeptide shared with the Fc domain subunit, as well as the Fab heavy chain of the second Fab molecule, carboxy-terminal peptide binding to the Fc domain subunit. Contains (VH ₍₂₎ -CH1 ₍₂ ) -CH2-CH3 (-CH4)) polypeptides shared with. In some embodiments, the (multispecific) antibody is a polypeptide (VL) in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule. ₍₁₎ -CH1 ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included. In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

In some embodiments, the (multispecific) antibody is such that the Fab light chain variable region of the first Fab molecule shares a carboxy-terminated peptide bond with the Fab heavy chain constant region of the first Fab molecule (ie, the first. The Fab molecule of 1 contains the crossover Fab heavy chain, the heavy chain variable region is replaced with the light chain variable region), and the Fab heavy chain constant region of the first Fab molecule is the second carboxy-terminal peptide bond. The Fab heavy chain of the Fab molecule shares with the Fab heavy chain of the Fab molecule, and the Fab heavy chain of the second Fab molecule shares the carboxy-terminal peptide bond with the Fc domain subunit (VL ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CH1. ₍₂₎ -CH2-CH3 (-CH4)) contains a polypeptide. In another embodiment, the (multispecific) antibody is such that the Fab heavy chain of the second Fab molecule shares a carboxy-terminated peptide bond with the Fab light chain variable region of the first Fab molecule and of the first Fab molecule. The Fab light chain variable region shares the carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (ie, the first Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region is light. The Fab heavy chain constant region of the first Fab molecule, which replaces the chain variable region), shares the carboxy-terminated peptide binding with the Fc domain subunit (VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -. Contains CH1 ₍₁₎ -CH2-CH3 (-CH4)) polypeptides. In some of these embodiments, the (multispecific) antibody is the first in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminated peptide bond with the Fab light chain constant region of the first Fab molecule. Fab molecule crossover Fab light chain polypeptide (VH ₍₁₎ -CL ₍₁₎ ) and second Fab molecule Fab light chain polypeptide (VL ₍₂₎ -CL ₍₂₎ ) are further included. In other aspects of these embodiments, if appropriate, the (multispecific) antibody comprises the Fab heavy chain variable region of the first Fab molecule and the carboxy-terminal peptide binding to the Fab light chain constant region of the first Fab molecule. The Fab light chain constant region of the first Fab molecule shares the carboxy-terminated peptide bond with the Fab light chain polypeptide of the second Fab molecule (VH ₍₁₎ -CL ₍₁₎ -VL _(2). -CL ₍₂₎ ) The polypeptide, or Fab light chain polypeptide of the second Fab molecule, shares the carboxy-terminated peptide binding with the Fab heavy chain variable region of the first Fab molecule and the Fab of the first Fab molecule. The heavy chain variable region further shares the carboxy-terminated peptide bond with the Fab light chain constant region of the first Fab molecule (VL ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ) polypeptide. include. In the (multispecific) antibody according to these embodiments, (i) the Fc domain subunit polypeptide (CH2-CH3 (-CH4)) or (ii) the Fab heavy chain of the third Fab molecule has a carboxy-terminal peptide bond. The polypeptide shared with the Fc domain subunit (VH ₍₃₎ -CH1 ₍₃ ) -CH2-CH3 (-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL ₍₃₎ -CL _(3). ) Can be further included. In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

In some embodiments, the (multispecific) antibody shares the Fab heavy chain variable region of the first Fab molecule with the Fab light chain constant region of the first Fab molecule (ie, the first). The Fab molecule of 1 contains the crossover Fab heavy chain, the heavy chain constant region is replaced with the light chain constant region), and the Fab light chain constant region of the first Fab molecule is the second carboxy-terminal peptide bond. The Fab heavy chain of the Fab molecule shares with the Fab heavy chain of the Fab molecule, and the Fab heavy chain of the second Fab molecule shares the carboxy-terminal peptide bond with the Fc domain subunit (VH ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CH1. ₍₂₎ -CH2-CH3 (-CH4)) contains a polypeptide. In another embodiment, the (multispecific) antibody is such that the Fab heavy chain of the second Fab molecule shares the carboxy-terminal peptide binding with the Fab heavy chain variable region of the first Fab molecule and of the first Fab molecule. The Fab heavy chain variable region shares the carboxy-terminal peptide binding with the Fab light chain constant region of the first Fab molecule (ie, the first Fab molecule contains the crossover Fab heavy chain and the heavy chain constant region is light. The Fab light chain constant region of the first Fab molecule, which replaces the chain constant region), forms a carboxy-terminated peptide bond with the Fc domain subunit (VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍ 1). ₎ -Contains a polypeptide shared with CH2-CH3 (-CH4)). In some of these embodiments, the (multispecific) antibody is the first in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminated peptide bond with the Fab heavy chain constant region of the first Fab molecule. Fab molecule crossover Fab light chain polypeptide (VL ₍₁₎ -CH1 ₍₁₎ ) and second Fab molecule Fab light chain polypeptide (VL ₍₂₎ -CL ₍₂₎ ). In other aspects of these embodiments, if appropriate, the (multispecific) antibody comprises the Fab light chain variable region of the first Fab molecule with the carboxy-terminal peptide binding to the Fab heavy chain constant region of the first Fab molecule. The Fab heavy chain constant region of the first Fab molecule shares the carboxy-terminated peptide bond with the Fab light chain polypeptide of the second Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ -VL _(2). -CL ₍₂₎ ) The polypeptide, or Fab light chain polypeptide of the second Fab molecule, shares the carboxy-terminated peptide binding with the Fab heavy chain variable region of the first Fab molecule and the Fab of the first Fab molecule. The heavy chain variable region further shares the carboxy-terminated peptide bond with the Fab light chain constant region of the first Fab molecule (VL ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ) polypeptide. include. In the (multispecific) antibody according to these embodiments, (i) the Fc domain subunit polypeptide (CH2-CH3 (-CH4)) or (ii) the Fab heavy chain of the third Fab molecule has a carboxy-terminal peptide bond. The polypeptide shared with the Fc domain subunit (VH ₍₃₎ -CH1 ₍₃ ) -CH2-CH3 (-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL ₍₃₎ -CL _(3). ) Can be further included. In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

In certain embodiments, the (multispecific) antibody does not contain the Fc domain. In such a preferred embodiment, the second and, if present, the third antigen-binding domain are conventional Fab molecules, respectively, and the first antigen-binding domain is the crossover Fab molecule described herein. That is, Fab molecules in which the variable domains VH and VL of the Fab heavy chain and the light chain or the constant domains CL and CH1 are exchanged / replaced with each other. In other such embodiments, the second and, if present, the third antigen-binding domain are crossover Fab molecules, respectively, and the first antigen-binding domain is a conventional Fab molecule.

In one such embodiment, the (multispecific) antibody consists substantially of a first and second antigen binding domain, and optionally one or more peptide linkers, the first and second antigens. The binding domains are both Fab molecules, and the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain. Such a structure is schematically depicted in FIGS. 1O and 1S (in these examples, the first antigen binding domain is a VH / VL crossover Fab molecule and the second antigen binding domain is conventional. Fab molecule).

In another such embodiment, the (multispecific) antibody consists substantially of a first and second antigen binding domain, and optionally one or more peptide linkers, the first and second antigens. The binding domains are both Fab molecules, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain. Such a structure is schematically depicted in FIGS. 1P and 1T (in these examples, the fir antigen binding domain is a VH / VL crossover Fab molecule and the second antigen binding domain is a conventional Fab molecule. Is).

In some embodiments, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the (multispecific) antibody is the third antigen. It further comprises a binding domain, particularly a third Fab molecule, which is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In certain such embodiments, the (multispecific) antibody consists substantially of a first, second and third Fab molecule, and optionally one or more peptide linkers, a second Fab. The molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is the Fab weight of the second Fab molecule at the C-terminus of the Fab heavy chain. It is fused to the N-terminus of the chain. Such structures are shown in FIGS. 1Q and 1U (in these examples, the first antigen binding domain is a VH / VL crossover Fab molecule and the second and third antigen binding domains are conventional Fab molecules, respectively. ), Or FIGS. 1X and 1Z (in these examples, the first antigen-binding domain is a conventional Fab molecule and the second and third antigen-binding domains are VH / VL crossover Fab molecules, respectively). It is depicted as a target.

In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the (multispecific) antibody is the third antigen. It further comprises a binding domain, particularly a third Fab molecule, which is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. In certain such embodiments, the (multispecific) antibody consists substantially of a first, second and third Fab molecule, and optionally one or more peptide linkers, the first Fab. The molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is the Fab weight of the second Fab molecule at the N-terminus of the Fab heavy chain. It is fused to the C-terminus of the chain. Such structures are shown in FIGS. 1R and 1V (in these examples, the first antigen binding domain is a VH / VL crossover Fab molecule and the second and third antigen binding domains are conventional Fab molecules, respectively. ), Or FIGS. 1W and 1Y (in these examples, the first antigen-binding domain is a conventional Fab molecule and the second and third antigen-binding domains are VH / VL crossover Fab molecules, respectively). It is depicted as a target.

In certain embodiments, in a (multispecific) antibody according to the invention, the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, the first. The Fab light chain variable region of the Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (ie, the first Fab molecule contains a crossover Fab heavy chain and is heavy chain variable. Regions have been replaced by light chain variable regions) (VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ ) contains polypeptides. In some embodiments, the (multispecific) antibody is a polypeptide (VH) in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule. ₍₁₎ -CL ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included.

In certain embodiments, the (multispecific) antibody according to the invention has a Fab light chain variable region of a first Fab molecule that shares a carboxy-terminated peptide bond with a Fab heavy chain constant region of a first Fab molecule (in certain embodiments). That is, the first Fab molecule contains the crossover Fab heavy chain and the heavy chain variable region is replaced with the light chain variable region), and the Fab heavy chain constant region of the first Fab molecule has a carboxy-terminal peptide bond. It contains a polypeptide shared with the Fab heavy chain of the second Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ ). In some embodiments, the (multispecific) antibody is a polypeptide (VH) in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule. ₍₁₎ -CL ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included.

In certain embodiments, in a (multispecific) antibody according to the invention, the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, the first. Fab heavy chain variable region of the Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (ie, the first Fab molecule contains a crossover Fab heavy chain and is a heavy chain constant). Regions have been replaced by light chain constant regions) (VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ) contains polypeptides. In some embodiments, the (multispecific) antibody is a polypeptide (VL) in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule. ₍₁₎ -CH1 ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included.

In certain embodiments, the (multispecific) antibody according to the invention has a Fab heavy chain variable region of a first Fab molecule that shares a carboxy-terminal peptide bond with the Fab light chain constant region of a first Fab molecule (in certain embodiments). That is, the first Fab molecule contains the crossover Fab heavy chain and the heavy chain constant region is replaced with the light chain constant region), and the Fab light chain constant region of the first Fab molecule has a carboxy-terminal peptide bond. It contains a polypeptide shared with the Fab heavy chain of the second Fab molecule (VH ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ ). In some embodiments, the (multispecific) antibody is a polypeptide (VL) in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule. ₍₁₎ -CH1 ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included.

In certain embodiments, the (multispecific) antibody according to the invention has a Fab heavy chain of a third Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule and a second Fab. The Fab heavy chain of the molecule shares the carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, and the Fab light chain variable region of the first Fab molecule binds the carboxy-terminal peptide bond to the first Fab molecule. (Ie, the first Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region is replaced by a light chain variable region) (VH ₍₃₎ -CH1 ₍ 3). ₎ -VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ ) Contains a polypeptide. In some embodiments, the (multispecific) antibody is a polypeptide (VH) in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule. ₍₁₎ -CL ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included. In some embodiments, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL ₍₃₎ -CL ₍₃₎ ) of a third Fab molecule.

In certain embodiments, in a (multispecific) antibody according to the invention, the Fab heavy chain of the third Fab molecule shares the carboxy-terminated peptide bond with the Fab heavy chain of the second Fab molecule and the second Fab. The Fab heavy chain of the molecule shares the carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, and the Fab heavy chain variable region of the first Fab molecule binds the carboxy-terminal peptide bond to the first Fab molecule. (Ie, the first Fab molecule contains the crossover Fab heavy chain and the heavy chain constant region is replaced by the light chain constant region) (VH ₍₃₎ -CH1 ₍ 3). ₎ -VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ) Contains a polypeptide. In some embodiments, the (multispecific) antibody is a polypeptide (VL) in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule. ₍₁₎ -CH1 ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included. In some embodiments, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL ₍₃₎ -CL ₍₃₎ ) of a third Fab molecule.

In certain embodiments, the (multispecific) antibody according to the invention has a Fab light chain variable region of a first Fab molecule that shares a carboxy-terminated peptide bond with a Fab heavy chain constant region of a first Fab molecule (in certain embodiments). That is, the first Fab molecule contains the crossover Fab heavy chain, and the heavy chain variable region is replaced with the light chain variable region), and the Fab heavy chain constant region of the first Fab molecule has a carboxy-terminal peptide bond. It shares with the Fab heavy chain of the second Fab molecule, and the Fab heavy chain of the second Fab molecule shares the carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule (VL ₍₁₎ -CH1 ₍ 1). ₎ -VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₃₎ -CH1 ₍₃₎ ) Contains a polypeptide. In some embodiments, the (multispecific) antibody is a polypeptide (VH) in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule. ₍₁₎ -CL ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included. In some embodiments, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL ₍₃₎ -CL ₍₃₎ ) of a third Fab molecule.

In certain embodiments, the (multispecific) antibody according to the invention has a Fab heavy chain variable region of a first Fab molecule that shares a carboxy-terminated peptide bond with the Fab light chain constant region of a first Fab molecule (in certain embodiments). That is, the first Fab molecule contains the crossover Fab heavy chain, and the heavy chain constant region is replaced with the light chain constant region), and the Fab light chain constant region of the first Fab molecule has a carboxy-terminal peptide bond. It shares with the Fab heavy chain of the second Fab molecule, and the Fab heavy chain of the second Fab molecule shares the carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule (VH ₍₁₎ -CL ₍ 1). ₎ -VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₃₎ -CH1 ₍₃₎ ) Contains a polypeptide. In some embodiments, the (multispecific) antibody is a polypeptide (VL) in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule. ₍₁₎ -CH1 ₍₁₎ ) and the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ) are further included. In some embodiments, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL ₍₃₎ -CL ₍₃₎ ) of a third Fab molecule.

In certain embodiments, in the (multispecific) antibody according to the invention, the Fab heavy chain of the first Fab molecule shares the carboxy-terminal peptide binding with the Fab light chain variable region of the second Fab molecule, and the second. The Fab light chain variable region of the Fab molecule shares the carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (ie, the second Fab molecule contains the crossover Fab heavy chain and is a heavy chain. The variable region replaces the light chain variable region), the Fab heavy chain constant region of the second Fab molecule shares the carboxy-terminal peptide binding with the Fab light chain variable region of the third Fab molecule, and the third Fab. The Fab light chain variable region of the molecule shares the carboxy-terminal peptide binding with the Fab heavy chain constant region of the third Fab molecule (ie, the third Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region It has been replaced by a light chain variable region) (VH ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CH1 ₍₂₎ -VL ₍₃₎ -CH1 ₍₃₎ ) contains a polypeptide. In some embodiments, the (multispecific) antibody is a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. ₍₂₎ -CL ₍₂₎ ) and the Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ) are further included. In some embodiments, the (multispecific) antibody is a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. ₍₃₎ -CL ₍₃₎ ) is further included.

In certain embodiments, in a (multispecific) antibody according to the invention, the Fab heavy chain of the first Fab molecule shares the carboxy-terminal peptide binding with the Fab heavy chain variable region of the second Fab molecule, and the second. The Fab heavy chain variable region of the Fab molecule shares the carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (ie, the second Fab molecule contains the crossover Fab heavy chain and is heavy chain defined. The normal region is replaced by the light constant region), the Fab light chain constant region of the second Fab molecule shares the carboxy-terminal peptide bond with the Fab heavy chain variable region of the third Fab molecule, and the third Fab molecule. Fab heavy chain variable region shares a carboxy-terminal peptide bond with the Fab light chain constant region of a third Fab molecule (ie, the third Fab molecule contains a crossover Fab heavy chain and the heavy chain constant region is light. It has been replaced by a chain constant region) (VH ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ -VH ₍₃₎ -CL ₍₃₎ ) contains polypeptides. In some embodiments, the (multispecific) antibody is a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. ₍₂₎ -CH1 ₍₂₎ ) and the Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ) are further included. In some embodiments, the (multispecific) antibody is a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule. ₍₃₎ -CH1 ₍₃₎ ) is further included.

In certain embodiments, the (multispecific) antibody according to the invention has a Fab light chain variable region of a third Fab molecule sharing a carboxy-terminal peptide binding with a Fab heavy chain constant region of a third Fab molecule (in certain embodiments). That is, the third Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced with the light chain variable region), and the Fab heavy chain constant region of the third Fab molecule has a carboxy-terminal peptide bond. It shares with the Fab light chain variable region of the second Fab molecule, and the Fab light chain variable region of the second Fab molecule shares the carboxy-terminal peptide binding with the Fab heavy chain constant region of the second Fab molecule (ie,). The second Fab molecule contains a crossover Fab heavy chain and the heavy chain variable region is replaced with a light chain variable region), and the Fab heavy chain constant region of the second Fab molecule is the first carboxy-terminal peptide bond. Includes a polypeptide shared with the Fab heavy chain of the Fab molecule (VL ₍₃₎ -CH1 ₍₃₎ -VL ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ ). In some embodiments, the (multispecific) antibody is a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. ₍₂₎ -CL ₍₂₎ ) and the Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ) are further included. In some embodiments, the (multispecific) antibody is a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. ₍₃₎ -CL ₍₃₎ ) is further included.

In certain embodiments, the (multispecific) antibody according to the invention has a Fab heavy chain variable region of a third Fab molecule that shares a carboxy-terminated peptide bond with the Fab light chain constant region of a third Fab molecule (in certain embodiments). That is, the third Fab molecule contains the crossover Fab heavy chain, the heavy chain constant region is replaced with the light chain constant region), and the Fab light chain constant region of the third Fab molecule has a carboxy-terminal peptide bond. The Fab heavy chain variable region of the second Fab molecule shares with the Fab heavy chain variable region of the second Fab molecule, and the Fab heavy chain variable region of the second Fab molecule shares the carboxy-terminal peptide binding with the Fab light chain constant region of the second Fab molecule (ie,). The second Fab molecule contains a crossover Fab heavy chain and the heavy chain constant region is replaced with a light chain constant region), the Fab light chain constant region of the second Fab molecule is the first carboxy-terminal peptide bond. Includes a polypeptide shared with the Fab heavy chain of the Fab molecule (VH ₍₃₎ -CL ₍₃₎ -VH ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ ). In some embodiments, the (multispecific) antibody is a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. ₍₂₎ -CH1 ₍₂₎ ) and the Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ) are further included. In some embodiments, the (multispecific) antibody is a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule. ₍₃₎ -CH1 ₍₃₎ ) is further included.

In one aspect, the invention
a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain or the constant domains CL and CH1 are replaced with each other. The heavy chain complementarity determining regions (HCDR) 1 of SEQ ID NO: 2, the heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 3 and HCDR3 of SEQ ID NO: 5, and the light chain complementarity determining regions of SEQ ID NO: 8. (LCDR) 1, a first antigen binding domain comprising a light chain variable region (VL) comprising LCDR2 of SEQ ID NO: 9 and LCDR3 of SEQ ID NO: 10.
b) A second antigen-binding domain that binds to a second antigen, particularly a target cell antigen, more specifically TYRP-1 or EGFRvIII, where the second antigen-binding domain is a (conventional) Fab molecule. , The second antigen-binding domain,
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
here,
(I) The first antigen-binding domain of (a) is fused to the N-terminal of the Fab heavy chain of the second antigen-binding domain of b) at the C-terminal of the Fab heavy chain, and the second antigen-binding domain of b). Is fused to the N-terminus of one of the Fc domain subunits of c) at the C-terminus of the Fab heavy chain, or (ii) the second antigen-binding domain of b) is at the C-terminus of the Fab heavy chain. The first antigen-binding domain of a) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and the first antigen-binding domain of a) is N at the C-terminus of the Fab heavy chain, which is one of the subunits of the Fc domain of c). Provided is a (multispecific) antibody fused to the terminal.

In a preferred embodiment, the invention
a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain or the constant domains CL and CH1 are replaced with each other. The heavy chain complementarity determining regions (HCDR) 1 of SEQ ID NO: 2, the heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 3 and HCDR3 of SEQ ID NO: 5, and the light chain complementarity determining regions of SEQ ID NO: 8. (LCDR) 1, a first antigen binding domain comprising a light chain variable region (VL) comprising LCDR2 of SEQ ID NO: 9 and LCDR3 of SEQ ID NO: 10.
b) Second and third antigen-binding domains that bind to a second antigen, particularly a target cell antigen, more specifically TYRP-1 or EGFRvIII, the second and third antigen-binding domains, respectively ( With the second and third antigen-binding domains, which are conventional) Fab molecules,
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
here,
(I) The first antigen-binding domain of (a) is fused to the N-terminal of the Fab heavy chain of the second antigen-binding domain of b) at the C-terminal of the Fab heavy chain, and the second antigen-binding domain of b). And b) the third antigen-binding domain is fused to the N-terminus of one of the subunits of the Fc domain of c) at the C-terminus of the Fab heavy chain, respectively, or (ii) the second antigen of b). The binding domain fuses at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a) the first antigen-binding domain, and the first antigen-binding domain of a) and the third antigen-binding domain of b). Provides (multispecific) antibodies fused to the N-terminus of one of the Fc domain subunits of c) at the C-terminus of the Fab heavy chain, respectively.

In another embodiment, the invention
a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain or the constant domains CL and CH1 are replaced with each other. The heavy chain complementarity determining regions (HCDR) 1 of SEQ ID NO: 2, the heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 3 and HCDR3 of SEQ ID NO: 5, and the light chain complementarity determining regions of SEQ ID NO: 8. (LCDR) 1, a first antigen binding domain comprising a light chain variable region (VL) comprising LCDR2 of SEQ ID NO: 9 and LCDR3 of SEQ ID NO: 10.
b) A second antigen-binding domain that binds to a second antigen, particularly a target cell antigen, more specifically TYRP-1 or EGFRvIII, where the second antigen-binding domain is a (conventional) Fab molecule. , The second antigen-binding domain,
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
here,
(I) The first antigen-binding domain of a) and the second antigen-binding domain of b) are fused to the N-terminus of one of the subunits of the Fc domain of c) at the C-terminus of the Fab heavy chain, respectively. , (Multispecific) antibodies are provided.

In all of the different structures of (multispecific) antibodies according to the invention, the amino acid substitutions (“charge modifications”) described herein are the second and (if present) third antigen bindings, if present. It may be in either the CH1 and CL domains of the domain / Fab molecule or the CH1 or CL domain of the first antigen binding domain / Fab molecule. Preferably, they are in the CH1 and CL domains of the second and (if any) third antigen binding domains / Fab molecules. According to the concepts of the invention, if the amino acid substitutions described herein are made in a second (and, if present, third) antigen-binding domain / Fab molecule, such amino acid substitutions are first. Not done in the antigen-binding domain / Fab molecule of. Conversely, if the amino acid substitutions described herein are made in the first antigen-binding domain / Fab molecule, such amino acid substitutions are in the second (and, if present, third) antigen-binding domain. / Not done in Fab molecules. Amino acid substitutions are preferably carried out in (multispecific) antibodies comprising Fab molecules in which the variable domains VL and VH1 of the Fab light chain and Fab heavy chain are replaced with each other.

In a preferred embodiment of the (multispecific) antibody according to the invention, especially if the amino acid substitutions described herein are made in the second (and, if present, third) antigen-binding domain / Fab molecule. The constant domain CL of the Fab molecule of 2 (and the 3rd, if any) is of the kappa isotype. In another embodiment of the (multispecific) antibody according to the invention, the first antigen binding domain / Fab, especially if the amino acid substitutions described herein are made in the first antigen binding domain / Fab molecule. The constant domain CL of the molecule is of the kappa isotype. In some embodiments, the second (and, if present, third) antigen-binding domain / Fab molecule constant domain CL and the first antigen-binding domain / Fab molecule constant domain CL are of the kappa isotype. be.

In one aspect, the invention
a) The first antigen-binding domain that binds to CD3, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and SEQ ID NO: 2 Heavy chain complementarity determining regions (HCDR) 1, heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 3 and HCDR3 of SEQ ID NO: 5, and light chain complementarity determining regions (LCDR) 1 of SEQ ID NO: 8, sequence. A first antigen binding domain comprising a light chain variable region (VL) comprising LCDR2 of No. 9 and LCDR3 of SEQ ID NO: 10.
b) A second antigen-binding domain that binds to a second antigen, particularly a target cell antigen, more specifically TYRP-1 or EGFRvIII, where the second antigen-binding domain is a (conventional) Fab molecule. , The second antigen-binding domain,
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
Here, in the constant domain CL of the second antigen-binding domain of b), the amino acid at position 124 is replaced by lysine (K) (numbering by Kabat), and the amino acid at position 123 is lysine (K) or arginine. The amino acid at position 147 is replaced by glutamic acid (E) in the constant domain CH1 of the second antigen-binding domain, which has been replaced by (R) (most preferably by arginine (R)) (numbering by Kabat). (Numbering by Kabat EU index), the amino acid at position 213 has been replaced by glutamic acid (E) (numbering by Kabat EU index).
here,
(I) The first antigen-binding domain of (a) is fused to the N-terminal of the Fab heavy chain of the second antigen-binding domain of b) at the C-terminal of the Fab heavy chain, and the second antigen-binding domain of b). Is fused to the N-terminus of one of the Fc domain subunits of c) at the C-terminus of the Fab heavy chain, or (ii) the second antigen-binding domain of b) is at the C-terminus of the Fab heavy chain. The first antigen-binding domain of a) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and the first antigen-binding domain of a) is N at the C-terminus of the Fab heavy chain, which is one of the subunits of the Fc domain of c). Provided is a (multispecific) antibody fused to the terminal.

In a preferred embodiment, the invention
a) The first antigen-binding domain that binds to CD3, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and SEQ ID NO: 2 Heavy chain complementarity determining regions (HCDR) 1, heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 3 and HCDR3 of SEQ ID NO: 5, and light chain complementarity determining regions (LCDR) 1 of SEQ ID NO: 8, sequence. A first antigen binding domain comprising a light chain variable region (VL) comprising LCDR2 of No. 9 and LCDR3 of SEQ ID NO: 10.
b) Second and third antigen-binding domains that bind to a second antigen, particularly a target cell antigen, more specifically TYRP-1 or EGFRvIII, the second and third antigen-binding domains, respectively ( With the second and third antigen-binding domains, which are conventional) Fab molecules,
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
Here, in the constant domain CL of the second antigen-binding domain of b) and the third antigen-binding domain of b), the amino acid at position 124 is replaced by lysine (K) (numbering by Kabat), and position 123. The amino acid of is replaced by lysine (K) or arginine (R) (most preferably by arginine (R)) (numbering by Kabat), the second antigen-binding domain of b) and the third antigen of b). In the constant domain CH1 of the binding domain, the amino acid at position 147 is replaced by glutamic acid (E) (numbering by Kabat EU index), and the amino acid at position 213 is replaced by glutamic acid (E) (numbering by Kabat EU index). ),
here,
(I) The first antigen-binding domain of (a) is fused to the N-terminal of the Fab heavy chain of the second antigen-binding domain of b) at the C-terminal of the Fab heavy chain, and the second antigen-binding domain of b). And b) the third antigen-binding domain is fused to the N-terminus of one of the subunits of the Fc domain of c) at the C-terminus of the Fab heavy chain, respectively, or (ii) the second antigen of b). The binding domain fuses at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a) the first antigen-binding domain, and the first antigen-binding domain of a) and the third antigen-binding domain of b). Provides (multispecific) antibodies fused to the N-terminus of one of the Fc domain subunits of c) at the C-terminus of the Fab heavy chain, respectively.

In another embodiment, the invention
a) The first antigen-binding domain that binds to CD3, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and SEQ ID NO: 2 Heavy chain complementarity determining regions (HCDR) 1, heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 3 and HCDR3 of SEQ ID NO: 5, and light chain complementarity determining regions (LCDR) 1 of SEQ ID NO: 8, sequence. A first antigen binding domain comprising a light chain variable region (VL) comprising LCDR2 of No. 9 and LCDR3 of SEQ ID NO: 10.
b) A second antigen-binding domain that binds to a second antigen, particularly a target cell antigen, more specifically TYRP-1 or EGFRvIII, where the second antigen-binding domain is a (conventional) Fab molecule. , The second antigen-binding domain,
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
Here, in the constant domain CL of the second antigen-binding domain of b), the amino acid at position 124 is replaced by lysine (K) (numbering by Kabat), and the amino acid at position 123 is lysine (K) or arginine. The amino acid at position 147 is replaced by glutamic acid (E) in the constant domain CH1 of the second antigen-binding domain, which has been replaced by (R) (most preferably by arginine (R)) (numbering by Kabat). (Numbering by Kabat EU index), the amino acid at position 213 has been replaced by glutamic acid (E) (numbering by Kabat EU index).
The first antigen-binding domain of a) and the second antigen-binding domain of b) are fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c) (multiplexing). Specificity) Provides an antibody.

According to any of the above embodiments, the components of the (multispecific) antibody (eg, Fab molecule, Fc domain) are described or said herein, in particular, either directly or via various linkers. It may be fused via a peptide linker containing one or more amino acids, typically about 2-20 amino acids known in the art. Suitable non-immunogenic peptide linkers include, for example, (G ₄ S) _n , (SG ₄ ) _n , (G ₄ S) _n or G ₄ (SG ₄ ) _n peptide linkers, where "n" is It is generally an integer of 1 to 10, typically 2 to 4.

In a preferred embodiment, the invention
a) The first antigen-binding domain that binds to CD3, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and SEQ ID NO: 2 Heavy chain complementarity determining regions (HCDR) 1, heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 3 and HCDR3 of SEQ ID NO: 5, and light chain complementarity determining regions (LCDR) 1 of SEQ ID NO: 8, sequence. A first antigen binding domain comprising a light chain variable region (VL) comprising LCDR2 of No. 9 and LCDR3 of SEQ ID NO: 10.
b) Second and third antigen binding domains that bind to TYRP-1, the second and third antigen binding domains are (conventional) Fab molecules, respectively, and the heavy chain complementarity determination of SEQ ID NO: 15. Heavy chain variable region (VH) containing region (HCDR) 1, HCDR2 of SEQ ID NO: 16 and HCDR3 of SEQ ID NO: 17, and light chain complementarity determining regions (LCDRs) 1 of SEQ ID NO: 19, LCDR2 and SEQ ID NO: 20. A second and third antigen binding domain comprising a light chain variable region (VL) comprising LCDR3 of number 21.
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
here,
In the constant domain CL of the second and third antigen-binding domains of b), the amino acid at position 124 is replaced by lysine (K) (numbering by Kabat), and the amino acid at position 123 is lysine (K) or arginine (numbering by Kabat). The amino acid at position 147 is glutamic acid (E) in the constant domain CH1 of the second and third antigen-binding domains that have been replaced (most preferably by arginine (R)) with R) (numbering by Kabat). Substituted by (Kabat EU index numbering), the amino acid at position 213 has been replaced by glutamic acid (E) (Kabat EU index numbering).
moreover,
The second antigen-binding domain of b) fuses at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a) the first antigen-binding domain, and the first antigen-binding domain of a) and b). Provides a (multispecific) antibody in which the third antigen binding domain of Fab is fused to the N-terminus of one of the subunits of the Fc domain of c) at the C-terminus of the Fab heavy chain, respectively.

In a more preferred embodiment, the invention
a) The first antigen-binding domain that binds to CD3, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and SEQ ID NO: 7 A first antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11.
b) Second and third antigen-binding domains that bind to TYRP-1, the second and third antigen-binding domains are (conventional) Fab molecules, respectively, and are heavy containing the amino acid sequence of SEQ ID NO: 18. A second and third antigen-binding domain comprising a chain variable region and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22.
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
here,
In the constant domain CL of the second and third antigen-binding domains of b), the amino acid at position 124 is replaced by lysine (K) (numbering by Kabat), and the amino acid at position 123 is lysine (K) or arginine (numbering by Kabat). The amino acid at position 147 is glutamic acid (E) in the constant domain CH1 of the second and third antigen-binding domains that have been replaced (most preferably by arginine (R)) with R) (numbering by Kabat). Substituted by (Kabat EU index numbering), the amino acid at position 213 has been replaced by glutamic acid (E) (Kabat EU index numbering).
moreover,
The second antigen-binding domain of b) fuses at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a) the first antigen-binding domain, and the first antigen-binding domain of a) and b). Provides a (multispecific) antibody in which the third antigen binding domain of Fab is fused to the N-terminus of one of the subunits of the Fc domain of c) at the C-terminus of the Fab heavy chain, respectively.

In a preferred embodiment, the invention
a) The first antigen-binding domain that binds to CD3, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and SEQ ID NO: 2 Heavy chain complementarity determining regions (HCDR) 1, heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 3 and HCDR3 of SEQ ID NO: 5, and light chain complementarity determining regions (LCDR) 1 of SEQ ID NO: 8, sequence. A first antigen binding domain comprising a light chain variable region (VL) comprising LCDR2 of No. 9 and LCDR3 of SEQ ID NO: 10.
b) Second and third antigen-binding domains that bind to EGFRvIII, where the second and third antigen-binding domains are (conventional) Fab molecules, respectively, and the heavy chain complementarity determining regions of SEQ ID NO: 85 ( HCDR) 1, heavy chain variable region (VH) containing HCDR2 of SEQ ID NO: 86 and HCDR3 of SEQ ID NO: 87, and light chain complementarity determining regions (LCDR) 1, SEQ ID NO: 90, LCDR2 and SEQ ID NO: 91. The second and third antigen binding domains, which include the light chain variable regions (VL) containing LCDR3,
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
here,
In the constant domain CL of the second and third antigen-binding domains of b), the amino acid at position 124 is replaced by lysine (K) (numbering by Kabat), and the amino acid at position 123 is lysine (K) or arginine (numbering by Kabat). The amino acid at position 147 is glutamic acid (E) in the constant domain CH1 of the second and third antigen-binding domains that have been replaced (most preferably by arginine (R)) with R) (numbering by Kabat). Substituted by (Kabat EU index numbering), the amino acid at position 213 has been replaced by glutamic acid (E) (Kabat EU index numbering).
moreover,
The second antigen-binding domain of b) fuses at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a) the first antigen-binding domain, and the first antigen-binding domain of a) and b). Provides a (multispecific) antibody in which the third antigen binding domain of Fab is fused to the N-terminus of one of the subunits of the Fc domain of c) at the C-terminus of the Fab heavy chain, respectively.

In a more preferred embodiment, the invention
a) The first antigen-binding domain that binds to CD3, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, and SEQ ID NO: 7 A first antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11.
b) Second and third antigen-binding domains that bind to EGFRvIII, the second and third antigen-binding domains being (conventional) Fab molecules, respectively, with variable heavy chains containing the amino acid sequence of SEQ ID NO: 88. A second and third antigen-binding domain comprising a region and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 92.
c) A (multispecific) antibody comprising an Fc domain consisting of the first and second subunits.
here,
In the constant domain CL of the second and third antigen-binding domains of b), the amino acid at position 124 is replaced by lysine (K) (numbering by Kabat), and the amino acid at position 123 is lysine (K) or arginine (numbering by Kabat). The amino acid at position 147 is glutamic acid (E) in the constant domain CH1 of the second and third antigen-binding domains that have been replaced (most preferably by arginine (R)) with R) (numbering by Kabat). Substituted by (Kabat EU index numbering), the amino acid at position 213 has been replaced by glutamic acid (E) (Kabat EU index numbering).
moreover,
The second antigen-binding domain of b) fuses at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a) the first antigen-binding domain, and the first antigen-binding domain of a) and b). Provides a (multispecific) antibody in which the third antigen binding domain of Fab is fused to the N-terminus of one of the subunits of the Fc domain of c) at the C-terminus of the Fab heavy chain, respectively.

In one aspect of these aspects of the invention, the threonine residue at position 366 is replaced with a tryptophan residue in the first subunit of the Fc domain (T366W), and the position in the second subunit of the Fc domain. The tyrosine residue at 407 is replaced with a valine residue (Y407V), the threonine residue at position 366 is optionally replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue. (L368A) (numbering by Kabat EU index).

In a further aspect of these embodiments of the invention, in the first subunit of the Fc domain, an additional serine residue at position 354 is replaced with a cysteine residue (S354C) or a glutamate residue at position 356. It replaces the cysteine residue (E356C) (in particular, the serine residue at position 354 replaces the cysteine residue), and in the second subunit of the Fc domain, an additional tyrosine residue at position 349 It replaces the cysteine residue (Y349C) (numbering by Kabat EU index).

In a further aspect of these embodiments of the invention, the leucine residue at position 234 is replaced with the alanine residue (L234A) in each of the first and second subunits of the Fc domain, leaving the leucine residue at position 235. The group has been replaced with an alanine residue (L235A) and the proline residue at position 329 has been replaced with a glycine residue (P329G) (numbering by Kabat EU index).

In yet a further aspect of these aspects of the invention, the Fc domain is a human IgG ₁ Fc domain.

In a preferred specific embodiment, the (multispecific) antibody is a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 23, the sequence of SEQ ID NO: 24. A polypeptide containing at least 95%, 96%, 97%, 98% or 99% identical amino acid sequence, at least 95%, 96%, 97%, 98% or 99% identical amino acid sequence to the sequence of SEQ ID NO: 25. Includes polypeptides comprising (particularly two polypeptides), and polypeptides comprising at least 95%, 96%, 97%, 98% or 99% identical amino acid sequences in the sequence of SEQ ID NO: 27. In a more preferred specific embodiment, the (multispecific) antibody is a polypeptide comprising the amino acid sequence of SEQ ID NO: 23, a polypeptide comprising the amino acid sequence of SEQ ID NO: 24, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 25 (particularly). Two polypeptides), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment, the invention comprises a polypeptide comprising at least 95%, 96%, 97%, 98% or 99% identical amino acid sequence in the sequence of SEQ ID NO: 23, at least 95%, 96% in the sequence of SEQ ID NO: 24. , 97%, 98% or 99% identical amino acid sequence, polypeptide containing at least 95%, 96%, 97%, 98% or 99% identical amino acid sequence to the sequence of SEQ ID NO: 25 (particularly). Binds to CD3 and TYRP-1 (multiple specificity) comprising a polypeptide containing at least 95%, 96%, 97%, 98% or 99% of the same amino acid sequence in the sequence of SEQ ID NO: 27). Gender) Provides an antibody. In one embodiment, the invention comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 23, a polypeptide comprising the amino acid sequence of SEQ ID NO: 24, a polypeptide comprising the amino acid sequence of SEQ ID NO: 25 (particularly two polypeptides), and. Provided are a (multispecific) antibody that binds to CD3 and TYRP-1, which comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

In a further preferred specific embodiment, the (multispecific) antibody of SEQ ID NO: 110 is a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 109. A polypeptide containing at least 95%, 96%, 97%, 98% or 99% identical amino acid sequence in the sequence, at least 95%, 96%, 97%, 98% or 99% identical amino acid in the sequence of SEQ ID NO: 111. It comprises a polypeptide comprising a sequence (particularly two polypeptides) and a polypeptide comprising at least 95%, 96%, 97%, 98% or 99% of the same amino acid sequence in the sequence of SEQ ID NO: 27. In a further specific embodiment, the (multispecific) antibody is a polypeptide comprising the amino acid sequence of SEQ ID NO: 109, a polypeptide comprising the amino acid sequence of SEQ ID NO: 110, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 111. In particular, it comprises two polypeptides), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment, the invention comprises a polypeptide comprising at least 95%, 96%, 97%, 98% or 99% identical amino acid sequence in the sequence of SEQ ID NO: 109, at least 95%, 96% in the sequence of SEQ ID NO: 110. , 97%, 98% or 99% identical amino acid sequence, polypeptide containing at least 95%, 96%, 97%, 98% or 99% identical amino acid sequence to the sequence of SEQ ID NO: 111 (particularly). Two polypeptides), and a polypeptide containing at least 95%, 96%, 97%, 98% or 99% identical amino acid sequence to the sequence of SEQ ID NO: 27, binding to CD3 and EGFRvIII (multispecific). Provide an antibody. In one embodiment, the invention comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 109, a polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a polypeptide comprising the amino acid sequence of SEQ ID NO: 111 (particularly two polypeptides), and. Provided are a (multispecific) antibody that binds to CD3 and EGFRvIII, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

8. Fc domain variant

In a preferred embodiment, the (multispecific) antibody of the invention comprises an Fc domain consisting of first and second subunits.

The Fc domain of an (multispecific) antibody consists of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit comprising CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one aspect, the (multispecific) antibody of the invention does not contain more than one Fc domain.

In one embodiment, the Fc domain of the (multispecific) antibody is an IgG Fc domain. In a preferred embodiment, the Fc domain is an IgG ₁ Fc domain. _In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG ₄ Fc domain comprising an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU index numbering). This amino acid substitution reduces the Fab arm exchange of IgG4 antibodies in vivo (see _Stevenrauch et al., Drag Metabolism and Disposition 38, 84-91 (2010)). In a more preferred embodiment, the Fc domain is a human Fc domain. In an even more preferred embodiment, the Fc domain is a human IgG ₁ Fc domain. An exemplary sequence of the human IgG ₁ Fc region is given by SEQ ID NO: 117.

a) Fc domain modification that promotes heterodimerization

The (multispecific) antibodies of the invention contain different antigen binding domains that can be fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically two. Included in non-identical polypeptide chains. Recombination co-expression of these polypeptides and subsequent dimerization results in some possible combinations of the two polypeptides. In order to improve the yield and purity of the (multispecific) antibody in recombinant production, it is advantageous to introduce modifications into the Fc domain of the (multispecific) antibody that promote association of the desired polypeptide.

Thus, in a preferred embodiment, the Fc domain of the (multispecific) antibody according to the invention comprises a modification that facilitates association of the first and second subunits of the Fc domain. The site of the longest protein-protein interaction between the two subunits of the human IgG Fc domain is within the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is in the CH3 domain of the Fc domain.

There are several methods for modifying the Fc domain in the CH3 domain to enhance heterodimerization, eg, WO 96/27011, WO 98/050431, European Patent No. 1870459. No., International Publication No. 2007/110205, International Publication No. 2007/147901, International Publication No. 2009/089004, International Publication No. 2010/129304, International Publication No. 2011/90754, International Publication No. 2011/143545, It is fully described in International Publication No. 2012058768, International Publication No. 2013157954, and International Publication No. 2013096291. Typically, in all such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both each CH3 domain (or a weight containing it). The chain) is not homodimerized with itself, but is manipulated in a complementary fashion that is directed to heterodimerize with other complementary CH3 domains (thus. The first and second CH3 domains are heterodimerized and no homodimer is formed between the two first or second CH3 domains). These different approaches for improved heavy chain heterodimerization reduce heavy chain / light chain mispairs and Bens-Jones type by-products, heavy chains in (multispecific) antibodies- Considered as different alternatives in combination with light chain modifications (eg, exchange / replacement of VH and VL in one binding arm, and introduction of replacement of charged amino acids with opposite charges at the CH1 / CL interface).

In a specific embodiment, the modification that facilitates association of the first and second subunits of the Fc domain is the so-called "knob-into-hole" modification, which is the two subunits of the Fc domain. One of the units contains a "knob" modification and the other of the two subunits of the Fc domain contains a "hole" modification.

The knob-in-to-hole technique is described, for example, in US Pat. No. 5,731,168, US Pat. No. 7,695,936, Ridgway et al. , Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In general, this method involves the introduction of a protrusion (“knob”) at the interface of the first polypeptide and a corresponding cavity (“hole”) at the interface of the second polypeptide, whereby the protrusion is heterozygous. It can be located within the cavity to promote dimer formation and interfere with homodimer formation. The protrusions are constructed by replacing the small amino acid side chains at the interface of the first polypeptide with larger side chains (eg, tyrosine or tryptophan). Compensatory cavities of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (eg, alanine or threonine).

Thus, in a preferred embodiment, in the CH3 domain of the first subunit of the Fc domain of the (multispecific) antibody, the amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby the first. Within the CH3 domain of the subunit, amino acid residues are more. It replaces amino acid residues with a small side chain volume, thereby creating a cavity within the CH3 domain of the second subunit, in which the protrusions within the CH3 domain of the first subunit can be located. It is possible.

Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

Protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, eg, by site-directed mutagenesis or by peptide synthesis.

In a specific embodiment, in the first subunit of the Fc domain (the "knob" subunit) (CH3 domain), the threonine residue at position 366 is replaced with the tryptophan residue (T366W), the first of the Fc domains. In the 2 subunits (“hole” subunits) (CH3 domain), the tyrosine residue at position 407 is replaced with the valine residue (Y407V). In one aspect, in the second subunit of the Fc domain, the threonine residue at position 366 is additionally replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue. (L368A) (numbering by Kabat EU index).

In a further embodiment, in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced with a cysteine residue (S354C) or the glutamate residue at position 356 is replaced with a cysteine residue. (E356C) (in particular, the serine residue at position 354 has been replaced with a cysteine residue), and in the second subunit of the Fc domain, an additional tyrosine residue at position 349 has been replaced with a cysteine residue (especially). Y349C) (numbering by Kabat EU index). The introduction of these two cysteine residues results in the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). ..

In a preferred embodiment, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (numbering by Kabat EU index). ..

In a preferred embodiment, the antigen binding domain that binds to CD3 comprises (optionally via a second antigen binding domain and / or a peptide linker) modification of the Fc domain that binds to the second antigen. ) Fuse with the first subunit. Without being bound by theory, fusion of the antigen-binding domain that binds to CD3 to the knob-containing subunit of the Fc domain produces an antibody that contains two antigen-binding domains that bind to CD3 (containing two knobs). Minimize (further) the steric flash of the polypeptide.

Other techniques for CH3 modification that enhance heterodimerization are considered as alternatives to the invention, eg, WO 96/27011, WO 98/050431, European Patent No. 1870459, International. Publication No. 2007/110205, International Publication No. 2007/147901, International Publication No. 2009/089004, International Publication No. 2010/129304, International Publication No. 2011/90754, International Publication No. 2011/143545, International Publication No. It is described in 2012/058768, International Publication No. 2013/157954, International Publication No. 2013/096291.

In one aspect, the heterodimerization technique described in European Patent No. 1870459 is used as an alternative. This approach is based on the introduction of charged amino acids with opposite charges to specific amino acid positions at the CH3 / CH3 domain interface between the two subunits of the Fc domain. A particular embodiment of the (multispecific) antibody of the invention is the amino acid mutations R409D, K370E in one of the two CH3 domains (of the Fc domain) and the amino acid mutations D399K, E357K in the other of the CH3 domain of the Fc domain (Kabat). Numbering by EU index).

In another embodiment, the (multispecific) antibody of the invention comprises an amino acid variant T366W in the CH3 domain of the first subunit of the Fc domain and an amino acid variant T366S, L368A in the CH3 domain of the second subunit of the Fc domain. It contains Y407V and additionally contains the amino acid variants R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid variants D399K, E357K in the CH3 domain of the second subunit of the Fc domain (according to the Kabat EU index). numbering).

In another embodiment, the (multispecific) antibody of the invention comprises the amino acid variants S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid variant Y349C in the CH3 domain of the second subunit of the Fc domain. The antibody comprising T366S, L368A, Y407V, or said (multispecific) antibody, has an amino acid mutation in the CH3 domain of the first subunit of the Fc domain, and an amino acid mutation in the CH3 domain of the second subunit of the Fc domain, Y349C, T366W. It contains S354C, T366S, L368A, Y407V, and additionally has amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain. Includes (all numbered by Kabat EU index).

In one aspect, the heterodimerization technique described in WO 2013/157953 is used as an alternative. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering by Kabat EU index). In a further embodiment, the first CH3 domain comprises an additional amino acid mutation L351K. In a further embodiment, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E (particularly L368E) (numbering by Kabat EU index).

In one aspect, the heterodimerization technique described in WO 2012/058768 is used in an alternative manner. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain is located at positions T411, D399, S400, F405, N390 or K392, eg, a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y. Or D399K, c) S400E, S400D, S400R or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392K Includes additional amino acid mutations (numbering by Kabat EU index). In a further embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further embodiment, the first CH3 domain comprises the amino acid mutation Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering by Kabat EU index).

In one aspect, the heterodimerization technique described in WO 2011/143545 is used in an alternative manner, for example having an amino acid modification at a position selected from the group consisting of 368 and 409 (according to the Kabat EU index). numbering).

In one aspect, the heterodimerization technique described in WO 2011/090762, which also uses the knob-in-to-hole technique described above, is used in an alternative manner. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering by Kabat EU index).

In one embodiment, the (multispecific) antibody or Fc domain thereof is of the IgG ₂ subclass and the heterodimerization technique described in WO 2010/129304 is used as an alternative.

In an alternative embodiment, modifications that facilitate association of the first and second subunits of the Fc domain include, for example, modifications that mediate the electrostatic steering effect described in PCT Application International Publication No. 2009/089004. include. In general, this method is electrostatically undesirable for homodimer formation, but one at the interface of two Fc domain subunits with charged amino acid residues so that heterodimerization is electrostatically desirable. Or with the replacement of multiple amino acid residues. In one such embodiment, the first CH3 domain comprises an amino acid substitution of K392 or N392 with a negatively charged amino acid (eg, with glutamic acid (E) or aspartic acid (D), especially K392D or N392D). The second CH3 domain is an amino acid substitution of D399, E356, D356 or E357 with a positively charged amino acid (eg, with lysine (K) or arginine (R), especially D399K, E356K, D356K or E357K, more specifically. Includes D399K and E356K). In a further embodiment, the first CH3 domain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (eg, with glutamic acid (E) or aspartic acid (D), especially K409D or R409D). In a further embodiment, the first CH3 domain further or optionally comprises amino acid substitutions of K439 and / or K370 with negatively charged amino acids (eg, glutamic acid (E) or aspartic acid (D)) (all Kabat EU). Numbering by index).

In still further embodiments, the heterodimerization technique described in WO 2007/147901 is used in an alternative manner. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K and K322D and the second CH3 domain comprises the amino acid mutations D239K, E240K and K292D (numbering by Kabat EU index).

In yet another embodiment, the heterodimerization technique described in WO 2007/110205 can be used as an alternative.

In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering by Kabat EU index).

b) Fc domain modification to reduce Fc receptor binding and / or effector function

The Fc domain imparts the (multispecific) antibody a favorable pharmacokinetic property and a preferred tissue blood distribution ratio, including a long serum half-life that contributes to good accumulation in the target tissue. However, at the same time, it may result in undesired targeting of (multispecific) antibodies to cells expressing the Fc receptor rather than the preferred antigen-carrying cells. In addition, co-activation of Fc receptor signaling pathways can result in cytokine release, combined with T cell activation properties and a long half-life of (multispecific) antibodies, when systemically administered results in an excess of cytokine receptors. Causes active activation and serious side effects. Activation of immune cells other than T cells (Fc receptor-bearing) can even reduce the effectiveness of (multispecific) antibodies, for example, due to the potential for destruction of T cells by NK cells.

Thus, in a preferred embodiment, the Fc domain of the (multispecific) antibody according to the invention exhibits reduced binding affinity to the Fc receptor and / or reduced effector function as compared to the native IgG ₁ Fc domain. In one such embodiment, the Fc domain (or the (multispecific) antibody comprising said Fc domain) is compared to the native IgG ₁ Fc domain (or the (multispecific) antibody comprising the native IgG ₁ Fc domain). And / or native IgG ₁ Fc domain domain (or native IgG ₁ Fc) exhibiting less than 50%, particularly less than 20%, more specifically less than 10%, particularly less than 5% binding affinity for Fc receptors. It exhibits less than 50%, particularly less than 20%, more specifically less than 10%, especially less than 5% effector function as compared to domain-containing (multispecific) antibodies. In one embodiment, the Fc domain domain (or (multispecific) antibody comprising said Fc domain) does not substantially bind to the Fc receptor and / or induce effector function. In a preferred embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, especially human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group of CDC, ADCC, ADCP and cytokine secretion. In a preferred embodiment, the effector function is ADCC. In one aspect, the Fc domain domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) as compared to the native IgG ₁ Fc domain domain. Substantially similar binding to FcRn is such that the Fc domain (or antibody containing said Fc domain (multispecific)) is a native IgG ₁ Fc domain (or antibody containing native IgG ₁ Fc domain (multispecific)). It is achieved when the FcRn exhibits a binding affinity of more than about 70%, particularly more than about 80%, more specifically more than about 90%.

In certain embodiments, the Fc domain is engineered to have reduced binding affinity to the Fc receptor and / or reduced effector function as compared to the unoperated Fc domain. In a preferred embodiment, the Fc domain of the (multispecific) antibody comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor. Typically, the same amino acid mutation is present in each of the two subunits of the Fc domain. In one embodiment, amino acid mutations reduce the binding affinity of the Fc domain for the Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least one-half, at least one-fifth, or at least one-tenth. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations will increase the binding affinity of the Fc domain to the Fc receptor by at least 10 minutes. 1. It can be reduced to at least 1/20 or even at least 1/50. In one embodiment, an antibody comprising an engineered Fc domain (multispecific) is less than 20%, particularly less than 10%, more specifically 5%, as compared to an antibody comprising a non-manipulated Fc domain (multispecific). It exhibits less than a binding affinity for the Fc receptor. In a preferred embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, especially human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity for complement components, specifically C1q, is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain to the receptor, is such that the Fc domain (or antibody containing the Fc domain (multispecific) antibody) does not manipulate the Fc domain. It is achieved if the FcRn exhibits a binding affinity of greater than about 70% of the morphology (or the (multispecific) antibody comprising the unoperated form of the Fc domain). The Fc domain or the (multispecific) antibody of the invention comprising said Fc domain can exhibit more than about 80% and even more than about 90% of such affinity. In certain embodiments, the Fc domain of a (multispecific) antibody is engineered to reduce effector function as compared to a non-manipulated Fc domain. Reduction of effector function includes reduction of complement-dependent cellular cytotoxicity (CDC), reduction of antibody-dependent cellular cytotoxicity (ADCC), reduction of antibody-dependent cellular cytotoxicity (ADCP), reduction of cytokine secretion, and antigen-presenting cells. Reduced immune complex-mediated antigen uptake, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monospheres, reduced binding to polymorphonuclear cells, direct signaling induction One or more of, but not limited to, reduction of sex apoptosis, reduction of cross-linking with target-binding antibody, reduction of dendritic cell maturation, or reduction of T cell priming. In one aspect, the reduction of effector function is one or more selected from the group of CDC reduction, ADCC reduction, ADCP reduction and cytokine secretion reduction. In a preferred embodiment, the reduction in effector function is a reduction in ADCC. In one aspect, the reduction of ADCC is less than 20% of ADCC induced by the unoperated Fc domain (or (multispecific) antibody comprising the unoperated Fc domain).

In one embodiment, the amino acid mutation that reduces the binding affinity and / or effector function of the Fc domain to the Fc receptor is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331 and P329 (numbering by Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group L234, L235 and P329 (numbering by Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering by Kabat EU index). In one such embodiment, the Fc domain is an IgG ₁ Fc domain, particularly a human IgG ₁ Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, in particular P329G (numbering by Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297 and P331 (numbering by Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a preferred embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbering by Kabat EU index). In a more preferred embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in a preferred embodiment, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., in each of the first and second subunits of the Fc domain. , The leucine residue at position 234 has been replaced with an alanine residue (L234A), the leucine residue at position 235 has been replaced with an alanine residue (L235A), and the proline residue at position 329 has been replaced with a glycine residue. (P329G) (numbering by Kabat EU index).

In one such embodiment, the Fc domain is an IgG ₁ Fc domain, particularly a human IgG ₁ Fc domain. The combination of amino acid substitutions "P329G LALA", as described in PCT Application International Publication No. 2012/130831, which is incorporated herein by reference in its entirety, is the Fcγ receptor of the human IgG ₁ Fc domain (similarly). In addition, the complement) bond is almost completely extinguished. WO 2012/130831 also describes how to prepare such mutant Fc domains and their properties, such as how to determine Fc receptor binding or effector function.

IgG ₄ antibody shows reduced binding affinity to Fc receptors and reduced effector function compared to IgG ₁ antibody. Thus, in some embodiments, the Fc domain of the ₍ multispecific) antibody of the invention is an IgG4 Fc domain, particularly a human _IgG4 Fc domain. _In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbering by Kabat EU index). To further reduce binding affinity to the Fc receptor and / or effector function, in one embodiment, the IgG ₄ Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (Kabat EU index). Numbering by). _In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbering by Kabat EU index). _In a preferred embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numbering by Kabat EU index). Such IgG4 Fc domain variants and their Fcγ receptor binding properties are described in PCT Application International Publication No. _2012/130831 , which is incorporated herein by reference in its entirety.

In a preferred embodiment, the Fc domain exhibiting reduced binding affinity to the Fc receptor and / or reduced effector function as compared to the native IgG ₁ Fc domain comprises humans containing amino acid substitutions L234A, L235A and optionally P329G. An IgG ₁ Fc domain, or a human IgG ₄ Fc domain containing amino acid substitutions S228P, L235E and optionally P329G (numbered by Kabat EU index).

In certain embodiments, N-glycosylation of the Fc domain is excluded. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution that replaces asparagine with alanine (N297A) or an amino acid substitution that replaces aspartic acid (N297D) (numbering by Kabat EU index). ..

In addition to the Fc domains described above and in PCT Application International Publication No. 2012/130831, Fc domains with reduced Fc receptor binding and / or effector function include Fc domain residues 238, 265, Also include those having one or more substitutions of 269, 270, 297, 327 and 329 (US Pat. No. 6,737,056) (numbering by Kabat EU index). Such Fc variants include Fc variants having substitutions at two or more of the amino acid positions 265, 269, 270, 297 and 327, with residues 265 and 297 substituted with alanine. Includes so-called "DANA" Fc variants (US Pat. No. 7,332,581).

Mutant Fc domains can be prepared by deletion, substitution, insertion or modification of amino acids using genetic or chemical methods well known in the art. Genetic methods include site-directed mutagenesis of coding DNA sequences, PCR, gene synthesis and the like. The exact nucleotide change can be confirmed, for example, by sequencing.

Binding to the Fc receptor is facilitated, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as BIAcore equipment (GE Healthcare) and Fc receptors that can be obtained, for example by recombinant expression. Can be decided. Alternatively, the binding affinity of the Fc domain or antibody containing the Fc domain (multispecific) to the Fc receptor expresses a cell line known to express a particular Fc receptor, eg, the FcγIIIa receptor. Human NK cells may be used for evaluation.

The effector function of an Fc domain or an antibody containing (multispecific) Fc domain can be measured by methods known in the art. Examples of in vitro assays for assessing ADCC activity of molecules of interest are described in US Pat. No. 5,500,362, Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al. , Proc Natl Acad Sci USA 82, 1499-1502 (1985), US Pat. No. 5,821,337, Bruggemann et al. , J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays can be used (eg, CellTechnology, Inc. Mountain View, CA) and CytoTox96® non-radioactive cell injury assay (Promega, ™) for flow cytometry. See Madison, WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be described, for example, in animal models, eg, Clynes et al. , Proc Natl Acad Sci USA 95, 652-656 (1998) may be evaluated in vivo.

In some embodiments, the binding of the Fc domain to the complement component, specifically C1q, is reduced. Therefore, in some embodiments where the Fc domain is engineered to reduce effector function, said reduction in effector function includes reduction of CDC. A C1q binding assay can be performed to determine if an Fc domain or an antibody containing the Fc domain (multispecific) can bind to C1q and therefore has CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. CDC assays may be performed to assess complement activation (eg, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Crag et al., Blood 101, 1045-1052 (2003). ); And Crag and Glennie, Blood 103, 2738-2743 (2004)).

FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (eg, Petkova, SB et al., Int'l. Immunol. 18 (12). ): 1759-1769 (2006); see International Publication No. 2013/120929).

B. Polynucleotide

The present invention further provides isolated polynucleotides encoding the antibodies of the invention. The isolated polynucleotide may be a single polynucleotide or a plurality of polynucleotides.

The polynucleotide encoding the (multispecific) antibody of the invention may be expressed as a single polynucleotide encoding a complete antibody, or co-expressed multiple (eg, two or more) polys. It may be expressed as a nucleotide. The polypeptide encoded by the co-expressed polynucleotide may be associated, for example, via a disulfide bond or other means to form a functional antibody. For example, the light chain portion of an antibody may be encoded by a separate polynucleotide derived from a portion of the antibody that comprises the heavy chain of the antibody. Upon co-expression, the heavy chain polypeptide associates with the light chain polypeptide to form an antibody. In another example, a portion of an antibody comprising one of two Fc domain subunits and optionally one or more Fab molecules (part of) is the other of the two Fc domain subunits. And optionally a Fab molecule, and may be encoded by a separate polynucleotide derived from a portion of the antibody comprising. Upon co-expression, the Fc domain subunits associate to form the Fc domain.

In some embodiments, the isolated polynucleotide encodes the entire antibody molecule according to the invention described herein. In another aspect, the isolated polynucleotide encodes a polypeptide contained in an antibody according to the invention described herein.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In another aspect, the polynucleotide of the invention is RNA, eg, RNA in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

C. Recombination method

The antibodies of the invention may be obtained, for example, by solid state peptide synthesis (eg, Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the antibody, eg, those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Will be done. Such polynucleotides may be readily isolated and sequenced using conventional procedures. In one aspect, an expression vector comprising a vector, in particular the polynucleotide of the invention (especially a single polynucleotide or a plurality of polynucleotides) is provided. Methods well known to those of skill in the art can be used to construct expression vectors containing the coding sequence of the antibody, along with appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / gene recombination. For example, Maniatis et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N. et al. Y. (1989); and Ausubel et al. , Current Protocols in Molecular Biology, Greene Publishing Associates and Willy Interscience, N.M. See technique described in Y (1989). The expression vector can be a plasmid, part of a virus, or even a nucleic acid fragment. The expression vector comprises an expression cassette in which the polynucleotide encoding the antibody (ie, the coding region) is operably associated and cloned with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is part of a nucleic acid consisting of codons that are translated into amino acids. A "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, but if present, it may be considered part of the coding region, but any flanking sequence, such as a promoter, ribosome binding. Sites, transcription terminators, introns, 5'and 3'untranslated regions, etc. are not part of the coding region. Two or more coding regions can be present in a single polynucleotide construct, eg, on a single vector, or in separate polynucleotide constructs, eg, on separate (different) vectors. be able to. Further, any vector may contain a single coding region or may contain more than one coding region, eg, the vector of the invention encodes one or more polypeptides. It may be separated into the final protein by cleavage by proteolysis after or at the same time as translation. In addition, the vector, polynucleotide or nucleic acid of the invention encodes a heterologous coding region and may or may not be fused to the polynucleotide encoding the antibody of the invention, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, special elements or motifs such as secretory signal peptides or heterologous functional domains. An operable association associates with one or more regulatory sequences in such a manner that the coding region of a gene product (eg, a polypeptide) expresses the gene product under the influence or control of the regulatory sequence. .. Two DNA fragments (eg, the polypeptide coding region and associated promoters) have the nature of binding between the two DNA fragments when the introduction of promoter function results in transcription of the mRNA encoding the desired gene product. If it does not interfere with the ability of the expression control sequence to direct the expression of the gene product, and does not interfere with the ability to transcribe the DNA template, it "operably associates". Thus, the promoter region may be operably associated with the nucleic acid encoding the polypeptide if the promoter is capable of transcribing the nucleic acid. The promoter may be a cell-specific promoter that directs only to substantial transcription of DNA within a given cell. Transcription control elements other than promoters, such as enhancers, operators, repressors, transcription arrest signals, may be operably associated with polynucleotides to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those of skill in the art. These include, but are not limited to, transcriptional regulatory regions that function within vertebrate cells, such as, but not limited to, promoter and enhancer segments derived from cytomegalovirus (eg, in combination with the earliest promoter, intron-A), Simian virus 40. (Eg early promoters) and retroviruses (eg Laus sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heatshock proteins, bovine growth hormone and rabbit β-globin, and other genes capable of controlling gene expression in eukaryotic cells. An array is mentioned. Further suitable transcriptional control regions include tissue-specific promoters and enhancers, and inducible promoters (eg, tetracycline-inducible promoters). Similarly, various translation control elements are known to those of skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and stop codons, and elements derived from the viral system (particularly internal ribosome entry sites, also referred to as IRESs, CITE sequences). The expression cassette also contains other features such as, for example, the origin of replication and / or chromosomal integration elements, such as the long terminal repeat (LTR) of a retrovirus, or the adeno-associated virus (AAV) terminal inversion sequence (ITR). You may.

The polynucleotide and nucleic acid coding regions of the invention may associate with additional coding regions encoding secretory or signal peptides, indicating the secretion of the polypeptide encoded by the polynucleotides of the invention. For example, if antibody secretion is desired, the DNA encoding the signal sequence may be located upstream of the nucleic acid encoding the antibody or fragment thereof of the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence, which are mature proteins as the protein chains grown through the coarse endoplasmic reticulum begin to exit. It cleaves from. For those skilled in the art, a polypeptide secreted by a vertebrate cell generally has a signal peptide that fuses to the N-terminus of the polypeptide, producing a secreted or "mature" form of the polypeptide. I know that it cleaves from the translated polypeptide. In certain embodiments, native signal peptides such as immunoglobulin heavy or light chain signal peptides are used, or functional derivatives of sequences that retain the ability to direct the secretion of operably associated polypeptides are used. To. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced with a human tissue plasminogen activator (TPA) or mouse β-glucuronidase leader sequence.

DNA that can be used to facilitate subsequent purification (eg, histidine tag) or that encodes a short protein sequence that is useful for labeling an antibody can be found in an antibody (fragment) that encodes a polynucleotide, or in an antibody (fragment) that encodes a polynucleotide. It may be contained at the end thereof.

In a further aspect, a host cell comprising the polynucleotide of the invention (ie, a single polynucleotide or a plurality of polynucleotides) is provided. A host cell containing the vector of the invention is provided in a particular embodiment. The polynucleotide and vector may incorporate any of the features described herein in connection with the polynucleotide and vector, respectively, alone or in combination. In one such embodiment, the host cell comprises (eg, transformed with) one or more vectors containing one or more polynucleotides encoding (part of) an antibody of the invention. Or transfected). As used herein, the term "host cell" refers to any type of cell line that can be engineered to produce an antibody or fragment thereof of the invention. Suitable host cells for replicating and assisting antibody expression are well known in the art. Such cells may, where appropriate, be transfected or transduced with a particular expression vector to grow cells containing large amounts of the vector for inoculation into a large fermenter. A sufficient amount of antibody can be obtained for clinical use. Suitable host cells include prokaryotic cells (eg, E. coli) or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells and the like. For example, the polypeptide may be produced in the bacterium if glycosylation is not particularly required. After expression, the polypeptide may be isolated from the bacterial cell paste in the soluble fraction or may be further purified. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for vectors encoding polypeptides, fungal strains and yeasts with "humanized" glycosylation pathways. It comprises a strain and, as a result, produces a polypeptide having a partially or completely human glycosylation pattern. Germanross, Nat Biotech 22, 1409-1414 (2004) and Li et al. , Nat Biotech 24, 210-215 (2006). Also, suitable host cells for the expression of (glycosylated) polypeptides are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and may be used in combination with insect cells, especially for transfection of Prodenia flugiperda cells. Plant cell cultures can also be used as hosts. For example, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (transformers). Describes PLANTIBODIES ™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as a host. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 strains transformed with SV40 (COS-7), human embryonic kidney strains (eg, Graham et al., J Gen Virol 36, 59 (1977)). , 293 cells or 293T cells as described), baby hamster kidney cells (BHK), mouse cell tricells (eg, TM4 cells as described in Mother, Biol Report 23, 243-251 (1980)), monkeys. Kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), humans Liver cells (Hep G2), mouse breast tumor cells (MMT 060562), TRI cells (eg, described in Mother et al., Anals NY Acad Sci 383, 44-68 (1982)), MRC 5 Cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (Urlab et al., Proc Natl Acad Sci USA 77, 4216 (1980)), myeloma cell lines, including dhfr ^- CHO cells. For example, YO, NS0, P3X63 and Sp2 / 0 can be mentioned. For a review of specific mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. See 255-268 (2003). Host cells include cultured cells, eg, cultured mammalian cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few, but are transgenic animals, transgenic plants or cultured plants or animals. It also includes cells contained in the tissue. In one embodiment, the host cell is a eukaryotic cell, in particular a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryo-renal (HEK) cell or a lymphocyte cell (eg, Y0, NS0, Sp20 cell). ). In one aspect, the host cell is not a cell in the human body.

Standard techniques for expressing foreign genes in these systems are known in the art. By manipulating cells expressing a polypeptide containing either a heavy chain or a light chain of an antigen-binding domain such as an antibody, the other antibody chain is also expressed and the expressed product is both heavy and light chains. Can be made to be an antibody having.

In one aspect, a method of producing an antibody according to the invention is provided, which method, as provided herein, is a host comprising a polynucleotide encoding the antibody, under conditions suitable for antibody expression. It involves culturing the cells and optionally recovering the antibody from the host cells (or host cell medium).

The components of the (multispecific) antibody of the invention may be genetically fused together. (Multispecific) antibodies can be designed so that their components are fused directly to each other or indirectly through a linker sequence. The composition and length of the linker may be determined according to methods well known in the art and may be tested for efficacy. Examples of linker sequences between different components of a (multispecific) antibody are provided herein. Also, if desired, additional sequences may be included to incorporate the cleavage site and separate the individual components of the fusion (eg, endopeptidase recognition sequence).

Antibodies prepared as described herein are purified by techniques known in the art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like. May be done. The actual conditions used to purify a particular protein depend in part on factors such as net charge, hydrophobicity, hydrophilicity and are apparent to those of skill in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens to which the antibody binds may be used. For example, a matrix containing protein A or protein G can be used for affinity chromatography purification of the antibodies of the invention. Antibodies can be isolated using continuous protein A or G affinity chromatography and size exclusion chromatography, as essentially described in the Examples. The purity of the antibody can be determined by any of a variety of well-known analytical methods such as gel electrophoresis, high performance liquid chromatography and the like.

D. Assay

The antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

1. 1. Binding assay

Binding (affinity) of an antibody to an Fc receptor or target antigen uses, for example, a standard instrument such as a BIAcore device (GE Healthcare), and a receptor or target protein available, for example by recombinant expression. It can be determined by surface plasmon resonance (SPR). Alternatively, antibody binding to different receptors or target antigens may be assessed, for example, by flow cytometry (FACS) using cell lines expressing a particular receptor or target antigen. Specific descriptive and exemplary embodiments for measuring binding activity to CD3 are described below.

In one aspect, the binding activity to CD3 is determined by SPR as follows.

SPR is performed on a Biacore T200 device (GE Healthcare). The anti-Fab capture antibody (GE Healthcare, # 28958325) is immobilized on the Series S Sensor Chip CM5 (GE Healthcare) with a surface density of 4000-6000 resonance units (RU) using standard amine coupling chemistry. .. HBS-P + (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) is used as the running and dilution buffer. A CD3 antibody at a concentration of 2 μg / ml (20 mM His, 140 mM NaCl, pH 6.0) is infused at a flow rate of 5 μl / min for about 60 seconds. The CD3 antigen used is a heterodimer of the CD3 delta and CD3 epsilon external domains fused to the human Fc domain with knob-in-to-hole modification and C-terminal Avi-tag (SEQ ID NOs: 28 and 29). Please refer to). The CD3 antigen is infused at a concentration of 10 μg / ml for 120 seconds and dissociation is monitored at a flow rate of 5 μl / min for about 120 seconds. The chip surface is regenerated by two consecutive injections of 10 mM glycine pH 2.1, each for about 60 seconds. The difference in volume index is corrected by subtracting the blank injection and by subtracting the response obtained from the blank control flow cell. For evaluation, the binding reaction is taken 5 seconds after the end of injection. To normalize the binding signal, CD3 binding is separated by an anti-Fab response (the signal (RU) obtained upon capture of the CD3 antibody on the immobilized anti-Fab antibody). The binding activity of an antibody to CD3 after a particular treatment, relative to the binding activity of an antibody to CD3 after different treatments (also referred to as relative activity concentration (RAC)), is after different treatments. It is calculated by reference to the binding activity of the antibody sample after a particular treatment with respect to the binding activity of the corresponding antibody sample of.

2. 2. Activity assay

The biological activity of the (multispecific) antibody of the invention can be measured by a variety of assays, as described in the Examples. Biological activity includes, for example, induction of T cell proliferation, induction of signal transduction in T cells, induction of expression of activation markers in T cells, induction of cytokine secretion by T cells, lysis of target cells such as tumor cells. And / or induction of tumor regression and / or improvement of survival may be included.

E. Compositions, formulations and routes of administration

In a further aspect, the invention provides a pharmaceutical composition comprising any of the antibodies provided herein for use, for example, in any of the following therapeutic methods. In one aspect, the pharmaceutical composition comprises an antibody according to the invention and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises an antibody according to the invention and at least one additional therapeutic agent (eg, as described below).

Further provided is a method of producing an antibody of the invention in a form suitable for administration in vivo, wherein the method comprises (a) obtaining the antibody according to the invention and (b) the antibody and at least. A method comprising blending with one pharmaceutically acceptable carrier, thereby formulating an antibody formulation for in vivo administration.

The pharmaceutical composition of the present invention comprises an effective amount of antibody dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" is generally non-toxic to the recipient at the dosage and concentration used, i.e., harmful when properly administered to an animal (eg, human). Refers to molecular elements and compositions that do not cause a reaction, allergic reaction or other adverse reaction. Preparation of pharmaceutical compositions comprising antibodies and optionally additional active ingredients is described in Remington's Pharmaceutical Sciences, 18th Ed. As exemplified by Mack Printing Company, 1990, those skilled in the art will know in view of the present disclosure. In addition, for animal (eg, human) administration, the pharmaceutical product establishes sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards or corresponding institutions in other countries. It will be understood that it should be met. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carriers" are known to those of skill in the art (eg, Remington's Pharmaceutical Sciences, 18th Ed. See Mack Printing Company, 1990, pp. 1289-1329), any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (eg, antibacterial agents, antifungal agents). , Isotonic agents, absorption retarders, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents , Dyes, such similar materials and combinations thereof. Use in pharmaceutical compositions is envisioned, except where any conventional carrier is incompatible with the active ingredient.

Antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if desired for topical treatment, intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be, for example, by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part whether the administration is short-lived or chronic. ..

Parenteral compositions include those designed for administration by injection (eg, by subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection). For injection, the antibodies of the invention may be formulated in aqueous solution, in particular in physiologically compatible buffers such as Hanks solution, Ringer's solution or physiological saline buffer. The solution may contain a compounding agent, such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the antibody may be in powder form for pre-use with a suitable vehicle, eg, sterile exothermic depleting water. Sterile injectable solutions are prepared by incorporating the antibodies of the invention in the required amounts into a suitable solvent, if necessary, along with various other components listed below. Sterilization can be easily achieved, for example, by filtration through a sterile filtration membrane. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and / or other ingredients. For sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred method of preparation is vacuum drying or vacuum drying to obtain powders of the active ingredient and any further desired ingredients from a liquid medium that has already been sterile filtered. It is a freeze-drying technique. The liquid medium should be adequately buffered if necessary and the liquid diluent should first be isotonic with sufficient saline or glucose prior to injection. The composition must be stable under manufacturing and storage conditions and must be protected from the contamination of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be maintained at safe levels, eg, below 0.5 ng / mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers, such as phosphates, citrates and other organic acids; antioxidants containing ascorbic acid and methionine; preservatives (eg, octadecyldimethylbenzylammonium chloride). Hexamethonium chloride; benzalconium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low Molecular weight (less than about 10 residues) polypeptide; protein, eg, serum albumin, gelatin or immunoglobulin; hydrophilic polymer, eg, polyvinylpyrrolidone; amino acids, eg, glycine, glutamine, asparagine, histidine, arginine or lysine; Sugars, disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents such as EDTA; saccharides such as sucrose, mannitol, trehalose or sorbitol; counterions forming salts such as sodium; Metal complexes (eg, Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). The aqueous injection suspension may contain compounds that increase the viscosity of the suspension (eg, sodium carboxymethyl cellulose, sorbitol, dextran, etc.). Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of highly concentrated solutions. In addition, suspensions of active compounds may be prepared as suitable oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (eg, sesame oil), or synthetic fatty acid esters (eg, ethyl cleats or triglycerides) or liposomes.

The active ingredient is also microcapsules prepared, for example by core selvation technology or by interfacial polymerization, eg hydroxymethylcellulose or gelatin microcapsules and polyemulsified microcapsules, colloidal drug delivery systems (eg, liposomes, albumin). It can also be incorporated into microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained release formulations may be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing polypeptides, which are in the form of molded articles such as, for example, films or microcapsules. In certain embodiments, sustained absorption of the injectable composition may be brought about by use in the composition of an agent that delays absorption (eg, aluminum monostearate, gelatin, or a combination thereof).

In addition to the compositions already described, the antibody may also be formulated as a depot formulation. Such long-acting formulations may be administered by implantation (eg, subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, the antibody may be formulated with a suitable polymer or hydrophobic material (eg, as an emulsion in an acceptable oil) or ion exchange resin, or as a slightly soluble derivative, eg, as a slightly soluble salt. May be done.

Pharmaceutical compositions containing the antibodies of the invention may be prepared by conventional mixing, dissolving, emulsifying, encapsulating, encapsulating or lyophilizing processes. The pharmaceutical composition uses a conventional mode with an adjunct that facilitates the processing of one or more physiologically acceptable carriers, diluents, excipients or proteins into a pharmaceutically usable formulation. It may be blended with. The appropriate formulation depends on the route of administration chosen.

The antibody may be incorporated into the composition in the form of a free acid or free base, neutral or salt. A pharmaceutically acceptable salt is a salt that substantially retains the biological activity of the free acid or free base. These include acid addition salts, such as those formed with free amino groups in protein compositions, or with inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid, oxalic acid, tartrate acid or mandelic acid, etc. Examples include those formed with the organic acids of. The salt formed with the free carboxyl group may also be derived from an inorganic base such as a hydroxide of sodium, potassium, ammonium, calcium or ferric hydroxide, or such as isopropylamine, trimethylamine, histidine or prokine. It may be derived from an organic base. Medicinal salts tend to be more soluble in aqueous and other protonic solvents than the corresponding free base forms.

F. Treatment method and composition

Any antibody provided herein may be used in a therapeutic method. The antibody of the present invention may be used as an immunotherapeutic agent, for example, in the treatment of cancer.

For use in therapeutic methods, the antibodies of the invention are formulated, divided into doses and administered in a manner consistent with the principles for good quality medicine. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the drug, the method of administration, the schedule of administration, and the healthcare professional. There are other factors known to the person.

In one aspect, an antibody of the invention for use as a pharmaceutical is provided. In a further aspect, the antibodies of the invention for use in the treatment of diseases are provided. In certain embodiments, the antibodies of the invention for use in therapeutic methods are provided. In one aspect, the invention provides an antibody of the invention for use in the treatment of a disease to an individual in need of treatment of the disease. In certain aspects, the invention provides an antibody for use in a method of treating an individual with a disease, comprising administering to the individual an effective amount of the antibody. In certain embodiments, the disease being treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (eg, an anti-cancer agent if the disease being treated is cancer). In a further aspect, the invention provides an antibody of the invention for use in inducing lysis of target cells, particularly tumor cells. In certain embodiments, the present invention is used in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual, comprising administering to the individual an effective amount of antibody to induce lysis of the target cell. The antibody of the present invention is provided. The "individual" according to any of the above embodiments is a mammal, preferably a human.

In a further aspect, the invention provides the use of the antibodies of the invention in the manufacture or preparation of a pharmaceutical. In one aspect, the pharmaceutical is for the treatment of a disease in an individual in need of treatment of the disease. In a further aspect, the drug is intended for use in a method of treating a disease, comprising administering to an individual with the disease an effective amount of the drug. In certain embodiments, the disease being treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In one aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (eg, an anti-cancer agent if the disease being treated is cancer). In a further aspect, the drug is for inducing lysis of target cells, especially tumor cells. Still in a further aspect, the pharmaceutical is to be used in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual, comprising administering to the individual an effective amount of the pharmaceutical to induce lysis of the target cell. belongs to. The "individual" according to any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating a disease. In one aspect, the method comprises administering to an individual with such a disease an effective amount of an antibody of the invention. In one aspect, the composition is administered to the individual and comprises the antibody of the invention in a pharmaceutically acceptable form. In certain embodiments, the disease being treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (eg, an anti-cancer agent if the disease being treated is cancer). The "individual" according to any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for inducing lysis of target cells, particularly tumor cells. In one aspect, the method comprises contacting a target cell with an antibody of the invention in the presence of T cells, particularly cytotoxic T cells. In a further aspect, a method for inducing lysis of target cells, particularly tumor cells, in an individual is provided. In one such embodiment, the method comprises administering to an individual an effective amount of an antibody of the invention to induce lysis of target cells. In one aspect, the "individual" is a human.

In certain embodiments, the disease being treated is a proliferative disorder, especially cancer. Non-limiting examples of cancer include bladder cancer, brain tumor, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colonic rectal cancer. Examples include cancer, rectal cancer, gastric cancer, prostate cancer, hematological cancer, skin cancer, squamous cell carcinoma, bone cancer and kidney cancer. Other cell proliferation disorders that can be treated with the antibodies of the invention include, but are not limited to, abdomen, bone, breast, digestive system, liver, pancreas, peritoneal gland, endocrine glands (adrenal, adrenal gland, pituitary gland, etc.). These include neoplasms located in the testicles, ovaries, thymus, thyroid glands), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissues, spleen, thoracic region and urogenital system. Precancerous conditions or lesions and cancer metastases are also included. In certain embodiments, the cancer is selected from the group consisting of kidney cancer, bladder cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer and prostate cancer. In particular, in one aspect in which the antibody is a multispecific antibody that binds to TYRP-1 as a second antigen, the cancer is a cancer that expresses TYRP-1. In particular, in one aspect in which the antibody is a multispecific antibody that binds to TYRP-1 as a second antigen, the cancer is skin cancer, especially melanoma. In particular, in one aspect in which the antibody is a multispecific antibody that binds to EGFRvIII as a second antigen, the cancer is a cancer that expresses EGFRvIII. In particular, in one aspect in which the antibody is a multispecific antibody that binds to EGFRvIII as a second antigen, the cancer is a brain tumor, especially glioblastoma. Those skilled in the art will readily recognize that antibodies often do not provide cure, but may provide only partial benefit. In some embodiments, physiological changes that have some effect are also considered therapeutically beneficial. Therefore, in some embodiments, the amount of antibody that gives a physiological change is considered an "effective amount." The subject, patient or individual in need of treatment is typically a mammal, more specifically a human.

In some embodiments, an effective amount of an antibody of the invention is administered to an individual to treat the disease.

Appropriate dosages of the antibodies of the invention (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of the disease are treated. Type of disease, route of administration, weight of patient, type of antibody, severity and course of disease, whether antibody is administered for prophylactic or therapeutic purposes, previous or current intervention, patient history and antibody It depends on the response and the discretion of the attending physician. In any case, the physician responsible for administration will determine the concentration of active ingredient in the composition, the appropriate dose for the individual subject. Various dosing schedules are contemplated herein, including, but not limited to, single doses or multiple doses, bolus doses, and pulse infusions over various time points.

The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg, 0.1 mg / kg to 10 mg / kg) of antibody may be administered, for example, by one or more separate doses. Alternatively, it may be an initial candidate dosage for administration to a patient, whether by continuous infusion or not. One typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. In repeated doses over several days or longer, depending on the condition, treatment is generally continued until the desired suppression of disease symptoms occurs. One exemplary dosage of antibody will be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, the dose is also about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 200 μg / kg body weight. 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight. It may include from kg body weight, from about 500 mg / kg body weight to about 1000 mg / kg body weight, or may be even higher per dose, and may be in any range derived from these. In a non-limiting example of the range derived from the numbers listed herein, the range from about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 μg / kg body weight to about 500 mg / kg body weight, etc., is described above. It may be administered based on the number of. Therefore, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, eg, weekly or every 3 weeks (eg, such that the patient receives about 2 to about 20 doses, or eg, about 6 doses of antibody). An initial higher dose may be given, followed by a single or multiple lower doses. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

The antibodies of the invention are generally used in an amount effective to achieve the intended purpose. For use in treating or preventing disease conditions, the antibodies of the invention, or pharmaceutical compositions thereof, are administered or applied in effective amounts.

For systemic administration, the effective dose can be initially estimated from an in vitro assay, eg, a cell culture assay. Dosages may then be formulated in animal models to achieve a blood concentration range containing _IC50 as determined by cell culture. Such information can be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, eg from animal models, using techniques well known in the art.

Dosing and dosing intervals may be adjusted individually to provide plasma concentrations of antibody sufficient to maintain therapeutic effect. Typical patient dosages for administration by injection range from about 0.1-50 mg / kg / day, typically about 0.5-1 mg / kg / day. A therapeutically effective plasma concentration may be achieved by administering multiple doses each day. The concentration in plasma may be measured, for example, by HPLC.

Effective doses of the antibodies of the invention generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the antibody can be determined by standard pharmaceutical procedures in cell cultures or laboratory animals. Cell culture assays and animal studies can be used to determine LD ₅₀ (50% of the population is lethal dose) and ED ₅₀ (50% of the population is therapeutically effective dose). The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio LD ₅₀ / ED ₅₀ . Antibodies with a large therapeutic index are preferred. In one aspect, the antibody according to the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal experiments can be used in determining dosage ranges suitable for human use. The dosage is preferably within the range of blood concentrations containing ED ₅₀ , with little or no toxicity. The dosage may vary within this range, depending on various factors such as, for example, the dosage form used, the route of administration used, the condition of the subject and the like. The exact formulation, route of administration and dosage may be selected by the individual physician in terms of the patient's condition (eg, which is incorporated herein by reference in its entirety, Finger et al., 1975, in: See The Pharmaceutical Basics of Therapeutics, Ch.1, p.1.).

The attending physician of a patient treated with the antibodies of the invention will know how and when to discontinue, discontinue, or adjust administration due to toxicity, organ failure, and the like. Conversely, the attending physician may also know to adjust treatment to higher levels if the clinical response is inadequate (without causing toxicity). The magnitude of the dose administered in the management of the disorder of interest will vary with the severity of the condition being treated, the route of administration, and the like. The severity of the condition may be assessed, for example, in part by standard prognostic methods. In addition, the dose, and possibly the frequency of dosing, will also vary with the age, weight and response of the individual patient.

The antibodies of the invention may be administered in combination with one or more other agents in treatment. For example, the antibodies of the invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may contain any active ingredient suitable for the particular disease to be treated, preferably those having complementary activities that do not adversely affect each other. good. In certain embodiments, the further therapeutic agent is an immunoregulatory agent, a cell proliferation inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of a cell to an apoptosis-inducing factor. .. In a preferred embodiment, the additional therapeutic agent is the activity of an anticancer agent, eg, a microtubule disruptor, a metabolic antagonist, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis. It is an agent or an anti-angiogenic agent.

Such other agents are appropriately present in combination in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of antibody used, the type of disorder or treatment, and the other factors mentioned above. Antibodies are generally used at the same dosage, on the route of administration described herein, or at about 1-99% of the dosage described herein, or empirically / clinically. Used in any dosage and any route determined to be appropriate.

Such combination therapies as described above include combined administration (two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case the administration of the antibodies of the invention is. , Further therapeutic agents and / or adjuvants may be performed before, at the same time as and / or after administration of the adjuvant. The antibodies of the invention may also be used in combination with radiation therapy.

G. Manufactured goods

In another aspect of the invention, there is provided a manufactured article containing materials useful for the treatment, prevention and / or diagnosis of the above-mentioned disorders. The manufactured article comprises a container and a label or package package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags and the like. The container may be made of various materials such as glass or plastic. The container may hold a composition that is effective in treating, preventing, and / or diagnosing the condition, either on its own or in combination with another composition, and may have a sterile access port (eg, the container). Can be a vial with a stopper that can be pierced by an IV solution bag or a hypodermic needle). At least one active agent in the composition is an antibody of the invention. Labels or package inserts indicate that the composition is used to treat the condition of choice. Further, the manufactured article contains (a) a first container containing the composition containing the antibody of the present invention, and (b) a composition containing a further cytotoxic agent or a therapeutic agent. It may include a second container contained. The manufactured article in this aspect of the invention may further include package inserts indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the manufactured article contains a second (or) pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. A third) container may be further included. Other materials may be further included, including other buffers, diluents, filters, needles, and syringes, which are desirable from a commercial and user standpoint.

H. Methods and compositions for diagnosis and detection

In certain embodiments, any of the antibodies provided herein is useful in detecting the presence of its target (eg, CD3, TYRP-1, EGFRvIII) in a biological sample. The term "detect", as used herein, includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as prostate tissue.

In one aspect, an antibody according to the invention for use in a diagnostic or detection method is provided. In a further embodiment, a method of detecting the presence of CD3, TYRP-1 or EGFRvIII in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with the antibody of the invention under conditions that allow the antibody to bind to CD3, TYRP-1 or EGFRvIII, and the complex comprises the antibody and CD3. , Detecting whether it is formed with TYRP-1 or EGFRvIII, and the like. Such a method may be an in vitro method or an in vivo method. In one aspect, the antibodies of the invention are used to select eligible subjects for treatment with antibodies that bind to CD3, TYRP-1 and / or EGFRvIII, eg, CD3, TYRP-1 and / or EGFRvIII. Is a biomarker for patient selection.

Exemplary disorders that can be diagnosed using the antibodies of the invention include cancers, especially skin cancers or brain tumors.

In a particular embodiment, an antibody according to the present invention, wherein the antibody is labeled, is provided. Labels include, but are not limited to, directly detected labels or moieties (eg, fluorescent, dye, electron-dense, chemiluminescent and radioactive labels) and indirectly by, for example, enzymatic reactions or molecular interactions. Examples include the detected moiety (eg, enzyme or ligand). Exemplary labels include, but are not limited to, radioactive isotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I, fluorophores such as rare earth chelate or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, un. Veriferon, luciferases, eg, firefly luciferase and bacterial luciferase (US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazindione, horseradish peroxidase (HRP), alkaline phosphatase, Concatenate with enzymes that use β-galactosidase, glucoamylase, lysozyme, glycooxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and hydrogen peroxide to form a dye precursor. Oxidizing xanthine oxidases include, for example, HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals and the like.

III. arrangement

IV.

The following are examples of the methods and compositions of the present invention. Given the general description given above, it is understood that various other embodiments may be implemented.

Example 1-Generation of optimized CD3 binder

Already described (eg, see International Publication No. 2014/131712, incorporated herein by reference), referred to herein as "CD3 _orig ", and the VHs of SEQ ID NOs: 6 and 11, respectively. Starting with a CD3 binder containing the VL sequence, we can optimize the properties of this binder by removing the two asparagine deamidation sequence motifs at Kabat positions 97 and 100 of the heavy chain CDR3. I aimed.

To this end, we generate a heavy chain antibody library from which asparagine at both Kabat positions 97 and 100 is depleted, suitable for phage display, and is further triggered by replacement of Asn97 and Asn100. Randomized CDRs H1, H2 and H3 were generated to compensate for the loss of affinity through the affinity maturation process.

This library was placed on linear phage via fusion to the minor coat protein p3 (Marks et al. (1991) J Mol Biol 2222,581-597) and was selected for binding to recombinant CD3ε.

Ten candidate clones were identified in the initial screening and showed acceptable binding on the recombinant antigen as measured by SPR as a Fab fragment (produced in E. coli).

However, only one of these clones showed acceptable binding activity to CD3 expressing cells as measured by flow cytometry after conversion to the IgG format.

Selected clones, referred to herein as "CD3 _opt " and containing the VH and VL sequences of SEQ ID NOs: 7 and 11, respectively, are further evaluated and into a bispecific format, as described below. It was converted.

Example 2-Binding of an optimized CD3 binder to CD3

Binding to recombinant CD3

For the optimized CD3 binder "CD3 _opt " and the original CD3 binder "CD3 _orig ", both of which are human IgG1 formats of P329G L234A L235A ("PGLALA", EU numbering) variants in the Fc region (SEQ ID NOs: 12 and 14). (CD3 _orig ) and SEQ ID NOs: 13 and 14 (CD3 _opt )), binding to recombinant CD3 was determined by surface plasmon resonance (SPR).

To assess the effect of deamidation site removal and its effect on antibody stability, binding of the original and optimized CD3 binders to recombinant CD3 at 37 ° C or 40 ° C for 14 days. Tested after temperature stress. A sample stored at -80 ° C was used as a reference. The reference sample and the sample stressed at 40 ° C were pH 6.0 in 20 mM His, 140 mM NaCl, and the sample stressed at 37 ° C was pH 7.4 in PBS, all. The concentration was 1.2 to 1.3 mg / ml. After a stress period (14 days), samples in PBS were dialyzed back to 20 mM His, 140 mM NaCl, pH 6.0 for further analysis.

The relative activity concentration (RAC) of the sample was determined by SPR as follows.

SPR was performed on a Biacore T200 device (GE Healthcare). The anti-Fab capture antibody (GE Healthcare, # 28958325) is fixed to the Series S Sensor Chip CM5 (GE Healthcare) with a surface density of 4000-6000 resonance units (RU) using standard amine coupling chemistry. It was made into. HBS-P + (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) was used as the running and dilution buffer. A CD3 antibody at a concentration of 2 μg / ml was injected at a flow rate of 5 μl / min for 60 seconds. The CD3 antigen (see below) was infused at a concentration of 10 μg / ml for 120 seconds and dissociation was monitored for 120 seconds at a flow rate of 5 μl / min. The chip surface was regenerated by two consecutive injections of 10 mM glycine pH 2.1 for 60 seconds each. The difference in volume index was corrected by subtracting the blank injection and by subtracting the response obtained from the blank control flow cell. For evaluation, the binding reaction was taken 5 seconds after the end of injection. To normalize the binding signal, CD3 binding was separated by an anti-Fab response (signal (RU) obtained upon capture of the CD3 antibody on the immobilized anti-Fab antibody). Relative activity concentrations were calculated by referencing each temperature stressed sample to the corresponding unstressed sample.

The antigen used was a heterodimer of the CD3 delta and CD3 epsilon external domains fused to the human Fc domain with knob-in-to-hole modification and C-terminal Avi-tag (SEQ ID NOs: 28 and 29). Please refer to).

The results of this experiment are shown in FIG. As can be seen, the optimized CD3 binder, CD3 _opt , is more resistant to CD3 after temperature stress (37 ° C, pH 7.4 for 2 weeks) compared to the original CD3 binder, CD3 _orig . Showed improved binding. The results showed that deamidation site removal was successful in relation to the formulation of the antibody at neutral pH with in vivo half-life, and that deamidation site removal yielded antibodies with excellent stability properties. It is shown that.

Binding to CD3 on Jurkat cells

For the optimized CD3 binder "CD3 _opt " and the original CD3 binder "CD3 _orig ", both of which are human IgG1 formats of P329G L234A L235A ("PGLALA", EU numbering) mutations in the Fc region (SEQ ID NOs: 12 and 14). (CD3 _orig ) and SEQ ID NOs: 13 and 14 (CD3 _opt )), binding to CD3 on the human reporter T cell line Jurkat NFAT was determined by FACS.
Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega # CS176501) are human acute lymphocytic leukemia reporter cell lines with an NFAT promoter and express human CD3. Cells were cultured in RPMI 1640, 2 g / l glucose, 2 g / l NaHCO ₃ , 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1x NEAA, 1 x sodium pyruvate in 0.1-0.5 mio cells per ml. .. A final concentration of 200 μg per ml of hygromycin B was added at any time of cell passage.

For the binding assay, Jurkat NFAT cells were harvested, washed with PBS and resuspended in FACS buffer. Antibody staining was performed in a 96-well round bottom plate. Therefore, 100,000 to 200,000 cells per well were seeded. The plate was centrifuged at 400 xg for 4 minutes and the supernatant was removed. The test antibody was diluted with FACS buffer and 20 μl of the antibody solution was added to the cells at 4 ° C for 30 minutes. Diluted secondary antibody (PE-conjugated AffiniPure F (ab') 2 Fragment goat anti-human IgG Fcg Fragment Specific; Jackson ImmunoResearch # 109-1 before addition to remove unbound antibody) Was washed twice with FACS buffer. After 30 minutes of incubation at 4 ° C, unbound secondary antibodies were washed away. Prior to measurement, cells were resuspended in 200 μl FACS buffer and then analyzed by flow cytometry using a BD Canto II appliance.

As shown in FIG. 3, the optimized CD3 binder "CD3 _opt " and the original CD3 binder "CD3 _orig " bound relatively well to CD3 on Jurkat cells.

Example 3-Functional activity of optimized CD3 binder

The functional activity of the optimized CD3 binder "CD3 _opt " was tested in a Jurkat reporter cell assay and compared to the activity of the original CD3 binder "CD3 _orig ". To test the functional activity of IgG, anti-PGLALA-expressing CHO cells were co-incubated with Jurkat NFAT reporter cells in the presence of increasing concentrations of CD3 _opt human IgG1 PGLALA or CD3 _orig human IgG1 PGLALA. Activation of CD3 on T cell cross-linked Jurkat NFAT reporter cells induces the production of luciferase, and luminescence can be measured as an activation marker. CD3 _orig human IgG 1 wt was included as a negative control, unable to bind to anti-PGLALA expressing CHO cells and thus unable to be crosslinked to Jurkat NFAT cells. A schematic diagram of the assay is provided in FIG.

Anti-PGLALA-expressing CHO cells are CHO-K1 cells engineered to express an antibody that specifically binds to human IgG ₁ Fc (PGLALA) on its surface (incorporated herein by reference). , International Publication No. 2017/072210). These cells were cultured in DMEM / F12 medium containing 5% FCS + 1% GluMax. Jurkat NFAT reporter cells are as described in Example 2.

When CD3 huIgG1 PGLALA simultaneously binds to CD3 expressed on anti-PGLALA expressed on CHO and Jurkat NFAT reporter cells, the NFAT promoter is activated to trigger the expression of active firefly luciferase. The intensity of the luminescent signal (obtained upon addition of the luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Jurkat-NFAT reporter cells grow in suspension and in RPMI1640, 2 g / l glucose, 2 g / l NaHCO3, 10% FCS, 25 mM HEEPS, 2 mM L-glutamin, 1x NEAA, 1x sodium pyruvate, The cells were cultured at 0.1-0.5 mio cells per 1 ml and 200 μg per 1 ml of hyglomycin. For the assay, CHO cells were harvested and ViCell was used to determine viability. In a flat-bottomed, white-walled 96-well plate (Greener bio-one # 655098), plate 30,000 target cells / well in 100 μl medium and add 50 μl / well diluted antibody or medium (for control) to CHO cells. Added. Subsequently, Jurkat-NFAT reporter cells were harvested and viability was assessed using ViCell. Cells were resuspended in cell medium at 1.2 mio cells / ml without hygromycin B and added to CHO cells at 60,000 cells / well (50 μl / well) to a final effector to target (E: T) ratio of 2. 1 and a final volume of 200 μl per well was obtained. 4 μl of GloSenser (Promega # E1291) was then added to each well (2% of final volume). The cells were incubated in a humidified incubator at 37 ° C. for 24 hours. At the end of the incubation time, luminescence was detected using TECAN Spark 10M.

As shown in FIG. 5, the optimized CD3 binder, CD3 _opt , had similar activity on Jurkat NFAT cells upon cross-linking as a CD3 _org .

Example 4-Generation of T cell bispecific antibody comprising optimized CD3 binder

TYRP1 TCB

Using the optimized CD3 binders identified in Example 1 (“CD3 _opt ”, SEQ ID NOs: 7 (VH) and 11 (VL)), T cell bispecific antibodies (TCBs) targeting CD3 and TYRP1. ) Was generated ("TYRP1 TCB").

The TYRP1 binder contained in this TCB is produced by humanization "TA99" of the TYRP1 binder (see GenBank entries AXQ57811 and AXQ57813 for heavy and light chains, respectively) and the weights set forth in SEQ ID NOs: 18 and 22, respectively. Includes chain and light chain variable region sequences.

A schematic diagram of the TCB molecule is provided in FIG. 6, the entire sequence thereof is given by SEQ ID NOs: 23, 24, 25 and 27.

Numerator similar to the original CD3 binding sequence was also prepared (SEQ ID NOs: 23, 24, 25 and 26).

Bispecific molecules were generated by transient transfection of HEK293 EBNA cells. Cells are 1: 2: 1: 1 depending on the corresponding expression vector (“Vector Heavy Chain (VH-CH1-VL-CH1-CH2-CH3)”: “Vector Light Chain (VL-CL)”: “Vector Heavy Chain”. (VH-CH1-CH2-CH3) ”:“ Vector light chain (VH-CL) ”)-transfected cells were centrifuged to warm the medium into pre-warmed CD CHO medium (Thermo Fisher, # 10743029). ). Expression vectors were mixed in CD CHO medium, PEI (polyethylenimine, Polysciences, # 23966-1) was added, the solution was vortexed and incubated for 10 minutes at room temperature. The cells (2 mio / ml) were then mixed with the vector / PEI solution, transferred to a flask and incubated at 37 ° C. for 3 hours in a shaking incubator with a 5% CO ₂ atmosphere. After incubation, Excell medium with replenisher (80% of total volume) was added. One day after transfection, a replenisher (Feed, 12% of total volume) was added. After 7 days, cell supernatants were collected by centrifugation and subsequent filtration (0.2 μm filter).

Proteins were purified from filtered cell culture supernatants by standard methods. Briefly, Fc-containing proteins were purified from cell culture supernatants by protein A affinity chromatography (MabSelectSure, GE Healthcare: equilibrium buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5). Elution buffer: 20 mM sodium citrate, 100 mM NaCl, 100 mM glycine pH 3.0). Elution was achieved at pH 3.0, followed by immediate pH neutralization of the sample. Proteins were concentrated by centrifugation (Millipore Amion® ULTRA-15 (# UFC903096)) and the aggregated proteins were subjected to size exclusion chromatography at 20 mM histidine, 140 mM sodium chloride, pH 6.0 (Superdex 200, GE Healthcare). Separated from the monomeric protein.

Pace et al. (1995), the concentration of purified protein was determined by measuring absorption at 280 nm using a mass attenuation coefficient calculated based on the amino acid sequence according to Protein Science 4,2411-23. Protein purity and molecular weight were analyzed by CE-SDS using LabChipGXII (PerkinElmer) in the presence and absence of a reducing agent (Table 1). Analytical size equilibrated in running buffer (25 mM K2 HPO ₄ , 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7, or 200 mM KH ₂ PO ₄ , 250 mM KCl, pH 6.2, _respectively ). Aggregation contents were determined at 25 ° C. by HPLC chromatography using an exclusion column (TSKgel G3000 SW XL or UP-SW3000) (Table 2).

Example 5-Binding of a T cell bispecific antibody comprising an optimized CD3 binder to CD3 and TYRP1

Binding to recombinant CD3

Binding of TYRP1 TCB to recombinant CD3 was evaluated by SPR using a TCB with either an optimized (TYRP1 TCB CD3 _opt ) or original (TYRP1 TCB CD3 _orig ) CD3 binding sequence.

SPR experiments were performed on Biacore T200 using HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 0.05% (v / v) detergent P20 (GE Healthcare)) as a running buffer. gone.

CM5 sensor chip surface with TYRP1 TCB, along with an immobilized antibody that specifically binds human IgG ₁ Fc (PGLALA) (see WO 2017/072210, incorporated herein by reference). Captured above. The capture antibody was coupled to the sensor chip surface using a standard amine coupling kit (GE Healthcare) by direct immobilization of approximately 8700 resonant units (RU) at pH 5.0. TCB molecules were captured at 5 nM for 30 seconds at a flow of 10 μl / min.

Human and cynomolgus monkey antigens (see below) were passed through the flow cell at a concentration of 12.35 to 3000 nM at a flow of 30 μl / min for 240 seconds. The dissociated phase was monitored for 240 seconds and triggered by switching from sample solution to HBS-EP. The chip surface was regenerated after each cycle using a single injection of 10 mM glycine pH 2.0 for 30 seconds.

The antigen used was a heterodimer of either the human or crab monkey CD3 delta and CD3 epsilon external domain fused to the human Fc domain with knob-in-to-hole modification and C-terminal Avi-tag. (See SEQ ID NOs: 28 and 29 (human CD3) and SEQ ID NOs: 30 and 31 (crab monkey CD3)).

The difference in volume index was corrected by subtracting the response obtained from the reference flow cell (TCB was not captured at all). The affinity constants were derived from the dynamic rate constants by fitting to a 1: 1 Langmuir bond using BIAever software (GE Healthcare).

KD values for binding to human and cynomolgus monkey CD3 were determined for _TYRP1 TCB CD3 _opt as 50 nM and 20 nM, respectively, and were similar to those for TYRP1 TCB CD3 _orig (50 nM and 40 nM, respectively).

This indicates that under non-stress conditions, both TCBs, including either CD3 _opt or CD3 _orig , bind relatively well to recombinant CD3.

Binding of TYRP1 TCBs to recombinant human CD3 was also assessed after 14 days of temperature stress at 37 ° C or 40 ° C using TCBs with either optimized or original CD3 binding sequences. .. The experiment was performed as described in Example 2 above, using TCB instead of IgG molecule.

The results of this experiment are shown in FIG.

As can be seen in FIG. 7, the TCB containing the optimized CD3 binder CD3 _opt is stressed (37 ° C, 2 at pH 7.4) as compared to the TCB containing the original CD3 binder CD3 _orig . After a week), it showed strongly improved binding to CD3. This result confirms that the improved properties of the optimized CD3 binder (see Example 2) are maintained at the TCB level.

Binding to recombinant TYRP1

Binding to recombinant TYRP1 was assessed by SPR using TYRP1 Fab fragments prepared by plasmin digestion of the corresponding antibody.

SPR experiments were performed on Biacore T200 using HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 0.05% (v / v) detergent P20 (GE Healthcare)) as a running buffer. gone.

Antibodies that specifically bind human IgG ₁ Fc (PGLALA) (see WO 2017/072210, which is incorporated herein by reference) with a standard amine coupling kit at pH 5.0. (GE Healthcare) was used to couple directly onto the CM5 sensor chip. Antigen (see below) was captured at a flow rate of 10 μl / min for 30 seconds. A 3-fold dilution series of TYRP1 Fab fragments was passed through a flow cell at 30 μl / min for 180 seconds and the associated phase was recorded. The dissociated phase was monitored for 180 or 1200 seconds and was triggered by switching from sample solution to HBS-EP. The chip surface was regenerated after each cycle using a single injection of 10 mM glycine pH 2 for 30 seconds at 30 μl / min.

The antigen used was a monomeric fusion of human, cynomolgus monkey or mouse TYRP1 extracellular domain (ECD) into a human Fc domain with knob-in-to-hole (and PG LALA) modifications and C-terminal Avi-tag. (See SEQ ID NOs: 32 and 35 (human TYRP1), SEQ ID NOs: 33 and 35 (cynomolgus monkey TYRP1), or SEQ ID NOs: 34 and 35 (mouse TYRP1)).

The difference in volume index was corrected by subtracting the response obtained from the reference flow cell (no antigen was captured). The affinity constant (KD) was derived from the dynamic rate constant by fitting to a 1: 1 Langmuir bond using _BIAever software (GE Healthcare).

KD values for binding to human, cynomolgus monkey or mouse _TYRP1 were determined as 130 pM, 180 pM and 530 pM, respectively, and were similar to those of the parent TA99 antibody (90 pM, 120 pM and 310 pM, respectively).

Binding of TYRP1 TCBs to recombinant TYRP1 was also assessed after 14 days of temperature stress at 37 ° C or 40 ° C using TCBs with either optimized or original CD3 binding sequences.

The experiment was performed as described above for binding to CD3 using recombinant TYRP1 (Sino Biologicals) as the antigen.

The results of this experiment are shown in FIG. These support that binding to human TYRP1 for both TCBs (and even for the corresponding TYRP1 binder in the IgG format) is unaffected by stress conditions.

Binding to CD3 on Jurkat cells

For TYRP1 TCBs containing the optimized CD3 binder "CD3 _opt " or the original CD3 binder "CD3 _orig " as described in Example 2 above, binding to CD3 on the human reporter T cell line Jurkat NFAT is by FACS. It has been determined.

As shown in FIG. 9, the TCB containing the optimized CD3 binder "CD3 _opt " binds to CD3 on Jurkat cells at least relatively well to the TCB containing the original CD3 binder "CD3 _orig ". ..

Example 6-Functional activity of T cell bispecific antibodies comprising optimized CD3 binders

CD3 activation

TYRP1 TCB (Example 4) containing either the optimized CD3 binder CD3 _opt or the original CD3 binder CD3 org, _TYRP1 -positive melanoma cell line M150543 (primary melanoma cell line, dermatology cell from Zurich University). Tested in the Jurkat NFAT reporter cell assay (see Example 3) in the presence of (obtained from bank).

When TYRP1 TCB simultaneously binds to TYRP1-positive target cells and CD3 antigens (expressed on Jurkat-NFAT reporter cells), the NFAT promoter is activated to trigger the expression of active firefly luciferase. The intensity of the luminescent signal (obtained upon addition of the luciferase substrate) is proportional to the intensity of CD3 activation and signaling. The assay was performed as described in Example 3 using M150543 instead of anti-PGLALA expressing CHO cells.

As seen in the case of IgG (Example 3), both TCBs containing either CD3 _opt or CD3 _org have similar functional activity on Jurkat NFAT reporter cells and are concentration dependent embodiments. Induced CD3 activation in (Fig. 10).

Target cell killing

In the next step, both TCB molecules were tested in a tumor cell killing assay using freshly isolated human PBMCs from three different donors co-incubated with human melanoma cell line M150543. Tumor cytolysis was measured by LDH release after 24 and 48 hours. Activation of CD4 and CD8 T cells was analyzed by upregulation of CD69 and CD25 on both cell subsets after 48 hours.

Briefly, target cells were harvested with trypsin / EDTA, washed and plated using a flat bottom 96-well plate at a density of 30,000 cells / well. The cells were left attached overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of fresh blood from healthy human donors. Fresh blood was diluted with sterile PBS and laminated on a Histopaque gradation (Sigma # H8889). After centrifugation (450 xg, 30 minutes, room temperature), plasma above the PBMC-containing interface was discarded and PBMC was transferred to a new Falcon tube, followed by filling with 50 ml of PBS. The mixture was centrifuged (400 xg, 10 minutes, room temperature), the supernatant was discarded, and the PBMC pellets were washed twice with sterile PBS (centrifugation step 350 xg, 10 minutes). The resulting PBMC population was automatically counted (ViCell) at 37 ° C., 5% CO ₂ in RPMI1640 medium containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom # K0302). Stored in the cell incubator for further use (not longer than 24 hours). Antibodies were added in triplicate at the indicated concentrations for the killing assay. PBMCs were added to target cells at a final effector to target (E: T) ratio of 10: 1. Target cell killing was assessed by quantification of LDH released into the cell supernatant by apoptotic / necrosis cells after 24 hours of incubation at 37 ° C., 5% CO ₂ (LDH Detection Kit, Roche Applied Science #). 11 644 793 001). Maximum lysis of target cells (= 100%) was achieved by incubation of target cells in 1% Triton X-100. Minimal lysis (= 0%) is relative to target cells co-incubated with effector cells that do not have bispecific constructs.

TCB-mediated activation of CD8 and CD4 T cells during T cell killing of target cells using antibodies that recognize the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). And evaluated by flow cytometry. After 48 hours of incubation, PBMCs were transferred to a round bottom 96-well plate, centrifuged at 350 xg for 5 minutes and washed twice with FACS buffer. Surface staining of CD4 APC (BioLegend # 300514), CD8 FITC (BioLegend # 344704), CD25 BV421 (BioLegend # 302630) and CD69 PE (BioLegend # 310906) was performed according to the supplier's instructions. Cells were washed twice with 150 μl / well FACS buffer and fixed at 4 ° C for 15 minutes using 100 μl / well fixed buffer (BD # 554655). After centrifugation, the sample was resuspended in 200 μl / well FACS buffer. Samples were analyzed by BD FACS Fortessa.

In all three donors, both TCBs, including either optimized or original CD3 binders, induced T cell activation and tumor cell lysis in a comparable manner (FIG. 11). The EC50 values for tumor cytolysis after 48 hours for all three donors are summarized in Table 3.

Example 7-PK study with T cell bispecific antibodies in mice

TYRP1 TCB pharmacokinetics (PK) on different CD3 binders (CD3 _orig and CD3 _opt ) to 1 mg / kg of human FcRn transgenics (line32, homo) and FcRn knockout mice (Jackson Laboratory story number 82 and 014565). Intravenous bolus administration was followed up in (n = 3 / strain / test compound). A series of blood microsamples were taken from human FcRn transgenic (tg) mice up to 672 hours (9 samples per mouse from 5 minutes to 672 hours post-dose) and up to 96 hours in FcRn knockout (ko) mice (5 post-dose). Eight samples per mouse) were taken from minutes to 96 hours. Serum was prepared and stored frozen until analysis. Mouse serum samples were analyzed under non-GLP conditions using a cobas® e411 (Roche) instrument in a genetic ECLIA method specific for the human Ig / Fab CH1 / kappa domain. Pharmacokinetic assessments were performed using standard non-partition analysis.

The results of this study are shown in Table 4. This indicates that manipulation of the CDRs did not cause other sequence liability that would affect antibody clearance. While CD3 _opt is as good as in CD3 _orig in terms of serum half-life, it has the additional advantage of increased CDR stability.

Example 8-Generation of additional T cell bispecific antibodies comprising an optimized CD3 binder

Using the optimized CD3 binders identified in Example 1 (“CD3 _opt ”, SEQ ID NOs: 7 (VH) and 11 (VL)), T cell bispecific antibodies (TCBs) targeting CD3 and EGFRvIII. ) Was generated (“EGFRvIII TCB”).

The EGFRvIII binder (P063.056) contained in this TCB was derived from a phage display followed by affinity maturation (see below), heavy chains and light chains shown in SEQ ID NOs: 88 and 92, respectively. Includes chain variable region sequences.

A schematic diagram of the TCB molecule is provided in FIG. 6, the entire sequence thereof is given by SEQ ID NOs: 109, 110, 111 and 27.

Numerator similar to the original CD3 binding sequence was also prepared (SEQ ID NOs: 109, 110, 111 and 26).

Bispecific molecules were generated by transient transfection of HEK293 EBNA cells and purified and analyzed as described in Example 4 above.

In addition, phage display-derived EGFRvIII antibodies transfect HEK EBNA cells in expression vectors in a 1: ₁ ratio for IgG heavy and light chains in a similar manner in human IgG1 format for use as described below. It was produced in (fect).

All IgG and TCB constructs were purified to comparable quality with a monomer content of 95% or higher as measured by size exclusion chromatography.

Selection of EGFRvIII antibody

The EGFRvIII antibody was derived from phage display and was affinity maturated. EGFRvIII (P056.021 (SEQ ID NOs: 40 and 44), P056.052 (SEQ ID NOs: 48 and 52), P047.019 (SEQ ID NOs: 56 and 60), P057.012 (SEQ ID NOs: 64 and 68), P057.011 (P. Antibodies that exhibit high affinity binding and specificity for SEQ ID NOs: 72 and 76), P056.027 (SEQ ID NOs: 80 and 84) are CHO cells that stably express EGFRvIII and EGFRvIII-positive human glioblastoma cell line DK. -MG was used to test for binding to EGFRvIII expressed on the cell surface. To support specificity and to eliminate cross-reactivity to wild-type EGFR (EGFRwt), selected antibodies were tested for binding to the EGFR wt-positive human tumor cell line MKN-45 (FIG. 12). Cetuximab was included as a positive control and non-target DP47 IgG was included as a negative control for binding to EGFRwt. All selected antibodies specifically bound to EGFRvIII without cross-reactivity to EGFRwt and further characterization was considered.

In the next step, the functional activity of these EGFRvIII antibodies as IgG1 PGLALA (human IgG1 format of P329GL L234A L235A (“PGLALA”, EU numbering) mutations within the Fc region) expresses the anti-PGLALA chimeric antigen receptor (CAR). Luminescence is measured on DK-MG cells co-incubated with Jurkat NFAT reporter cells (see CAR J Assay, international application PCT / EP2018 / 086038, which is incorporated herein by reference in its entirety). Was evaluated by that. DP47 IgG1 PGLALA was included as a negative control. All tested EGFRvIII antibodies induced strong activation of CAR-expressing Jurkat NFAT reporter cells (FIG. 13). All tested EGFRvIII antibodies were selected for conversion to the TCB format (with CD _orig as a CD3 binder), except for P047.019, which showed the weakest binding and activation.

Binding of the selected EGFRvIII antibody to the TCB format to CHO-EGFRvIII cells was compared to the binding of the corresponding IgG to support that conversion to the TCB format did not affect the binding capacity of the EGFRvIII antibody. (Fig. 14). Most of the EGFRvIII clones tested retained their ability to bind to EGFRvIII when converted to TCB format, and only clone P057.011 had a slight reduction compared to the corresponding IgG binding in TCB format. Binding to EGFRvIII is shown (Table 5).

Subsequently, the functional activity of EGFRvIII TCB was tested in the Jurkat NFAT reporter cell assay of EGFRvIII positive DK-MG cells (FIG. 15). All tested EGFRvIII TCBs had activity in the Jurkat NFAT reporter cell assay, with P056.021 being the most predominant, with similar activity P056.027, P056.052 and This was followed by P057.012, as well as P057.011, which had the lowest activity. Next, in order to eliminate the cross-reactivity of EGFRvIII TCB to EGFRwt, EGFRvIII TCB was tested in a tumor cytolysis assay on PBMCs co-cultured with either DK-MG or MKN-45 cells (FIG. 16). .. In this assay, T cell activation (FIG. 17) and cytokine release (FIG. 18) were measured as additional readouts apart from tumor cell lysis. As already seen in the reporter cell assay, EGFRvIII TCB P056.021 had the highest activity on EGFRvIII positive cells without any activity on EGFRwt positive cells. EGFRvIII TCB P057.011 showed non-specific activity on EGFR wt cells and was therefore excluded. EGFRvIII TCB P056.027, P056.052 and P057.012 had comparable activity. Based on these results, EGFRvIII binders P056.021 and P057.012 were selected for a further round of affinity maturation.

No good binder could be derived from P057.012 (results not shown). Table 6 shows the affinity and specificity of the selected EGFRvIII binder derived from P056.021 for EGFRvIII as measured by SPR.

Affinity matured EGFRvIII binders (P063.056 (SEQ ID NOs: 88 and 92), P064.078 (SEQ ID NOs: 96 and 100), P065.036 (SEQ ID NOs: 104 and 108)) are also provided with U87MG-EGFRvIII and MKN-. Specific binding to EGFRvIII on 45 cells was compared to the parent binder (FIG. 19). The EGFRvIII binder, P063.056, which has the best affinity and specificity for EGFRvIII, was selected for conversion to the TCB format with either CD3 _orig or CD3 _opt as the CD3 binder.

The functional activity of EGFRvIII TCB P063.056 (with CD3 _opt or CD3 _orig ) was compared to the parent EGFRvIII TCB P056.021 in the Jurkat NFAT reporter cell assay of U87MG-EGFRvIII, DK-MG and MKN-45 cells (FIG. 20). ). All three TCBs induce specific Jurkat NFAT activation only in the presence of EGFRvIII positive cells. EGFRvIII TCB P063.056 had slightly higher activity than the parent EGFRvIII TCB P056.021.

(Method)

Surface plasmon resonance

The affinity of the EGFRvIII antibody for EGFRvIII was measured using HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 0.005% (v / v) surfactant P20; GE Healthcare) as a running buffer. Measured by surface plasmon resonance with Biacore T200 at 25 ° C. Anti-EGFRvIII PGLALA IgG in an antibody that specifically binds human IgG ₁ Fc (PGLALA) immobilized on a CM5 chip (see WO 2017/072210, incorporated herein by reference). , 25 nM for 30 seconds. The EGFRvIII-ECD avi his antigen (see Example 9 below) was passed through all flow cells at a concentration of 12.4 to 1000 nM at a flow of 30 μl / min for 200 seconds. The dissociated phase was monitored for 300 seconds and triggered by switching from sample solution to HBS-EP. The chip surface was regenerated after each cycle using two injections of 10 mM glycine pH 2.0 for 30 seconds. The difference in refractive index was corrected by subtracting the response obtained from the reference flow cell. Affinity constants were derived from dynamic rate constants by fitting to a 1: 1 Langmuir bond using BIAever software (GE Healthcare).

For specificity determination, EGFRvIII and EGFRwt ECD antigens were captured at 100 nM for 40 seconds using anti-his (PentaHis, Qiagen) immobilized on a CM5 chip. Prior to regeneration at 10 mM glycine pH 2.0 for 60 seconds, a single injection of 500 nM of anti-EGFRvIII antibody for 60 seconds was performed. Over 50 reaction units were observed for EGFRvIII binding. Reactions above 5 reaction units (RU) were considered positive for EGFR wt binding and IgG was classified as specific for reactions less than 5 RU for EGFR wt.

Cell line

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega # CS176501) are human acute lymphocytic leukemia reporter cell lines with an NFAT promoter and express human CD3. Cells were cultured in RPMI 1640, 2 g / l glucose, 2 g / l NaHCO ₃ , 10% FCS, 25 mM HEPES, 1% GlutaMAX, 1x NEAA, 1x sodium pyruvate in 0.1-0.5 mio cells per ml. A final concentration of 200 μg per ml of hygromycin B was added at any time of cell passage.

Jurkat NFAT cells with PGLALA CAR were generated in-house. The original cell line (Jurkat NFAT, Signosis) is a human acute lymphocytic leukemia reporter cell line with an NFAT promoter that causes luciferase expression upon activation via human CD3. They were engineered to express a chimeric antigen receptor capable of recognizing the P293G LALA mutation. When cultured, cells grow in suspension in RPMI 1640 supplemented with 10% FCS and 1% glutamine and maintained at 0.4-1.5 mio cells per ml.

CHO-EGFRvIII cells were generated in-house. CHO-K1 cells were stably transduced with EGFRvIII. Cells were cultured in DMEM / F12 medium containing 5% FCS, 1% GlutaMAX and 6 μg / ml puromycin.

DK-MG (DSMZ # ACC 277) is a human glioblastoma cell line. DK-MG cells were enriched by cell sorting for EGFRvIII expression. Cells were cultured in RPMI1860, 10% FCS and 1% GlutaMAX.

U87MG-EGFRvIII (ATCC HTB-14) is a human glioblastoma cell line stably transduced with EGFRvIII. Cells were cultured in DMEM, 10% FCS and 1% GlutaMAX.

MKN-45 (DSMZ ACC 409) is a human gastric adenocarcinoma cell expressing high levels of EGFRwt. Cells were cultured in high RPMI 1640 containing 2% FCS and 1% GlutaMAX.

Target binding by flow cytometry

The cells used in the binding experiment were harvested, washed with PBS and resuspended in FACS buffer. Antibody staining was performed in a 96-well round bottom plate. Cells were harvested, counted and seeded with 100,000 to 200,000 cells per well. The plate was centrifuged at 400 xg for 4 minutes and the supernatant was removed. The test antibody was diluted with FACS buffer and 20 μl of the antibody solution was added to the cells at 4 ° C for 30 minutes. Diluted secondary antibody PE-conjugated AffiniPure F (ab') 2 Fragment goat anti-human IgG Fcg Fragment Specific (Jackson ImmunoResearch, # 109-1-16- The cells were washed twice with FACS buffer prior to the addition of 098). After 30 minutes of incubation at 4 ° C, unbound secondary antibodies were washed away. Prior to measurement, cells were resuspended in 200 μl FACS buffer and analyzed by flow cytometry using BD Canto II or BD FACS Fortessa.

CAR J NFAT Reporter Cell Assay with EGFRvIII PGLALA IgG

The efficacy of inducing T cell activation of EGFRvIII PGLALA IgG was evaluated using the CAR J NFAT reporter cell assay. The principle of the assay is to co-culture Jurkat-NFAT engineered effector cells with cancer cells expressing tumor antigens. Only when IgG binds to CAR simultaneously via the PGLALA mutation to the target antigen EGFRvIII, the NFAT promoter is activated to cause increased expression of luciferase in Jurkat effector cells. When sufficient substrate is added, the active firefly luciferase causes the emission of luminescence, which can be measured as a signal of CAR-mediated activation. Briefly, target cells were harvested and viability determined. The day before the start of the assay, 30,000 target cells / well were plated in 100 μl medium in a flat-bottomed, white-walled 96-well plate (Greener bio-one, # 655098). The next day, the medium was removed and 25 μl / well diluted antibody or medium (for control) was added to the target cells. Subsequently, Jurkat-NFAT reporter cells were harvested and viability was assessed using ViCell. The cells were resuspended in cell medium at 1.5 mio cells / ml and added to the tumor cells at 75,000 cells / well (50 μl / well), with a final effector to target (E: T) ratio of 2.5: 1. And a final volume of 75 μl per well was obtained. 4 μl of GloSenser (Promega, # E1291) was then added to each well (2% of finished volume). The cells were incubated in a humidified incubator at 37 ° C. for 24 hours. At the end of the incubation time, the plates were allowed to room temperature (about 15 minutes). 25 μl / well of One-Glo Luciferase (Promega, # E6120) was then added and the plates were incubated in the dark for 15 minutes before detecting luminescence using TECAN Spark.

Jurkat NFAT Reporter Cell Assay with EGFRvIII TCB

The ability of EGFRvIII TCB with either improved CD3 or the original CD3 binder to induce T cell cross-linking and subsequently T cell activation was evaluated using EGFRvIII positive cells and Jurkat-NFAT reporter cells. When EGFRvIII TCB simultaneously binds to EGFRvIII-positive target cells and CD3 antigens (expressed on Jurkat-NFAT reporter cells), the NFAT promoter is activated to trigger the expression of active firefly luciferase. The intensity of the luminescent signal (obtained upon addition of the luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Target cells were harvested and viability determined for the assay. In a flat-bottomed, white-walled 96-well plate (Greener bio-one, # 655098), plate 30,000 target cells / well in 100 μl medium and apply 50 μl / well diluted antibody or medium (for control) to the target cells. Was added to. Subsequently, Jurkat-NFAT reporter cells were harvested and viability was assessed using ViCell. Cells were resuspended in cell medium at 1.2 mio cells / ml without hygromycin B and added to tumor cells at 60,000 cells / well (50 μl / well) with a final effector to target (E: T) ratio of 2. 1 and a final volume of 200 μl per well was obtained. 4 μl of GloSenser (Promega, # E1291) was then added to each well (2% of finished volume). The cells were incubated in a humidified incubator at 37 ° C. for 24 hours. At the end of the incubation time, luminescence was detected using TECAN Spark.

T cell-mediated tumor cell killing

Target cells were harvested with trypsin / EDTA, washed and plated using a flat bottom 96-well plate at a density of 30,000 cells / well. The cells were left attached overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of fresh blood from healthy human donors. Fresh blood was diluted with sterile PBS and laminated on a Histopaque gradation (Sigma, # H8889). After centrifugation (450 xg, 30 minutes, room temperature), plasma above the PBMC-containing interface was discarded and PBMC was transferred to a new falcon tube, followed by filling with 50 ml of PBS. The mixture was centrifuged (400 xg, 10 minutes, room temperature), the supernatant was discarded, and the PBMC pellets were washed twice with sterile PBS (centrifugation step 350 xg, 10 minutes). The resulting PBMC population was automatically counted (ViCell) in RPMI1640 medium containing 10% FCS and 1% GlutaMAX at 37 ° C., 5% CO ₂ until further use in the cell incubator. Stored (not longer than 24 hours). Antibodies were added in triplicate at the indicated concentrations for the killing assay. PBMCs were added to target cells at a final effector to target (E: T) ratio of 10: 1. Target cell killing was assessed by quantification of LDH released into the cell supernatant by apoptotic / necrosis cells after 24 hours of incubation at 37 ° C., 5% CO ₂ (LDH Detection Kit, Roche Applied Science, # 11 644 793 001). Maximum lysis of target cells (= 100%) was achieved by incubation of target cells in 1% Triton X-100. Minimal lysis (= 0%) is relative to target cells co-incubated with effector cells that do not have bispecific constructs.

TCB-mediated activation of CD8 and CD4 T cells during T cell killing of target cells using antibodies that recognize the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). And evaluated by flow cytometry. After 48 hours of incubation, PBMCs were transferred to a round bottom 96-well plate, centrifuged at 350 xg for 5 minutes and washed twice with FACS buffer. Surface staining for CD4 APC (BioLegend, # 300514), CD8 FITC (BioLegend, # 344704), CD25 BV421 (BioLegend, # 302630) and CD69 PE (BioLegend, # 310906) was performed according to the supplier's instructions. Cells were washed twice with 150 μl / well FACS buffer and fixed at 4 ° C for 15 minutes using 100 μl / well fixed buffer (BD, # 554655). After centrifugation, the sample was resuspended in 200 μl / well FACS buffer. Samples were analyzed by BD FACS Fortessa.

Cytokine secretion in the supernatant was measured by flow cytometry using a cytometric bead array (CBA) according to the manufacturer's instructions, but only 25 μl supernatant and beads were used instead of 50 μl beads and sample. .. The following CBA kit (BD Biosciences) was used. CBA human interferon gamma (IFNγ) Flex Set, CBA human Granzyme ss Flex Set and CBA human TNF Flex Set. Samples were measured using BD FACS Canto II or BD FACS Fortessa and analysis was performed using Diva Software (BD Biosciences).

Example 9-Binding of a T cell bispecific antibody comprising an optimized CD3 binder to CD3 and EGFRvIII

Binding to recombinant CD3

Binding of EGFRvIII TCB to recombinant CD3 by SPR using a TCB with either an optimized (EGFRvIII TCB CD3 _opt ) or original (EGFRvIII TCB CD3 _orig ) CD3 binding sequence for the TYRP1 TCB above. The evaluation was performed as described in Example 5. The capture antibody is coupled to the sensor chip surface by direct immobilization of approximately 5200 resonant units (RU) at pH 5.0 using a standard amine coupling kit (GE Healthcare) and the TCB molecule is applied for 30 seconds. It was captured at 20 nM with a flow of 10 μl / min.

KD values for binding to human and cynomolgus monkey CD3 were determined for _TYRP1 TCB CD3 _opt as 30 nM and 20 nM, respectively, and were similar to those for TYRP1 TCB CD3 _orig (40 nM and 30 nM, respectively).

This indicates that under non-stress conditions, both TCBs, including either CD3 _opt or CD3 _orig , bind relatively well to recombinant CD3.

Binding of EGFRvIII TCBs to recombinant human CD3 was also assessed after 14 days of temperature stress at 37 ° C or 40 ° C using TCBs with either optimized or original CD3 binding sequences. .. The experiment was performed as described in Example 2 above, using TCB instead of IgG molecule.

The results of this experiment are shown in FIG.

As can be seen in FIG. 21, the TCB containing the optimized CD3 binder CD3 _opt is stressed (37 ° C, 2 at pH 7.4) as compared to the TCB containing the original CD3 binder CD3 _orig . After a week), it showed strongly improved binding to CD3. This result again confirms that the improved properties of the optimized CD3 binder (see Example 2) are maintained at the TCB level.

Binding to recombinant EGFRvIII

Binding of EGFRvIII TCB to recombinant EGFRvIII was evaluated by SPR.

SPR experiments were performed on Biacore T200 using HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 0.05% (v / v) detergent P20 (GE Healthcare)) as a running buffer. gone.

Anti-Fc antibody (GE Healthcare) was coupled directly onto the CM5 sensor chip at pH 5.0 using a standard amine coupling kit (GE Healthcare). EGFRvIII TCB (5 nM) was captured at a flow rate of 10 μl / min for 30 seconds. A 3-fold dilution series of EGFRvIII antigen was passed through a flow cell at 30 μl / min for 200 seconds and the associated phase was recorded. The dissociated phase was monitored for 300 seconds and triggered by switching from sample solution to HBS-EP. The chip surface was regenerated after each cycle using a single 20 μl / min injection of 3M MgCL ₂ for 30 seconds.

The antigen used comprises the extracellular domain of human EGFRvIII fused to the C-terminal Avi-tag and His-tag (EGFRvIII-ECD avihis; SEQ ID NO: 36).

The difference in volume index was corrected by subtracting the response obtained from the reference flow cell (TCB was not captured at all). The affinity constant (KD) was derived from the dynamic rate constant by fitting to a 1: 1 Langmuir bond using _BIAever software (GE Healthcare). The apparent _avidity constant KD was approximately determined by dynamic analysis using the 1: 1 binding fitting to this 2: 1 interaction via the rate constant.

The KD value (affinity) for binding to human _EGFRvIII was determined to be 6 nM for both EGFRvIII TCBs containing either CD3 _opt or CD3 _orig .

Binding of EGFRvIII TCBs to recombinant EGFRvIII was also assessed after 14 days of temperature stress at 37 ° C or 40 ° C using TCBs with either optimized or original CD3 binding sequences. The experiment was performed as described in Example 5 above, using EGFRvIII-ECD avihis as the antigen.

The results of this experiment are shown in FIG. These support that binding to human EGFRvIII for both TCBs is unaffected by stress conditions.

Binding to CD3 on Jurkat cells

For EGFRvIII TCBs containing the optimized CD3 binder "CD3 _opt " or the original CD3 binder "CD3 _orig " as described in Example 2 above, binding to CD3 on the human reporter T cell line Jurkat NFAT is by FACS. It has been determined.

As shown in FIG. 23, TCBs containing either the optimized CD3 binder "CD3 _opt " or the original CD3 binder "CD3 _orig " bound relatively well to CD3 on Jurkat cells.

Binding to EGFRvIII on U87MG-EGFRvIII cells

Binding to EGFRvIII on the human glioblastoma cell line U87MG-EGFRvIII for EGFRvIII TCBs containing EGFRvIII binder P063.056 with either CD3 _opt or CD3 _orig or EGFRvIII clone P056.021 with CD3 _orig by FACS. It was measured. EGFRvIII binder P063.056 was also included in the IgG format.

As shown in FIG. 24, all three TCBs bind with high affinity to EGFRvIII expressed on U87MG-EGFRvIII cells, and the binding is not compromised by conversion to the TCB format.

Example 10-Functional activity of T cell bispecific antibodies comprising optimized CD3 binders

CD3 activation

EGFRvIII TCB containing the selected EGFRvIII binder (P063.056) and either the optimized CD3 binder CD3 _opt or the original CD3 binder CD3 _orig , EGFRvIII positive glioblastoma cells DK-MG, U87MG-huEGFRvIII and EGFRwt positive MKN45. In the presence of cells, it was tested with a Jurkat NFAT reporter cell assay as described in Example 8 above.

As seen in the case of IgG (Example 3), both TCBs containing either CD3 _opt or CD3 _org have similar functional activity on Jurkat NFAT reporter cells and are concentration dependent embodiments. Induced CD3 activation in (Fig. 20).

Target cell killing

The EGFRvIII TCB containing either the selected EGFRvIII binder ( _P063.056 ) and the optimized CD3 binder CD3 _opt or the original CD3 binder CD3 org was performed above in the presence of the glioblastoma cell lines U87MG-EGFRvIII and PBMC. Tumor cell killing tests as described in Example 8 were compared to EGFRvIII TCBs containing parent EGFRvIII binder P056.021 and CD3 binder CD3 _orig . As seen on Jurkat NFAT reporter cells, the functional activity of TCB with either CD3 _opt or CD3 _orig is similar with respect to the induction of tumor cell lysis and activation of CD4 and CD8 T cells as measured by CD69 upregulation. (Fig. 25).

In addition, the functional activity of EGFRvIII TCB with EGFRvIII binder P063.056 and optimized CD3 binder CD3 _opt in "2 + 1 format" (as shown in FIG. 6), the same EGFRvIII and CD3 binder in "1 + 1 head-to-tail format". ”(Graphically shown in FIG. 1G) compared to EGFRvIII TCB. These two EGFRvIII TCBs were tested with the glioblastoma cell line U87MG-EGFRvIII in the Jurkat NFAT reporter cell assay and in the tumor cell killing assay as described in Example 8. The 2 + 1 format EGFRvIII TCB is excellent in both CD3 activation measured in the Jurkat NFAT reporter cell assay (FIG. 26) and inducing tumor cell killing and T cell activation in the killing assay using PBMC (FIG. 27). Had functional activity.

Example 11-Functional characterization of T cell bispecific antibodies comprising optimized CD3 binders

T cell proliferation and activation by EGFRvIII TCB

Functional activity of EGFRvIII TCB, including the selected EGFR binder ( _P063.056 ) and either the optimized CD3 binder CD3 _opt or the original CD3 binder CD3 org, in a T cell proliferation assay on U87MG-EGFRvIII cells, parental EGFRvIII. Comparison was made with EGFRvIII TCB containing binder P056.021 and CD3 binder CD3 _orig (FIG. 28). All three TCBs induced strong proliferation and activation of CD4 T cells and CD8 T cells. The P063.056 EGFRvIII TCB with CD3 _opt had higher activity than the other two EGFRvIII TCBs.

Tumor cytolysis by EGFRvIII TCB

Next, the EGFRvIII TCB containing the selected EGFR binder (P063.056) and the optimized CD3 binder CD3 _opt , and the EGFRvIII TCB containing the parent EGFRvIII binder P056.021 and the CD3 binder CD3 _orig were co-cultured with DK-MG cells. The PBMC was tested in a tumor cytolysis assay (FIG. 29). In this assay, apart from tumor cell lysis, T cell activation and cytokine release were measured as additional readouts. As has already been seen, P063.056 EGFRvIII TCB with CD3 _opt has higher activity than P056.021 EGFRvIII TCB with CD3 _orig in tumor cytolysis, T cell activation and release of IFNγ and TNFα. Was there.

T cell activation and tumor cell lysis by TYRP1 TCB

The functional properties of TYRP1 TCB that induce cytokine release were tested by co-culture of the primary melanoma cell line M150543 with PBMCs isolated from healthy donors. T cell-mediated tumor cytolysis via TYRP1 TCB was analyzed 24 and 48 hours after treatment (FIG. 30). Release of IFNγ and TNFα into the supernatant, as well as CD4 and CD8 T cell activation, was analyzed 48 hours after treatment. TYRP1 TCB was already able to induce strong tumor cytolysis after 24 hours. This was accompanied by upregulation of CD25 and strong activation of CD4 and CD8 T cells as measured by significant release of IFNγ and TNFα.

(Method)

PBMC isolation

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of fresh blood from healthy human donors. Fresh blood was diluted with sterile PBS and laminated on a Histopaque gradation (Sigma, # H8889). After centrifugation (450 xg, 30 minutes, room temperature), plasma above the PBMC-containing interface was discarded and PBMC was transferred to a new falcon tube, followed by filling with 50 ml of PBS. The mixture was centrifuged (400 xg, 10 minutes, room temperature), the supernatant was discarded, and the PBMC pellets were washed twice with sterile PBS (centrifugation step 350 xg, 10 minutes). The resulting PBMC population was automatically counted (ViCell) in RPMI1640 medium containing 10% FCS and 1% GlutaMAX at 37 ° C., 5% CO ₂ until further use in the cell incubator. Stored (not longer than 24 hours) or frozen in liquid nitrogen until further use. The day before use, frozen PBMCs were thawed and cultured overnight at 37 ° C in medium.

T cell proliferation
Briefly, target cells were harvested, counted and washed twice with PBS. The cells were resuspended in PBS with 5 mio cells per ml. Cells were stained with the cell proliferation dye eFluor670 (eBioscience, # 65-0840-85) at a final concentration of 5 μM at 37 ° C. for 10 minutes. To stop the staining reaction, 4 volumes of cold complete cell culture medium were added to the cell suspension, incubated for 5 minutes at 4 ° C., and then washed 3 times with medium. Labeled target cells were counted and adjusted to 0.1 mio cells per ml in RPMI 1640, 10% FCS and 1% GlutaMax. 10,000 target cells per well were seeded on 96-well plates. The treatment agent was then added at the indicated concentration and finally 100,000 PBMC isolated from a healthy donor was added per well. The cells were incubated at 37 ° C for 5 days, then PBMCs were harvested and collected from CD3 BUV395 (BioLegend, # 563548), CD4 PE (BioLegend, # 300508), CD8 APC (BioLegend, # 344722), CD25 PE / Cy7. It was stained with (BioLegend, # 302612). Proliferation is determined by dilution of the eFluor670 dye in CD4 T cells and CD8 T cells as measured by flow cytometry (FACS Fortessa, BD Biosciences), and activation of CD4 and CD8 T cells is measured by CD25 upregulation. It was decided by that.

T cell-mediated tumor cell killing

Target cells were harvested with trypsin / EDTA, washed and plated using a flat bottom 96-well plate at a density of 30,000 cells / well. The cells were left attached overnight. Antibodies were added in triplicate at the indicated concentrations for the killing assay. PBMCs were added to target cells at a final effector to target (E: T) ratio of 10: 1. Target cell killing was assessed by quantification of LDH released into the cell supernatant by apoptotic / necrosis cells after 24 hours of incubation at 37 ° C., 5% CO ₂ (LDH Detection Kit, Roche Applied Science, # 11 644 793 001). Maximum lysis of target cells (= 100%) was achieved by incubation of target cells in 1% Triton X-100. Minimal lysis (= 0%) is relative to target cells co-incubated with effector cells that do not have bispecific constructs.

T cell activation

TCB-mediated activation of CD8 and CD4 T cells during T cell killing of target cells using antibodies that recognize the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). And evaluated by flow cytometry. After 48 hours of incubation, PBMCs were transferred to a round bottom 96-well plate, centrifuged at 350 xg for 5 minutes and washed twice with FACS buffer. Surface staining for CD4 APC (BioLegend, # 300514), CD8 FITC (# 344704, BioLegend), CD25 BV421 (BioLegend, # 302630) and CD69 PE (BioLegend, # 310906) was performed according to the supplier's instructions. Cells were washed twice with 150 μl / well FACS buffer and fixed at 4 ° C for 15 minutes using 100 μl / well fixed buffer (BD, # 554655). After centrifugation, the sample was resuspended in 200 μl / well FACS buffer. Samples were analyzed by BD FACS Fortessa.

Cytokine secretion

Cytokine secretion in the supernatant was measured by flow cytometry using a cytometric bead array (CBA) according to the manufacturer's instructions, but only 25 μl supernatant and beads were used instead of 50 μl beads and sample. .. The following CBA kit (BD Biosciences) was used. CBA human interferon gamma (IFNγ) Flex Set and CBA human TNF Flex Set. Samples were measured using BD FACS Canto II or BD FACS Fortessa and analysis was performed using Diva Software (BD Biosciences).

Example 13-In vivo efficacy of a T cell bispecific antibody comprising an optimized CD3 binder

TYRP1 TCB (including the optimized CD3 binder identified in Example 1) was tested for its antitumor efficacy in a human tumor cell line xenograft mouse model, IGR-1 melanoma xenograft model.

IGR-1 cells (human melanoma) were cultured in DMEM medium containing 10% FCS (Sigma). Cells were cultured at 37 ° C. in a water saturated atmosphere at 5% CO ₂ . Subculture 6 was used for transplantation. The cell viability was 96.7%. 2 × 10 ⁶ cells per animal were injected subcutaneously into the flanks of mice in 100 μl RPMI cell medium (Gibco) using a 1 ml tuberculin syringe (BD Biosciences, Germany).

Fully humanized NSG female mice (Roche Glycart AG, Switzerland) are subjected to specific pathogen-free conditions in a daily cycle of 12 hours light / 12 hours dark according to the entrusted guidelines (GV-Solas; Ferrasa; TierschG). Maintained in. This experimental research protocol was investigated and approved by the local government (ZH223 / 2017). Continuous health monitoring was performed on a regular basis.

Mice were injected subcutaneously with 2 × ¹⁰⁶ IGR-1 cells on day 0 in the study, randomized and weighed. Twenty days after tumor cell injection (tumor volume> 200 mm ³ ), mice were intravenously infused with 10 μg (0.5 mg / kg) TYRP1 TCB twice weekly for 5 weeks. All mice were intravenously infused with 200 μl of the appropriate solution. Mice in the vehicle group were infused with histidine buffer and mice in the treatment group were infused with the TYRP1 TCB construct. The stock solution was diluted with histidine buffer, if necessary, to obtain the appropriate amount of antibody per 200 μl. Tumor size was measured three times a week with calipers and plotted with GrahPad Prism software as a volume +/- SEM of mm ³ . Statistical analysis was performed with JMP12 software.

FIG. 31 shows that TYRP1 TCB mediated significant efficacy in suppressing tumor growth compared to the vehicle group (68% TGI, p = 0.0058 ^* ).

EGFRvIII TCB (including the optimized CD3 binder identified in Example 1) was similarly tested for its antitumor efficacy in a human tumor cell line xenograft mouse model, U87-EGFRvIII glioblastoma xenograft model. ..

U87 cells (human glioblastoma) were first obtained from ATCC (Manassas, USA) and stably transfected to express human EGFRvIII protein (Roche Glycart AG, Switzerland). After expansion, the cells were deposited in the Roche Glycart internal cell bank. U87-EGFRvIII cell lines were cultured in DMEM medium containing 10% FCS (Sigma) and 0.5 μg / ml puromycin (Invitrogen). Cells were cultured at 37 ° C. in a water saturated atmosphere at 5% CO ₂ . Subculture 8 was used for transplantation. The cell viability was 94.7%. ⁵ × 105 cells per animal were injected subcutaneously into the flanks of mice in 100 μl RPMI cell medium (Gibco) using a 1 ml tuberculin syringe (BD Biosciences, Germany).

Fully humanized NSG female mice (Roche Glycart AG, Switzerland) are subjected to specific pathogen-free conditions in a daily cycle of 12 hours light / 12 hours dark according to the entrusted guidelines (GV-Solas; Ferrasa; TierschG). Maintained in. This experimental research protocol was investigated and approved by the local government (ZH223 / 2017). Continuous health monitoring was performed on a regular basis.

Mice were injected subcutaneously with ⁵ × 105 U87-EGFRvIII cells on day 0 in the study, randomized and weighed. Two weeks after tumor cell injection (tumor volume> 200 mm ³ ), mice were intravenously infused with 10 μg (0.5 mg / kg) EGFRvIII TCB twice weekly for 3 weeks. All mice were intravenously infused with 200 μl of the appropriate solution. Mice in the vehicle group were infused with histidine buffer and mice in the treatment group were infused with the EGFRvIII TCB construct. The stock solution was diluted with histidine buffer, if necessary, to obtain the appropriate amount of antibody per 200 μl. Tumor size was measured three times a week with calipers and plotted with GrahPad Prism software as a volume +/- SEM of mm ³ .

FIG. 32 shows that EGFRvIII TCB mediated significant efficacy in controlling tumor growth and all mice achieved complete remission.

Example 12-PK study with EGFRvIII TCB in mice

The pharmacokinetics (PK) of EGFRvIII TCB, including optimized CD3 binder, CD3 _opt , was studied following intravenous bolus administration at 1 mg / kg to human FcRn transgenic (line32, homo) and NOD-SCID mice. A series of blood microsamples were taken from human FcRn transgenic (tg) mice and NOD-SCID mice for up to 672 hours (9 samples per mouse from 5 minutes to 672 hours after dosing). Samples of mouse serum treated with EGFRvIII TCB were analyzed under non-GLP conditions using a specific enzyme-linked immunosorbent assay (ELISA). Capture of EGFRvIII TCB was performed using a biotinylated EGFRvIII antigen (huEGFRvIII his biotin) on a streptavidin-coated microtiter plate (SA-MTP). Bound EGFRvIII TCB was detected using a digoxigenin-labeled monoclonal antibody against human IgG1 Fc (PGLALA) (see Example 3), followed by addition of anti-digoxigenin-POD secondary detection antibody. The signal was generated by the addition of a peroxidase substrate (ABTS). The calibration range was 2.35 ng / ml to 150 ng / ml, and 2.5 ng / ml was the lower limit of quantification (LLOQ).

The results of this study are shown in Table 7. The PK profile of EGFRvIII TCB is within expectations for both tested mouse strains. This indicates that manipulation of the CDRs of the CD3 binder did not cause other sequence liability that would affect antibody clearance.

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Although the above invention has been described in some detail by description and examples for the purpose of clarifying understanding, the description and examples should not be construed as limiting the scope of the present invention. All patent and scientific literature disclosures cited herein are expressly incorporated by reference in their entirety.

Claims (41)

An antibody that binds to CD3 and EGFRvIII.
(A) A first antigen-binding domain that binds to CD3, including the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11, and (b) a second antigen-binding domain that binds to EGFRvIII, optionally. , An antibody comprising a third antigen binding domain that binds to EGFRvIII. The antibody according to claim 1, wherein the first antigen-binding domain is a Fab molecule. The antibody according to claim 1 or 2, which comprises an Fc domain composed of the first and second subunits. The antibody according to any one of claims 1 to 3, wherein the second antigen-binding domain and / or, if present, the third antigen-binding domain is a Fab molecule. The first aspect of any one of claims 1 to 4, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain or the constant domains CL and CH1 are replaced with each other. Antigen. The antibody according to any one of claims 1 to 5, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other. The antibody according to any one of claims 1 to 6, wherein the second antigen-binding domain and, if present, the third antigen-binding domain are conventional Fab molecules. The second antigen-binding domain and, if present, the third antigen-binding domain is a Fab molecule, and in the constant domain CL, the amino acid at position 124 independently contains lysine (K) and arginine (R). ) Or histidine (H) (numbering by Kabat), the amino acid at position 123 is independently replaced by lysine (K), arginine (R) or histidine (H) (numbering by Kabat), constant domain. In CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by the Kabat EU index) and the amino acid at position 213 is independently glutamic acid (E) or aspartic acid (E) or aspartic acid. The antibody according to any one of claims 1 to 7, which is substituted with acid (D) (numbering according to Kabat EU index). The antibody according to any one of claims 1 to 8, wherein the first antigen-binding domain and the second antigen-binding domain are optionally fused to each other via a peptide linker. The first antigen-binding domain and the second antigen-binding domain are Fab molecules, respectively, and (i) the second antigen-binding domain is the C-terminal of the Fab heavy chain, and the first antigen-binding domain. Is fused to the N-terminus of the Fab heavy chain, or (ii) the first antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain. The antibody according to any one of claims 1 to 9, which is one of the above-mentioned antibodies. The first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain, if present, are Fab molecules, respectively, and the antibody comprises the first and second subunits. (I) The second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain. The binding domain is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, or (ii) the first antigen binding domain is the C-terminus of the Fab heavy chain. The second antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain and the N-terminus of the first subunit of the Fc domain. The third antigen-binding domain is at the C-terminus of the Fab heavy chain, at the C-terminus of the Fc domain, at the N-terminus of the second subunit of the Fc domain. The antibody according to any one of claims 1 to 10, which is fused. The antibody comprises the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, the Fc domain, and optionally one or more peptide linkers. The antigen-binding domain is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain at the C-terminus of the Fab heavy chain, and the first antigen-binding domain is at the C-terminus of the Fab heavy chain, said Fc. The third antigen-binding domain is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. The antibody according to claim 11. The antibody according to any one of claims 3 to 12, wherein the Fc domain is an IgG Fc domain. The antibody according to any one of claims 3 to 13, wherein the Fc domain is the Fc domain of IgG ₁ . The antibody according to any one of claims 3 to 14, wherein the Fc domain is a human Fc domain. The antibody according to any _one of claims 3 to 15, wherein the Fc domain is the Fc domain of human IgG1. The antibody according to any one of claims 3 to 16, wherein the Fc domain comprises a modification that promotes association of the first subunit and the second subunit of the Fc domain. In the CH3 domain of the first subunit of the Fc domain, the amino acid residue replaces the amino acid residue with the larger side chain volume, thereby the second subunit within the CH3 domain of the first subunit. In the CH3 domain of the second subunit of the Fc domain, the amino acid residue replaces the amino acid residue with the smaller side chain volume, which creates a protrusion that can be located in the cavity within the CH3 domain of the Fc domain. The antibody according to any one of claims 3 to 17, which creates a cavity in the CH3 domain of the second subunit in which a protrusion in the CH3 domain of the first subunit can be located. .. In the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue. (Y407V) (numbering by Kabat EU index), the antibody according to any one of claims 3 to 18. In the second subunit of the Fc domain, the threonine residue at position 366 is additionally replaced with the serine residue (T366S) and the leucine residue at position 368 is replaced with the alanine residue (L368A) ( The antibody according to claim 19), numbered by the Kabat EU index. In the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C). The antibody according to claim 19 or 20, wherein in the second subunit of the Fc domain, the tyrosine residue at position 349 is additionally replaced with a cysteine residue (Y349C) (numbering by Kabat EU index). The antibody according to any one of claims 3 to 21, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to the Fc receptor and / or reduce effector function. The antibody according to any one of claims 3 to 22, wherein the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering by Kabat EU index). The antibody according to any one of claims 3 to 23, wherein each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (numbering according to the Kabat EU index). The second antigen-binding domain and, if present, the third antigen-binding domain are VH containing HCDR1 of SEQ ID NO: 85, HCDR2 of SEQ ID NO: 86, and HCDR3 of SEQ ID NO: 87, and LCDR1 of SEQ ID NO: 89. , The antibody of any one of claims 1-24, comprising VL comprising LCDR2 of SEQ ID NO: 90 and LCDR3 of SEQ ID NO: 91. The second antigen-binding domain and, if present, the third antigen-binding domain are at least about 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence of SEQ ID NO: 88. A VH containing the same amino acid sequence and / or a VL containing at least about 95%, 96%, 97%, 98%, 99% or 100% of the same amino acid sequence with respect to the amino acid sequence of SEQ ID NO: 92. The antibody according to claim 25 . A polypeptide containing at least 95%, 96%, 97%, 98%, 99% or 100% of the same amino acid sequence in the sequence of SEQ ID NO: 109, and at least 95%, 96%, 97%, 98 in the sequence of SEQ ID NO: 110. %, 99% or 100% of the polypeptide containing the same amino acid sequence, a polypeptide containing at least 95%, 96%, 97%, 98%, 99% or 100% of the same amino acid sequence in the sequence of SEQ ID NO: 111 (particularly). 25 or 26 , comprising two polypeptides), and a polypeptide comprising at least 95%, 96%, 97%, 98%, 99% or 100% of the same amino acid sequence in the sequence of SEQ ID NO: 27. The antibody described. An isolated polynucleotide encoding the antibody according to any one of claims 1 to 27. A host cell comprising the isolated polynucleotide according to claim 28. A method for producing an antibody that binds to CD3 and EGFRvIII , wherein (a) the step of culturing the host cell according to claim 29 under conditions suitable for expression of the antibody, and optionally (b). A method comprising the steps of recovering the antibody. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 27 and a pharmaceutically acceptable carrier. A pharmaceutical agent for treating a disease, which comprises the antibody according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 31. The medicine according to claim 32, wherein the disease is cancer. 33. The apparatus according to claim 33, wherein the cancer is a cancer expressing EGFRvIII. The medicine according to claim 33 or 34, wherein the cancer is a brain tumor. The drug according to any one of claims 33 to 35, wherein the cancer is glioblastoma. Use of the antibody according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 31 in the manufacture of a pharmaceutical. Use of the antibody according to any one of claims 1 to 27 or the pharmaceutical composition according to claim 31 in the manufacture of a pharmaceutical agent for treating cancer. 38. The use according to claim 38, wherein the cancer is a cancer expressing EGFRvIII. The use according to claim 38 or 39, wherein the cancer is a brain tumor. The use according to any one of claims 38-40, wherein the cancer is glioblastoma.

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