Antibodies that bind to CD3 and FolR1

Abstract

The present invention relates generally to bispecific antibodies that bind to CD3 and folate receptor 1 (FolR1), for example to activate T cells. The invention further relates to polynucleotides encoding such antibodies, as well as vectors and host cells comprising such polynucleotides. The invention further relates to methods of producing the antibodies and their use in the treatment of disease.

Description

[Technical Field]

[0001] The present invention generally relates to bispecific antibodies that bind to CD3 and folate receptor 1 (FolR1), for example, to activate T cells. Furthermore, the present invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells containing such polynucleotides. The present invention further relates to methods for producing antibodies and methods for using them in the treatment of diseases. [Background technology]

[0002] The selective destruction of individual cells or specific cell types is often desirable in a variety of clinical settings. For example, a primary goal of cancer treatment is to specifically destroy tumor cells while leaving healthy cells and tissues intact.

[0003] An attractive way to achieve this is to induce an immune response against the tumor, allowing immune effector cells such as natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) to attack and destroy the tumor cells. While CTLs constitute the most potent effector cells of the immune system, they cannot be activated by the effector mechanism mediated by the Fc domain of conventional therapeutic antibodies.

[0004] In this regard, bispecific antibodies designed to bind to the surface antigen of target cells with one "arm" and to the activation-invariant component of the T cell receptor (TCR) complex with the second "arm" have recently attracted attention. When such antibodies bind to both targets simultaneously, a transient interaction occurs between the target cells and T cells, activating cytotoxic T cells, and subsequently lysing the target cells. Thus, the immune response is redirected to the target cells, independent of the presentation of peptide antigens by the target cells or the T cell specificity associated with the normal MHC restriction activation of CTLs. In this context, it is important that CTLs are activated only when the target cells present bispecific antibodies to them, i.e., when immunological synapses are mimicked. Bispecific antibodies that do not require lymphocyte conditioning or co-stimulation are particularly desirable to induce efficient lysis of target cells.

[0005] CD3 is a widely studied drug target. Monoclonal antibodies targeting CD3 have been used as immunosuppressive therapies in the treatment of autoimmune diseases such as type 1 diabetes and graft rejection. The CD3 antibody muromonab-CD3 (OKT3) was the first monoclonal antibody approved for clinical use in humans in 1985.

[0006] Recent applications of CD3 antibodies take the form of bispecific antibodies that bind to CD3 on one hand and to tumor cell antigens on the other. When such antibodies bind to both targets simultaneously, a transient interaction occurs between the target cells and T cells, activating cytotoxic T cells, which then lyse the target cells.

[0007] FOLR1 is expressed in epithelial tumor cells of various origins, such as ovarian cancer, lung cancer, breast cancer, kidney cancer, colorectal cancer, and endometrial cancer. Several approaches targeting FOLR1 with therapeutic antibodies such as farletuzumab, antibody-drug conjugates, or adoptive T-cell therapy for tumor imaging have been described (Kandalaft et al., J Transl Med. 2012 Aug 3;10:157. doi:10.1186 / 1479-5876-10-157; van Dam et al., Nat Med. 2011 Sep 18;17(10):1315-9. doi: 10.1038 / nm.2472; Cliftonet al., Hum Vaccin. 2011 Feb;7(2):183-90. Epub 2011 Feb 1; Kelemen et al., Int J Cancer. 2006 Jul 15;119(2):243-50; Vaitilingam et al., J Nucl Med. 2012 Jul;53(7);Teng et al., 2012 Aug;9(8):901-8. doi:10.1517 / 17425247.2012.694863. Epub 2012 Jun 5. Several attempts have been made to target folate receptor-positive tumors using constructs that target folate receptors and CD3 (Kranz et al., Proc Natl Acad Sci US A. Sep 26, 1995;92(20):9057-9061;Roy et al., Adv Drug Deliv Rev. 2004 Apr 29;56(8):1219-31;Huiting Cui et al Biol Chem. Aug 17, 2012;287(34):28206-28214;Lamers et al., Int. J. Cancer. 60(4):450 (1995); Thompson et al., MAbs. 2009 Jul-Aug;1(4):348-56. Epub 2009 Jul 19;Mezzanzanca et al., Int. J. Cancer, 41, 609-615(1988). However, previous approaches have many shortcomings.The molecules used to date required chemical crosslinking, making them difficult to produce easily and reliably. Similarly, hybrid molecules cannot be produced on the same scale as human proteins, and require the use of rat, mouse, or other proteins that are highly immunogenic when administered to humans and therefore have limited therapeutic value. Furthermore, many existing molecules retained FcgR bonds.

[0008] More recently, WO2016 / 079076 describes a T cell-activating bispecific antigen-binding molecule that targets CD3 and FolR1.

[0009] For therapeutic purposes, a crucial requirement that antibodies must meet is sufficient stability both in vitro (for drug storage) and in vivo (after administration to the patient).

[0010] Modifications such as deamidation of asparagine are typical degradations of recombinant antibodies and can affect both in vitro stability and in vivo biological function.

[0011] Given the tremendous therapeutic potential of antibodies, particularly bispecific antibodies against T cell activation, there is a need for bispecific CD3 / FolR1 antibodies with optimized properties. [Overview of the project]

[0012] The present invention provides antibodies, including multispecific (e.g., bispecific) antibodies, that bind to CD3, are resistant to degradation by, for example, asparagine deamidation, and are therefore particularly stable when required for therapeutic purposes. The provided (multispecific) antibodies further combine excellent efficacy and productivity with low toxicity and favorable pharmacokinetic properties.

[0013] As shown herein, the CD3-binding antibodies provided by the present invention, including multispecific antibodies, retain approximately 90% or more of their binding activity to CD3 after two weeks at pH 7.4 and 37°C, compared to the binding activity after two weeks at pH 6 and -80°C, as determined by surface plasmon resonance (SPR).

[0014] In certain embodiments, the present invention provides a bispecific antibody that binds to CD3 and folate receptor 1 (FolR1) and, as determined by surface plasmon resonance (SPR), retains more than 90% of the binding activity to CD3 after 2 weeks at pH 7.4 and 37°C compared to the binding activity after 2 weeks at pH 6 and -80°C.

[0015] In one embodiment, a bispecific antibody that binds to CD3 and folate receptor 1 (FolR1) is provided, and the bispecific antibody is (i) A first antigen-binding domain that can specifically bind to CD3, comprising a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 5, and a light chain variable region (VL) including light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10, (ii) A second antigen-binding domain that can specifically bind to FolR1 and Includes.

[0016] In one embodiment, a bispecific antibody is provided in which the VH of the first antigen-binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and / or the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11.

[0017] In one embodiment, the bispecific antibody binds to CD3 and FolR1, and the bispecific antibody is (i) A first antigen-binding domain that can specifically bind to CD3, comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11, (ii) A second antigen-binding domain that can specifically bind to FolR1 and Includes.

[0018] In one embodiment, the first antigen-binding domain is a Fab molecule.

[0019] In one embodiment, the bispecific antibody includes an Fc domain composed of a first and a second subunit.

[0020] In one embodiment, the bispecific antibody includes a third antigen-binding domain that can specifically bind to FolR1.

[0021] In one embodiment, the second and / or third antigen-binding domain, if present, is a Fab molecule.

[0022] In one embodiment, the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain, particularly the variable domains VL and VH, are substituted for each other.

[0023] In one embodiment, the second and, if present, the third antigen-binding domain is a conventional Fab molecule.

[0024] In one embodiment, the second and, if present, third antigen-binding domains are Fab molecules, and in the constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), the bispecific antibody according to any one of claims 6 to 9.

[0025] In one embodiment, the first and second antigen-binding domains are optionally fused to each other via a peptide linker.

[0026] In one embodiment, the first and second antigen-binding domains are each a Fab molecule, and (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain of the first antigen-binding domain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, or (ii) 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.

[0027] In one embodiment, the first, second, and, if present, third antigen-binding domains are each Fab molecules, and the bispecific antibody includes an Fc domain composed of first and second subunits, wherein (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding domain, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding domain, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and, if present, the 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.

[0028] In one embodiment, the Fc domain is IgG, particularly the IgG1 Fc domain.

[0029] In one embodiment, the Fc domain is a human Fc domain.

[0030] In one embodiment, Fc includes modifications that facilitate the association of the first and second subunits of the Fc domain.

[0031] In one embodiment, the Fc domain includes one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

[0032] In one embodiment, the second and, if present, third antigen-binding domains include a VH comprising HCDR1 of SEQ ID NO: 124, HCDR2 of SEQ ID NO: 125, and HCDR3 of SEQ ID NO: 126, and a VL comprising LCDR1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10.

[0033] In one embodiment, the provided is a bispecific antibody as described herein, comprising a second, VH, which, if present, has an amino acid sequence identical at least about 95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 123, and / or VL, which has an amino acid sequence identical at least about 95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 11.

[0034] In one embodiment, isolated polynucleotides encoding the bispecific antibody of the present invention are provided.

[0035] In one embodiment, a host cell containing isolated polynucleotides is provided.

[0036] In one embodiment, a method is provided for producing a bispecific antibody that binds to CD3 and FolR1, comprising (a) culturing the host cells described in claim 21 under conditions suitable for the expression of a bispecific antibody, and (b) optionally recovering the bispecific antibody.

[0037] In one embodiment, the provided antibody is a bispecific antibody that binds to CD3 and FolR1, produced by the method described herein.

[0038] In one embodiment, a pharmaceutical composition comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier is provided.

[0039] In one embodiment, a bispecific antibody or a pharmaceutical composition of the present invention for use as a pharmaceutical is provided.

[0040] In one embodiment, a bispecific antibody or a pharmaceutical composition of the present invention is provided for use in the treatment of cancer.

[0041] In one embodiment, the use of the bispecific antibody or pharmaceutical composition of the present invention in the manufacture of a drug is provided.

[0042] In one embodiment, the use of the bispecific antibody or pharmaceutical composition of the present invention in the manufacture of a pharmaceutical product for the treatment of cancer is provided.

[0043] In one embodiment, a method is provided for treating a disease in an individual, comprising administering an effective amount of the bispecific antibody or the pharmaceutical composition of the present invention to the individual.

[0044] In one aspect, the disease is cancer. [Brief explanation of the drawing]

[0045] [Figure 1A-F] Exemplary configurations of the (multispecific) antibody of the present invention. (A, D) Diagram of the "1+1 CrossMab" molecule. (B, E) Diagram of the "2+1 IgG Crossfab" molecule with the order of the Crossfab and Fab components reversed ("inverted"). (C, F) Diagram of the "2+1 IgG Crossfab" molecule. [Figure 1G-N] Exemplary configurations of the (multispecific) antibody of the present invention. (G, K) Diagram of a "1+1 IgG Crossfab" molecule with alternating Crossfab and Fab components ("inverted"). (H, L) Diagram of a "1+1 IgG Crossfab" molecule. (I, M) Diagram of a "2+1 IgG Crossfab" molecule with two CrossFabs. (J, N) Diagram of a "2+1 IgG Crossfab" molecule containing two CrossFabs and alternating Crossfab and Fab components ("inverted"). [Figure 10-V]Exemplary configuration of the (multispecific) antibody of the present invention. (O, S) Diagram of the "Fab-Crossfab" molecule. (P, T) Diagram of the "Crossfab-Fab" molecule. (Q, U) Diagram of the "(Fab)2-Crossfab" molecule. (R, V) Diagram of the "Crossfab-(Fab)2" molecule. [Figure 1W-Z] Exemplary configurations of the (multispecific) antibody of the present invention. (W, Y) Diagram of the "Fab-(Crossfab)2" molecule. (X, Z) Diagram of the "(Crossfab)2-Fab" molecule. Black circles: Optional modification of the Fc domain to promote heterodimerization. ++, --: Reversely charged amino acids optionally introduced into the CH1 and CL domains. The Crossfab molecule is depicted to include the exchange of the VH and VL regions, but in embodiments where charge modifications are not introduced into the CH1 and CL domains, it may instead include the exchange of the CH1 and CL domains. [Figure 2] The relative binding activity (IgG format) of the original and optimized CD3 binders, CD3orig and CD3opt, to recombinant CD3 was measured by SPR under stress-free conditions after 14 days at 40°C, pH 6, or after 14 days at 37°C, pH 7.4. [Figure 3] Binding of original and optimized CD3 binders, CD3orig and CD3opt, to Jurkat NFAT cells was measured by flow cytometry (IgG format). Antibodies bound to Jurkat NFAT cells were detected with fluorescently labeled anti-human Fc-specific secondary antibodies. [Figure 4] Schematic diagram of the CD3 activation assay used in Example 3. [Figure 5] Jurkat NFAT activation using original and optimized CD3 binders, CD3orig and CD3opt (IgG format). Jurkat NFAT reporter cells were co-cultured with anti-PGLALA expressing CHO (CHO-PGLALA) cells in the presence of CD3orig or CD3opt IgG PGLALA, or CD3opt IgG wt as a negative control. CD3 activation was quantified by measuring luminescence after 24 hours. [Figure 6] Schematic diagram of the T cell bispecific antibody (TCB) molecules prepared in the examples. All TCB antibody molecules tested were generated as "2+1 IgG CrossFab, inverted" with charge modifications (VH / VL exchange of CD3 binder, charge modification of target antigen binder, EE=147E, 213E; RK=123R, 124K). [Figure 7] The relative binding activity of TYRP1 TCBs containing the original or optimized CD3 binder, CD3orig or CD3opt, to recombinant CD3, measured by SPR under stress-free conditions after 14 days at 40°C, pH 6, or after 14 days at 37°C, pH 7.4. [Figure 8] The relative binding activity of TYRP1 TCBs containing the original or optimized CD3 binder, CD3orig or CD3opt, or the corresponding TYRP1 IgG, to recombinant TYRP1, measured by SPR under stress-free conditions after 14 days at 40°C, pH 6, or after 14 days at 37°C, pH 7.4. [Figure 9] Binding of TYRP1 TCBs containing original and optimized CD3 binders, CD3orig or CD3opt, to Jurkat NFAT cells was measured by flow cytometry. Antibodies bound to the TCBs were detected with fluorescently labeled anti-human Fc-specific secondary antibodies. [Figure 10] Jurkat NFAT activation by TYRP1 TCBs containing original or optimized CD3 binders. Jurkat NFAT reporter cells were co-cultured with melanoma cell line M150543 in the presence of TYRP1 TCB CD3orig or TYRP1 TCB D3opt. CD3 activation in the presence of TCBs was quantified by measuring luminescence after 24 hours. [Figure 11A-B]Figures 11A and B show tumor cell killing and T cell activation by TYRP1 TCB containing the original or optimized CD3 binder. Killing of melanoma cell line M150543 by treatment with TYRP1 TCB CD3orig and TYRP1 TCB CD3opt by PBMCs from three different healthy donors (AF: Donor 1, GL: Donor 2, MR: Donor 3) was determined by LDH release at 24 hours (A, G, M) and 48 hours (B, H, N). In parallel, T cells in PBMCs upregulated by CD25 (C, E, I, K, O, Q) and CD69 (D, F, J, L, P, R) relative to CD8 (E, F, K, L, Q, R) and CD4 (C, D, I, J, O, P) were measured by flow cytometry as a marker of T cell activation at 48 hours. [Figure 11C-F] Figures 11C-F show tumor cell killing and T cell activation by TYRP1 TCB containing the original or optimized CD3 binder. Killing of melanoma cell line M150543 by treatment with TYRP1 TCB CD3orig and TYRP1 TCB CD3opt by PBMCs from three different healthy donors (AF: Donor 1, GL: Donor 2, MR: Donor 3) was determined by LDH release at 24 hours (A, G, M) and 48 hours (B, H, N). In parallel, T cells in PBMCs upregulated by CD25 (C, E, I, K, O, Q) and CD69 (D, F, J, L, P, R) relative to CD8 (E, F, K, L, Q, R) and CD4 (C, D, I, J, O, P) were measured by flow cytometry as a marker of T cell activation at 48 hours. [Figure 11G-H]Figures 11G and H show tumor cell killing and T cell activation by TYRP1 TCB containing the original or optimized CD3 binder. Killing of melanoma cell line M150543 by treatment with TYRP1 TCB CD3orig and TYRP1 TCB CD3opt by PBMCs from three different healthy donors (AF: Donor 1, GL: Donor 2, MR: Donor 3) was determined by LDH release at 24 hours (A, G, M) and 48 hours (B, H, N). In parallel, T cells in PBMCs upregulated by CD25 (C, E, I, K, O, Q) and CD69 (D, F, J, L, P, R) relative to CD8 (E, F, K, L, Q, R) and CD4 (C, D, I, J, O, P) were measured by flow cytometry as a marker of T cell activation at 48 hours. [Figure 11I-L] Figures 11, I–L, show tumor cell killing and T cell activation by TYRP1 TCB containing the original or optimized CD3 binder. Killing of melanoma cell line M150543 by treatment with TYRP1 TCB CD3orig and TYRP1 TCB CD3opt by PBMCs from three different healthy donors (AF: Donor 1, GL: Donor 2, MR: Donor 3) was determined by LDH release at 24 hours (A, G, M) and 48 hours (B, H, N). In parallel, T cells in PBMCs upregulated by CD25 (C, E, I, K, O, Q) and CD69 (D, F, J, L, P, R) relative to CD8 (E, F, K, L, Q, R) and CD4 (C, D, I, J, O, P) were measured by flow cytometry as a marker of T cell activation at 48 hours. [Figure 11M-N]Figures 11, I–L, show tumor cell killing and T cell activation by TYRP1 TCB containing the original or optimized CD3 binder. Killing of melanoma cell line M150543 by treatment with TYRP1 TCB CD3orig and TYRP1 TCB CD3opt by PBMCs from three different healthy donors (AF: Donor 1, GL: Donor 2, MR: Donor 3) was determined by LDH release at 24 hours (A, G, M) and 48 hours (B, H, N). In parallel, T cells in PBMCs upregulated by CD25 (C, E, I, K, O, Q) and CD69 (D, F, J, L, P, R) relative to CD8 (E, F, K, L, Q, R) and CD4 (C, D, I, J, O, P) were measured by flow cytometry as a marker of T cell activation at 48 hours. [Figure 11O-R] Figure 11, O-R, shows tumor cell killing and T cell activation by TYRP1 TCB containing the original or optimized CD3 binder. Killing of melanoma cell line M150543 by treatment with TYRP1 TCB CD3orig and TYRP1 TCB CD3opt by PBMCs from three different healthy donors (AF: Donor 1, GL: Donor 2, MR: Donor 3) was determined by LDH release at 24 hours (A, G, M) and 48 hours (B, H, N). In parallel, T cells in PBMCs upregulated by CD25 (C, E, I, K, O, Q) and CD69 (D, F, J, L, P, R) relative to CD8 (E, F, K, L, Q, R) and CD4 (C, D, I, J, O, P) were measured by flow cytometry as a marker of T cell activation at 48 hours. [Figure 12A-B] Figures 12A and 12B show the specific binding of EGFRvIII IgG PGLALA antibodies. Specific binding of EGFRvIII IgG PGLALA antibodies to EGFRvIII (without cross-reactivity with EGFRwt) was tested by flow cytometry using CHO-EGFRvIII (A) and EGFRvIII-positive DK-MG (B). Cetuximab was included as a positive control for EGFRwt expression. [Figure 12C]Figure 12C shows the specific binding of EGFRvIII IgG PGLALA. Specific binding of EGFRvIII IgG PGLALA antibody to EGFRvIII (without cross-reactivity with EGFRwt) was tested by flow cytometry on EGFRwt-expressing MKN-45(C). Cetuximab was included as a positive control for EGFRwt expression. [Figure 13] Activation of CAR J by EGFRvIII IgG PGLALA. Jurkat NFAT reporter cells expressing anti-PGLALA CAR were co-cultured with EGFRvIII-expressing DK-MG cells and EGFRvIII IgG PGLALA antibody or DP47 IgG PGLALA as a negative control. Activation of Jurkat NFAT cells was quantified by measuring luminescence after 22 hours. [Figure 14A-B] Binding of EGFRvIII IgG PGLALA and its corresponding TCB to EGFRvIII. Specific binding of the EGFRvIII binder as IgG PGLALA and its conversion to TCB to CHO-EGFRvIII(A) and MKN-45(B) cells was measured by flow cytometry. [Figure 15] Activation of Jurkat NFAT by EGFRvIII TCBs. Jurkat NFAT activation was determined as a marker of CD3 binding with EGFRvIII TCBs in the presence of EGFRvIII-positive DK-MG cells. DP47 TCBs were included as a negative control. [Figure 16A-D] Activation of tumor cells by EGFRvIII TCBs. Specific induction of tumor cell lysis by EGFRvIII TCBs was determined during co-culture of newly isolated PBMCs with either EGFRvIII-positive DK-MG cells (A, B) or EGFRwt-positive MKN-45 cells (C, D) for 24 hours (A, C) or 48 hours (B, D). [Figure 17A-D]T cell activation by EGFRvIII TCBs. The induction of T cell activation by EGFRvIII TCBs was determined using the activation markers CD25 (A, C, E, G) or CD69 (B, D, F, H) on CD4 T cells (A-D) or CD8 T cells (E-H) when co-culturing newly isolated PBMCs with either EGFRvIII-positive DK-MG cells (A, B, E, F) or EGFRwt-positive MKN-45 cells (C, D, G, H). [Figure 17E-H] T cell activation by EGFRvIII TCBs. The induction of T cell activation by EGFRvIII TCBs was determined using the activation markers CD25 (A, C, E, G) or CD69 (B, D, F, H) on CD4 T cells (A-D) or CD8 T cells (E-H) when co-culturing newly isolated PBMCs with either EGFRvIII-positive DK-MG cells (A, B, E, F) or EGFRwt-positive MKN-45 cells (C, D, G, H). [Figure 18A-F] Citkine release by EGFRvIII TCBs. Induction of IFN (A, D), TNF (B, E), and granzyme B (C, F) release by EGFRvIII TCBs was determined during co-culture of newly isolated PBMCs with either EGFRvIII-positive DK-MG cells (AC) or EGFRwt-positive MKN-45 cells (DF). [Figure 19A-B] Specific binding of affinity-matured EGFRvIII IgG PGLALA. The specific binding of affinity-matured EGFRvIII antibodies to EGFRvIII was compared with that of a parent EGFRvIII binder in U87MG-EGFRvIII cells (A) and the EGFRwt-positive cell line MKN-45 (B). [Figure 20A] Activation of Jurkat NFAT by EGFRvIII TCBs. Activation of Jurkat NFAT was determined as a marker of CD3 binding with EGFRvIII TCBs in the presence of EGFRvIII-positive DK-MG cells (A). DP47 TCBs were included as a negative control. [Figure 20B]Activation of Jurkat NFAT by EGFRvIII TCBs. Activation of Jurkat NFAT was determined as a marker of CD3 binding with EGFRvIII TCBs in the presence of U87MG-EGFRvIII cells (B). DP47 TCBs were included as a negative control. [Figure 20C] Activation of Jurkat NFAT by EGFRvIII TCBs. Activation of Jurkat NFAT was determined as a marker of CD3 binding with EGFRvIII TCBs in the presence of MKN-45 cells (C). DP47 TCBs were included as a negative control. [Figure 21] The relative binding activity of EGFRvIII TCBs containing the original or optimized CD3 binder, CD3orig or CD3opt, to recombinant CD3, measured by SPR under stress-free conditions after 14 days at 40°C, pH 6, or after 14 days at 37°C, pH 7.4. [Figure 22] The relative binding activity of EGFRvIII TCBs containing the original or optimized CD3 binder, CD3orig or CD3opt, to recombinant CD3, measured by SPR under stress-free conditions after 14 days at 40°C, pH 6, or after 14 days at 37°C, pH 7.4. [Figure 23] Binding of EGFRvIII TCBs containing original and optimized CD3 binders, CD3orig or CD3opt, to Jurkat NFAT cells was measured by flow cytometry. Antibodies bound to the TCBs were detected with fluorescently labeled anti-human Fc-specific secondary antibodies. [Figure 24] Binding of EGFRvIII TCBs containing P063.056 or P056.021 EGFRvIII binders to U87MG-EGFRvIII cells was measured by flow cytometry. TCBs bound to U87MG-EGFRvIII cells were detected with a fluorescently labeled anti-human Fc-specific secondary antibody. [Figure 25A-B]Tumor cell lysis and T cell activation by EGFRvIII TCBs. Induction of specific tumor cell lysis (A, B) and T cell activation (C, D) by EGFRvIII TCBs was determined during 24-hour (A, C) or 48-hour (B, D) co-culture of newly isolated PBMCs with U87MG-EGFRvIII cells. DP47 TCBs were included as a negative control. [Figure 25C-D] Tumor cell lysis and T cell activation by EGFRvIII TCBs. Induction of specific tumor cell lysis (A, B) and T cell activation (C, D) by EGFRvIII TCBs was determined during 24-hour (A, C) or 48-hour (B, D) co-culture of newly isolated PBMCs with U87MG-EGFRvIII cells. DP47 TCBs were included as a negative control. [Figure 26] Jurkat NFAT activation compared to EGFRvIII TCB 2+1 and 1+1 formats. Jurkat NFAT activation was determined to be a marker of CD3 binding to EGFRvIII TCBs in 2+1 inverted and 1+1 head-tail formats in the presence of EGFRvIII-positive U87MG-EGFRvIII cells. [Figure 27A-B] Tumor cell lysis and T cell activation compared with EGFRvIII TCB 2+1 and 1+1 formats. Specific induction of tumor cell lysis (A, B) and T cell activation (C, D) by 2+1 inverted format and 1+1 head-tail format EGFRvIII TCBs was determined during 24-hour (A, C) or 48-hour (B, D) co-culture of newly isolated PBMCs with U87MG-EGFRvIII cells. [Figure 27C-D] Tumor cell lysis and T cell activation compared with EGFRvIII TCB 2+1 and 1+1 formats. Specific induction of tumor cell lysis (A, B) and T cell activation (C, D) by 2+1 inverted format and 1+1 head-tail format EGFRvIII TCBs was determined during 24-hour (A, C) or 48-hour (B, D) co-culture of newly isolated PBMCs with U87MG-EGFRvIII cells. [Figure 28A-B]T cell activation and proliferation by EGFRvIII TCBs. The induction of T cell proliferation (A, C) and T cell activation of CD4 T cells (A, B) and CD8 T cells (C, D) by EGFRvIII TCBs was determined during co-culture of U87MG-EGFRvIII with PBMCs isolated from healthy donors. [Figure 28C-D] T cell activation and proliferation by EGFRvIII TCBs. The induction of T cell proliferation (A, C) and T cell activation of CD4 T cells (A, B) and CD8 T cells (C, D) by EGFRvIII TCBs was determined during co-culture of U87MG-EGFRvIII with PBMCs isolated from healthy donors. [Figure 29A-B] Evolution of tumor cell lysis, T cell activation, and cytokine release by EGFRvIII TCBs. The induction of tumor cell lysis (A, B), T cell activation (C, D), and IFN and TNF release (E, F) by EGFRvIII TCBs was determined during co-culture of U87MG-EGFRvIII cells with PBMCs. Tumor cell lysis was measured 24 and 48 hours after treatment, while T cell activation and cytokine release were determined at 48 hours. [Figure 29C-D] Evolution of tumor cell lysis, T cell activation, and cytokine release by EGFRvIII TCBs. The induction of tumor cell lysis (A, B), T cell activation (C, D), and IFN and TNF release (E, F) by EGFRvIII TCBs was determined during co-culture of U87MG-EGFRvIII cells with PBMCs. Tumor cell lysis was measured 24 and 48 hours after treatment, while T cell activation and cytokine release were determined at 48 hours. [Figure 29E-F] Evolution of tumor cell lysis, T cell activation, and cytokine release by EGFRvIII TCBs. The induction of tumor cell lysis (A, B), T cell activation (C, D), and IFN and TNF release (E, F) by EGFRvIII TCBs was determined during co-culture of U87MG-EGFRvIII cells with PBMCs. Tumor cell lysis was measured 24 and 48 hours after treatment, while T cell activation and cytokine release were determined at 48 hours. [Figure 30A-B]Induction of tumor cell lysis, T cell activation, and cytokine release by TYRP-1 TCBs. The induction of tumor cell lysis (A, B), T cell activation (C, D), and IFNγ and TNFα release (E, F) by TYRP-1 TCBs was determined during co-culture of patient-derived melanoma cell line M150543 with PBMCs. Tumor cell lysis was measured 24 and 48 hours after treatment, while T cell activation and cytokine release were determined at 48 hours. [Figure 30C-D] Induction of tumor cell lysis, T cell activation, and cytokine release by TYRP-1 TCBs. The induction of tumor cell lysis (A, B), T cell activation (C, D), and IFNγ and TNFα release (E, F) by TYRP-1 TCBs was determined during co-culture of patient-derived melanoma cell line M150543 with PBMCs. Tumor cell lysis was measured 24 and 48 hours after treatment, while T cell activation and cytokine release were determined at 48 hours. [Figure 30E-F] Induction of tumor cell lysis, T cell activation, and cytokine release by TYRP-1 TCBs. The induction of tumor cell lysis (A, B), T cell activation (C, D), and IFNγ and TNFα release (E, F) by TYRP-1 TCBs was determined during co-culture of patient-derived melanoma cell line M150543 with PBMCs. Tumor cell lysis was measured 24 and 48 hours after treatment, while T cell activation and cytokine release were determined at 48 hours. [Figure 31] In vivo efficacy of TYRP-1 TCB. The IGR-1 human melanoma cell line was subcutaneously injected into humanized NSG mice to study tumor growth inhibition in a subcutaneous xenograft model of melanoma. Significant tumor growth inhibition (TGI) was observed in the TYRP-1 TCB group compared to the vehicle group (68% TGI, p=0.0058*). [Figure 32] In vivo efficacy of EGFRvIII TCB. The U87-huEGFRvIII human glioblastoma cell line was subcutaneously injected into humanized NSG mice to study tumor growth inhibition in a subcutaneous xenograft model of glioblastoma. Significant tumor control was observed in the EGFRvIII TCB group, and all mice achieved complete remission. [Figure 33A-B] Formats of FolR1 TCB molecules. A is a classic 2+1 TCB molecule with CD3Fab fused to the VH of the internal FOLR1 Fab via a (G4S)2 linker. Heterodimerization via knob-into-hole technique, PGLALA mutation of Fc. B is an inverted 2+1 FOLR1 TCB with CD3optFab inside the Fc knob chain. C is a classic 1+1 head-tail FOLR1 TCB molecule containing CD3optFab fused to the VH of the internal FOLR1 Fab via a (G4S)2 linker. Heterodimerization via knob-into-hole technique, PGLALA mutation of Fc. D is an inverted 1+1 head-tail FOLR1 TCB with CD3optFab inside the Fc knob chain. E is a 1+1 IgG-like FOLR1 TCB molecule with CD3optFab on the Fc knob chain and FOLR1 Fab on the Fc hole chain. Heterodimerization of Fc via Fab knob-into-hole technology, PGLALA mutation. [Figure 33C-D] Formats of FolR1 TCB molecules. A is a classic 2+1 TCB molecule with CD3Fab fused to the VH of the internal FOLR1 Fab via a (G4S)2 linker. Heterodimerization via knob-into-hole technique, PGLALA mutation of Fc. B is an inverted 2+1 FOLR1 TCB with CD3optFab inside the Fc knob chain. C is a classic 1+1 head-tail FOLR1 TCB molecule containing CD3optFab fused to the VH of the internal FOLR1 Fab via a (G4S)2 linker. Heterodimerization via knob-into-hole technique, PGLALA mutation of Fc. D is an inverted 1+1 head-tail FOLR1 TCB with CD3optFab inside the Fc knob chain. E is a 1+1 IgG-like FOLR1 TCB molecule with CD3optFab on the Fc knob chain and FOLR1 Fab on the Fc hole chain. Heterodimerization of Fc via Fab knob-into-hole technology, PGLALA mutation. [Figure 33E]Formats of FolR1 TCB molecules. A is a classic 2+1 TCB molecule with CD3Fab fused to the VH of the internal FOLR1 Fab via a (G4S)2 linker. Heterodimerization via knob-into-hole technique, PGLALA mutation of Fc. B is an inverted 2+1 FOLR1 TCB with CD3optFab inside the Fc knob chain. C is a classic 1+1 head-tail FOLR1 TCB molecule containing CD3optFab fused to the VH of the internal FOLR1 Fab via a (G4S)2 linker. Heterodimerization via knob-into-hole technique, PGLALA mutation of Fc. D is an inverted 1+1 head-tail FOLR1 TCB with CD3optFab inside the Fc knob chain. E is a 1+1 IgG-like FOLR1 TCB molecule with CD3optFab on the Fc knob chain and FOLR1 Fab on the Fc hole chain. Heterodimerization of Fc via Fab knob-into-hole technology, PGLALA mutation. [Figure 34] Activation of Jurkat NFAT via FOLR1-TCB (CD3opt). Activation of Jurkat NFAT mediated by FOLR1-TCB using CD3opt is demonstrated. FOLR1-TCB was incubated with huFOLR1-coated beads and Jurkat NFAT effector cells at 37°C for 5.5 hours. The dotted line represents beads containing Jurkat cells without TCB. Each dot represents the mean of technical triplicates. The standard deviation is shown by error bars (n=1). [Figure 35A]Tumor cell lysis and T cell activation by FOLR1(pro-)TCB(CD3opt). Human PBMCs (effector cells) and FOLR1-positive target cells (Ovcar-3) were co-cultured with FOLR1 TCBs after 24 hours, and dose-dependent tumor cell lysis and T cell activation were analyzed. Induction of tumor cell lysis at 24 and 48 hours (A). T cell activation was measured after 48 hours of treatment by quantification of CD69 in CD4 and CD8 T cells by FACS. CD69-positive CD4 T cells (C) and CD8 T cells (D) are shown in the upper panel. In the lower panel, the median CD69 {PE} is plotted for CD4 (E) and CD8 (F)-positive T cells. Each point represents the mean of technical triplicates. Standard deviation is shown by error bars. [Figure 35B] Tumor cell lysis and T cell activation by FOLR1(pro-)TCB(CD3opt). Human PBMCs (effector cells) and FOLR1-positive target cells (Ovcar-3) were co-cultured with FOLR1 TCBs after 24 hours, and dose-dependent tumor cell lysis and T cell activation were analyzed. Induction of tumor cell lysis at 24 and 48 hours (A). T cell activation was measured after 48 hours of treatment by quantification of CD69 in CD4 and CD8 T cells by FACS. CD69-positive CD4 T cells (C) and CD8 T cells (D) are shown in the upper panel. In the lower panel, the median CD69 {PE} is plotted for CD4 (E) and CD8 (F)-positive T cells. Each point represents the mean of technical triplicates. Standard deviation is shown by error bars. [Figure 35C-F]Tumor cell lysis and T cell activation by FOLR1(pro-)TCB(CD3opt). Human PBMCs (effector cells) and FOLR1-positive target cells (Ovcar-3) were co-cultured with FOLR1 TCBs after 24 hours, and dose-dependent tumor cell lysis and T cell activation were analyzed. Induction of tumor cell lysis at 24 and 48 hours (A). T cell activation was measured after 48 hours of treatment by quantification of CD69 in CD4 and CD8 T cells by FACS. CD69-positive CD4 T cells (C) and CD8 T cells (D) are shown in the upper panel. In the lower panel, the median CD69 {PE} is plotted for CD4 (E) and CD8 (F)-positive T cells. Each point represents the mean of technical triplicates. Standard deviation is shown by error bars. [Modes for carrying out the invention]

[0046] I. Definition Unless otherwise defined below, this specification uses terms commonly used in the art.

[0047] As used herein, the terms “first,” “second,” or “third” with respect to antigen-binding domains, etc., are used for convenience in distinguishing between two or more parts of each type. The use of these terms is not intended to assign a particular order or orientation to the parts unless expressly stated otherwise.

[0048] The terms "anti-CD3 antibody" and "CD3-binding antibody" refer to an antibody that can bind to CD3 with sufficient affinity so that it is useful as a diagnostic and / or therapeutic agent targeting CD3. In one embodiment, the degree of binding of an anti-CD3 antibody to unrelated non-CD3 proteins is less than about 10% of the antibody's binding to CD3, as measured, for example, by surface plasmon resonance (SPR). In certain contexts, an antibody that binds to CD3 has a dissociation constant (K) of ≤1 μM, ≤500 nM, ≤200 nM, or ≤100 nM. D ) has. The antibody is measured, for example, by SPR, and the antibody has a K content of 1 μM or less. DWhen an antibody has this property, it is said to "specifically bind" to CD3. In certain embodiments, anti-CD3 antibodies bind to CD3 epitopes that are conserved among CD3s from different species.

[0049] The term "antibody" as used herein is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0050] An "antibody fragment" refers to a molecule other than the intact antibody, including a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (such as scFv or scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments. For an overview of specific antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0051] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody.

[0052] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in the population are identical and / or bind to the same epitope, except for possible variant antibodies, and may include, for example, spontaneously occurring mutations or those arising during the production of a monoclonal antibody preparation; such variants are generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by a specific method. For example, monoclonal antibodies can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0053] "Isolated" antibodies are those separated from components of their natural environment. In some embodiments, antibodies are purified to a purity of 95% or more than 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC, affinity chromatography, size exclusion chromatography). For a review of antibody purity evaluation methods, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some embodiments, the antibodies provided by the present invention are isolated antibodies.

[0054] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, and the remainder of the heavy chain and / or light chain originates from a different source or species.

[0055] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, where all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as “humanized variable regions.” A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. In some embodiments, some FR residues of a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residues are derived) to restore or improve the specificity or affinity of the antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0056] A “human antibody” is one that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues. In certain embodiments, a human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain embodiments, a human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from a human antibody library are also considered human antibodies or human antibody fragments in this specification.

[0057] The term "antigen-binding domain" refers to a portion of an antibody that contains a region that binds to and is complementary to a part or all of an antigen. The antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In a preferred embodiment, the antigen-binding domain includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0058] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and complementarity-determining regions (CDRs). For example, Kindt et al., Kuby Immunology, 6 th See ed., WH Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain from the antigen-binding antibody, and libraries of complementary VL or VH domains can be screened, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). Where used herein in relation to variable region sequences, “Kabat numbering” refers to the numbering system defined by Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0059] As used herein, the amino acid positions of all constant regions and domains of the 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), and are referred herein to as "Kabat numbering" or "Kabat numbering." Specifically, the Kabat numbering system (see pages 647–660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661–723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), in which case further clarification is provided herein by reference to the "Kabat EU index numbering system" or "Kabat EU index numbering".

[0060] As used herein, the terms “hypervariable region” or “HVR” refer to each region of the antibody variable domain that has a highly variable sequence and determines antigen-binding specificity, such as “complementarity-determining regions” (“CDR”). Generally, an antibody contains six CDRs; three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Illustrative CDRs as used herein are: (a) Hypervariable loops generated at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs generated at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (a) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)) Includes.

[0061] Unless otherwise specified, the CDR is determined according to Kabat et al. as cited above. Those skilled in the art will understand that the CDR designation may also be determined according to Chothia as cited above, McCallum as cited above, or any other scientifically recognized nomenclature.

[0062] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR typically consists of the following four FR domains: FR1, FR2, FR3, and FR4. It is composed of the following. Therefore, the HVR and FR sequences are generally represented in VH (or VL) in the following order: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0063] Unless otherwise specified, CDR residues and other residues (e.g., FR residues) within variable domains are numbered herein in accordance with Kabat et al.

[0064] For the purposes of this specification, “receptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A receptor human framework “derived” from a human immunoglobulin framework or a human consensus framework may include the same amino acid sequence or may include amino acid sequence variations. In some embodiments, the number of amino acid variations 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 receptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0065] The "Human Consensus Framework" is a framework representing the most commonly present amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from subgroups of variable domain sequences. Generally, these sequence subgroups are as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0066] In this specification, the term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, IgG-class immunoglobulins are heterotetrameric glycoproteins with approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin is assigned to one of five types called α(IgA), δ(IgD), ε(IgE), γ(IgG), or μ(IgM), some of which are further divided into subtypes, e.g., γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), α1(IgA1), and α2(IgA2). The light chain of an immunoglobulin can be assigned to one of two types called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin basically consists of two Fab molecules and one Fc domain linked via the hinge region of the immunoglobulin.

[0067] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region in its heavy chain. Antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0068] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of the immunoglobulin heavy chain ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain").

[0069] A "crossover" Fab molecule (also called a "Crossfab") refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., substituted for each other). Specifically, a crossover Fab molecule includes a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1CH1 (VL-CH1, N-terminal to C-terminal direction), and a peptide chain composed of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, N-terminal to C-terminal direction). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain variable domain VH is referred herein as the "heavy chain" of the (crossover) Fab molecule.

[0070] In contrast, a “conventional” Fab molecule refers to a Fab molecule in its natural form, that is, a heavy chain consisting of a variable domain and a constant domain (VH-CH1, N-terminus to C-terminus), and a light chain consisting of a variable domain and a constant domain (VL-CL, N-terminus to C-terminus).

[0071] In certain embodiments, the present invention relates to a bispecific molecule having at least two binding sites that confer specific binding to their respective antigens (e.g., CD3 and FolR1), consisting of an identical light chain and a corresponding modified heavy chain. This so-called "common light chain" principle, that is, combining two or more binders that share one light chain but have individual specificities, prevents light chain mispairing. Consequently, byproducts during production are reduced, and the uniform preparation of bispecific molecules is facilitated.

[0072] In this specification, the terms “Fc domain” or “Fc region” are used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids, particularly one or two, from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by the expression of a particular nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain or cleaved variants of the full-length heavy chain. This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to 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. The amino acid sequence of the heavy chain containing the Fc region (or a subunit of the Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide unless otherwise shown. In one embodiment, the heavy chain containing the Fc region (subunit) specified herein, as contained in the antibody according to the present invention, includes an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment, the heavy chain containing the Fc region (subunit) specified herein, as contained in the antibody according to the present invention, includes an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above).As used herein, the term "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide containing the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-assembly. For example, a subunit of an IgG Fc domain includes the IgG CH2 and IgG CH3 constant domains.

[0073] "Fusion" means that the constituent elements (e.g., the Fab molecule and the Fc domain subunit) are linked together by peptide bonds, either directly or via one or more peptide linkers.

[0074] The term "multispecificity" means that an antibody can specifically bind to at least two different antigenic determinants. A multispecific antibody may be, for example, a bispecific antibody. Typically, a bispecific antibody contains two antigen-binding sites, each of which is specific to a different antigenic determinant. In certain embodiments, a multispecific (e.g., bispecific) antibody can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

[0075] As used herein, the term “valence” means the presence of a specified number of antigen-binding sites within an antigen-binding molecule. Therefore, the term “monovalent binding to an antigen” means the presence of one (and no more than one) antigen-specific antigen-binding sites in the antigen-binding molecule.

[0076] An "antigen-binding site" refers to the portion of an antigen-binding molecule that provides interaction with an antigen, i.e., one or more amino acid residues. For example, the antigen-binding site of an antibody includes amino acid residues from the complementarity-determining region (CDR). Natural immunoglobulin molecules typically have two antigen-binding sites, while Fab molecules typically have a single antigen-binding site.

[0077] As used herein, the terms “antigenic determinant” or “antigen” refer to a site on a polypeptide macromolecule to which an antigen-binding domain binds (e.g., a structural configuration consisting of a continuous stretch of amino acids or different regions of discontinuous amino acids) to form an antigen-binding domain-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of other diseased cells, on the surface of immune cells, in the serum and / or in the extracellular matrix (ECM). In a preferred embodiment, the antigen is a human protein.

[0078] "CD3" refers to native CD3 derived from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. This term includes not only "full-length" untreated CD3 but also any form of CD3 resulting from intracellular processing. The term also includes naturally occurring variants of CD3, such as splice variants or allele variants. In one embodiment, CD3 refers to human CD3, particularly the epsilon subunit (CD3) of human CD3. ε ) is human CD3 ε The amino acid sequence is shown in SEQ ID NO: 112 (excluding the signal peptide). (www.uniprot.org) Accession number P07766 (version 189), or See also NCBI (www.ncbi.nlm.nih.gov / )RefSeq NP_000724.1. In another aspect, CD3 is cynomolgus macaque (Macaca fascicularis) CD3, in particular cynomolgus macaque CD3. ε This is the Crab-eating macaque CD3. ε The amino acid sequence is shown in SEQ ID NO: 113 (excluding the signal peptide). See also NCBI GenBank no. BAB71849.1. In certain embodiments, the antibody of the present invention binds to CD3 epitopes that are conserved among CD3 antigens from different species, particularly humans and cynomolgus monkeys. In preferred embodiments, the antibody binds to human CD3.

[0079] As used herein, “target cell antigen” refers to an antigenic determinant presented on the surface of a target cell, such as a cell in a tumor, such as a cancer cell or a cell in the tumor stroma (in which case, “tumor cell antigen”). Preferably, the target cell antigen is not and / or expressed on cells other than CD3. In one embodiment, the target cell antigen is TYRP-1, particularly human TYRP-1. In another embodiment, the target cell antigen is EGFRvIII, particularly human EGFRvIII. In a preferred embodiment, the target antigen is folate receptor 1 (FolR1).

[0080] "FolR1" refers to folate receptor 1 (synonyms include, but are not limited to, folate receptor alpha (FRA)), a folate-binding protein (FBP), MOv18, P15328, FRA1, FRAI), which is a protein receptor that mediates the intracellular renewal of folate and reduced folate derivatives. The sequence of human FolR1 is shown in Sequence ID No. 137. See also UniProt entry number P15328. As used herein, "FolR1" refers to native FolR1 from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term includes not only "full-length" untreated FolR1 but also any form of FolR1 resulting from intracellular processing. The term also includes naturally occurring variants of FolR1, such as splice variants or allele variants. In one embodiment, FolR1 is human FolR1.

[0081] "TYRP1" or "TYRP-1" represents tyrosine-related protein 1, an enzyme involved in melanin synthesis. The mature form of TYRP1, also known 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 bank P17643 (version 185). As used herein, "TYRP1" refers to native TYRP1 from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. This term includes not only "full-length" untreated TYRP1 but also any form of TYRP1 resulting from intracellular processing. The term also includes naturally occurring variants of TYRP1, such as splice variants or allele variants. In one embodiment, TYRP1 is human TYRP1.

[0082] "EGFRvIII" represents epidermal growth factor receptor variant III, a variant of EGFR, formed by in-frame deletion of exons 2-7, resulting in a 267-amino acid deletion with a glycine substitution at the junction. The sequence of human EGFRvIII is shown in SEQ ID NO: 115 (without signal peptide). The sequence of human EGFR is shown in SEQ ID NO: 116 (without signal peptide). See also UniProt entry bank P00533 (version 258). As used herein, "EGFRvIII" refers to native EGFRvIII from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term includes "full-length" unprocessed EGFRvIII (but not wild-type EGFR), as well as any form of EGFRvIII resulting from intracellular processing (e.g., EGFRvIII without signal peptide). In one embodiment, EGFRvIII is human EGFRvIII.

[0083] "Affinity" refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by its 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).

[0084] A "affinity-matured" antibody refers to an antibody that has one or more changes in one or more complementarity-determining regions (CDRs) compared to a parent antibody that does not have such changes, and such changes result in an improved affinity of the antibody to the antigen.

[0085] "Decreased binding," for example, decreased binding to the Fc receptor, refers to a decrease in affinity for each interaction, as measured, for example, by SPR. To clarify, this term also includes reducing affinity to zero (or below the detection limit of the analytical method), i.e., eliminating the interaction completely. Conversely, "increased binding" refers to an increase in binding affinity for each interaction.

[0086] As used herein, “T cell activation” refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Appropriate assays for measuring T cell activation are known in the art and are described herein.

[0087] "Modifications that promote the association of the first and second subunits of the Fc domain" are modifications of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of the polypeptide containing the Fc domain subunits, thereby forming homodimers with the same polypeptide. Modification-promoting associations as used herein preferably involve distinct modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, wherein the modifications are complementary to each other in order to promote the association of the two Fc domain subunits. For example, the association-promoting modifications can alter the structure or charge of one or both of the Fc domain subunits to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing the first Fc domain subunit and a polypeptide containing the second Fc domain subunit, which may not be identical in the sense that the further components fused to each subunit (e.g., antigen-binding domains) are not the same. In some embodiments, modifications that promote the association of the first and second subunits of the Fc domain include amino acid mutations, specifically amino acid substitutions, in the Fc domain. In preferred embodiments, modifications that promote the association of the first and second subunits of the Fc domain include distinct amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

[0088] The term "effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, antigen uptake by antigen-presenting cells via immune complexes, downregulation of cell surface receptors (such as B cell receptors), and B cell activation. Includes.

[0089] An "activated Fc receptor" is an Fc receptor that, following the involvement of the antibody's Fc domain, triggers a signaling event that stimulates receptor-hosting cells to perform effector functions. Human activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0090] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which antibodies or derivatives containing an Fc region specifically bind, generally via the protein portion at the N-terminus of the Fc region. As used herein, the term “reduced ADCC” is defined by either a decrease in the number of target cells lysed within a given time at a given concentration of antibody in the culture medium surrounding the target cells by the ADCC mechanism as defined above, and / or an increase in the concentration of antibody in the culture medium surrounding the target cells required to achieve the lysis of a given number of target cells within a given time by the ADCC mechanism. Reduced ADCC is compared to ADCC mediated by the same antibody produced by the same type of host cells using the same standard production, purification, preparation, and storage methods (known to those skilled in the art), but which is not manipulated. For example, reduced ADCC mediated by an antibody containing an amino acid substitution in its Fc domain that reduces ADCC is compared to ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Appropriate assays for measuring ADCC are well known in the art (see, for example, PCT publication numbers WO2006 / 082515 or WO2012 / 130831).

[0091] As used herein, the terms “manipulated” are understood to include any manipulation of the peptide backbone, or post-translational modification of a native or recombinant polypeptide or fragment thereof. Manipulations include modification of the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, and combinations of these approaches.

[0092] As used herein, the term “amino acid mutation” encompasses the substitution, deletion, insertion, and modification of amino acids. Any combination of substitutions, deletions, insertions, and modifications can be performed to arrive at a final construct, provided that the final construct has the desired characteristics, e.g., decreased binding to the Fc receptor or increased association with another peptide. Deletions and insertions of amino acid sequences include deletions and insertions of the amino-terminus and / or carboxy-terminus of amino acids. Preferred amino acid mutations are amino acid substitutions. For example, non-conservative amino acid substitutions, i.e., substitution of one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred for the purpose of altering the binding properties of the Fc region. Amino acid substitutions include substitutions with amino acids that do not exist naturally, or with naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is intended that methods of modifying the side chain groups of amino acids by means other than genetic engineering, such as chemical modification, may also be useful. In this specification, various designations may be used to indicate the same amino acid mutation. For example, a substitution of proline to glycine at position 329 of the Fc domain can be represented as 329G, G329, G329, P329G, or Pro329Gly.

[0093] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum percentage sequence identity, and without considering conservative substitutions as part of the sequence identity. Alignment for determining amino acid sequence identity percentage can be achieved in various ways within the scope of the art using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or FASTA program packages. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. Alternatively, the percentage identity value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, was filed with the U.S. Copyright Office (Washington, DC, 20559) and registered under U.S. Copyright Registration No. TXU510087, as described in WO2001 / 007611.

[0094] Unless otherwise specified, for the purposes of this specification, % amino acid sequence identity values ​​are generated using the ggsearch program in FASTA package version 36.3.8c or later, along with the BLOSUM50 comparison matrix. The FASTA program package was authored by WR Pearson and DJ Lipman ("Improved Tools for Biological Sequence Analysis," PNAS 85(1988)2444-2448), WR Pearson ("Effective protein sequence comparison," MethEnzymol. 266 (1996)227-258), and Pearson et al. (Genomics 46(1997)24-36), and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used for sequence comparison using the ggsearch(global protein:protein) program with default options (BLOSUM50;open:-10;ext:-2;Ktup=2) to ensure that global rather than local alignment is performed. The amino acid identity percentage is displayed in the output alignment header.

[0095] The terms “polynucleotide” or “nucleic acid molecule” include any compound and / or substance containing polymers of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by a sequence of bases, thereby representing the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually represented from 5' to 3'. In this specification, the term nucleic acid molecule includes, for example, deoxyribonucleic acid (DNA) including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may include natural or non-natural nucleotides. Examples of nucleotides not found in nature include modified nucleotide bases having derivatized sugar or phosphate backbone links or chemically modified residues. The nucleic acid molecules also include DNA and RNA molecules suitable as vectors for directly expressing the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that it can be injected to a target to generate an antibody in vivo (see, e.g., Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2 101 823 B1).

[0096] "Isolated" nucleic acid molecules refer to nucleic acid molecules that have been separated from components in their natural environment. Isolated nucleic acid molecules include those normally found in cells containing nucleic acid molecules, but these nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0097] "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, and includes polynucleotide molecules such as those in a single vector or separate vectors, and polynucleotide molecules present at one or more locations within a host cell.

[0098] As used herein, the term “vector” refers to a nucleic acid molecule capable of replicating another nucleic acid it is bound to. The term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of a operably linked nucleic acid. Such vectors are referred to herein as “expression vectors.”

[0099] 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 the offspring of such cells. Host cells include “transformers” and “transformed cells,” and include primary transformed cells and their offspring, regardless of passage number. Offspring may contain mutations, although their nucleic acid content may not be exactly the same as that of the parent cells. Mutant offspring having the same function or biological activity as those screened or selected in the original transformed cells are included herein. Host cells are any type of cell line that can be used to produce the antibodies of the present invention. Host cells include cultured cells, mammalian cultured cells such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, plant cells, and, to name just a few, cells contained within transgenic animals, transgenic plants, or cultured plants or animal tissues. In one embodiment, the host cells of the present invention are eukaryotic cells, particularly mammalian cells. In one embodiment, the host cell is not a cell from the human body.

[0100] The term "pharmaceutical composition" or "pharmaceutical preparation" refers to a preparation that is in a form that enables the biological activity of the active ingredient contained therein, and that does not contain any additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0101] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient that is non-toxic to the target substance. Pharmaceutically acceptable carriers include, but are not limited to, buffers, vehicles, stabilizers, or preservatives.

[0102] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease in an individual being treated, which may be performed for prevention or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or mitigation of the disease state, remission or improvement of prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of a disease or to slow the progression of a disease.

[0103] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0104] The “effective dose” of a drug, such as a pharmaceutical composition, refers to the amount that is effective in the dose and duration required to achieve the desired therapeutic or preventive outcome.

[0105] The term “package insert” is used to refer to the instructions that are customarily included in the market packaging of a therapeutic product, including information relating to indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings for the use of such therapeutic product.

[0106] II. Compositions and Methods This invention provides a bispecific antibody that binds to CD3 and FolR1. The antibody exhibits excellent stability, coupled with other properties advantageous for therapeutic application, such as efficacy, safety, pharmacokinetics, and productivity. The antibody of this invention is useful for treating diseases such as cancer.

[0107] A. Bispecific anti-CD3 anti-FolR1 antibody In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1. In one embodiment, isolated bispecific antibodies that bind to CD3 and FolR1 are provided. In one embodiment, the present invention provides a bispecific antibody that specifically binds to CD3 and FolR1. In a particular embodiment, the bispecific anti-CD3 anti-FolR1 antibody retains more than 90% of the binding activity to CD3 after 2 weeks at pH 7.4 and 37°C compared to the binding activity after 2 weeks at pH 6 and -80°C, as determined by surface plasmon resonance (SPR).

[0108] In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a first antigen-binding domain comprising a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 5, and a light chain variable region (VL) including light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10.

[0109] In one embodiment, the antibody is a humanized antibody. In one embodiment, the antigen-binding domain is a humanized antigen-binding domain (i.e., the antigen-binding domain of a humanized antibody). In one embodiment, VH and / or VL are humanized variable regions.

[0110] In one embodiment, VH and / or VL include an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0111] In one embodiment, VH includes one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO: 7. In one embodiment, VH includes 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 embodiment, VH includes an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. In one embodiment, VH includes 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 includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody containing that sequence retains the ability to bind to CD3. In certain embodiments, a total of 1 to 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 region outside the CDR (i.e., FR). In one embodiment, VH contains the amino acid sequence of SEQ ID NO: 7. Optionally, VH contains the amino acid sequence of SEQ ID NO: 7, including post-translational modifications of that sequence.

[0112] In one embodiment, the VL includes one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO: 11. In one embodiment, the VL includes 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 embodiment, the VL includes an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 11. In one embodiment, the VL includes 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 includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody containing that sequence retains the ability to bind to CD3. In certain embodiments, a total of 1 to 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 region outside the CDR (i.e., FR). In one embodiment, VL contains the amino acid sequence of SEQ ID NO: 11. Optionally, VL contains the amino acid sequence of SEQ ID NO: 11, including post-translational modifications of that sequence.

[0113] In one embodiment, VH includes 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, and / or VL includes 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 embodiment, VH includes the amino acid sequence of SEQ ID NO: 7, and VL includes the amino acid sequence of SEQ ID NO: 11.

[0114] In a further embodiment, the present invention provides an antibody that binds to CD3, the antibody comprising a first antigen-binding domain comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 11.

[0115] In a further embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, wherein the antibody comprises a first antigen-binding domain including the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11.

[0116] In another embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a first antigen-binding domain comprising VH, which contains the heavy chain CDR sequence of SEQ ID NO: 7, and VL, which contains the light chain CDR sequence of VL, SEQ ID NO: 11.

[0117] In a further embodiment, the first antigen-binding domain includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH in SEQ ID NO: 7 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL in SEQ ID NO: 11.

[0118] In one embodiment, VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 7 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 7. In another embodiment, VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 7 and a framework having at least 95% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 7.

[0119] In one embodiment, the VL includes the light chain CDR sequence of the VL of SEQ ID NO: 11 and a framework with at least 95%, 96%, 97%, 98%, or 99% sequence identity to the framework sequence of the VL of SEQ ID NO: 11. In another embodiment, the VL includes the light chain CDR sequence of the VL of SEQ ID NO: 11 and a framework with at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO: 11.

[0120] In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, wherein the antibody comprises a first antigen-binding domain comprising a VH sequence as in any of the embodiments provided above, and a VL sequence as in any of the embodiments provided above.

[0121] In one embodiment, the bispecific antibody includes a human constant region. In one embodiment, the bispecific antibody is an immunoglobulin molecule including a human constant region, particularly an IgG class immunoglobulin molecule including human CH1, CH2, CH3 and / or CL domains. Exemplary sequences of human constant domains are shown in SEQ ID NOs: 120 and 121 (human kappa and lambda CL domains, respectively) and SEQ ID NOs: 122 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one embodiment, the bispecific antibody includes a light chain constant region including an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 120 or 121, particularly the amino acid sequence of SEQ ID NOs: 120. In one embodiment, the bispecific antibody includes a heavy chain constant region including an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 122. In particular, the heavy chain constant region may contain amino acid mutations in the Fc domain as described herein.

[0122] In one embodiment, the first antigen-binding domain includes a human constant region. In one embodiment, the first antigen-binding portion is a Fab molecule including a human constant region, particularly a human CH1 and / or CL domain. In one embodiment, the first antigen-binding domain includes a light chain constant region, particularly the amino acid sequence of SEQ ID NO: 120, which includes an amino acid sequence that 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. In particular, the light chain constant region may include amino acid mutations as described herein under “charge modification”, and / or, in the case of a crossover Fab molecule, one or more (particularly two) N-terminal amino acid deletions or substitutions. In some embodiments, the first antigen-binding domain includes a heavy chain constant region, which includes an amino acid sequence that 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. In particular, the heavy chain constant region (specifically the CH1 domain) may contain amino acid mutations as described under "charge modification" in this specification.

[0123] In one embodiment, the bispecific antibody is a monoclonal antibody.

[0124] In one embodiment, the bispecific antibody is IgG, particularly IgG1 antibody. In another embodiment, the bispecific antibody is a full-length antibody.

[0125] In another embodiment, the first and / or second and / or further antigen-binding domain is an antibody fragment selected from the group consisting of Fv molecules, (a)scFv molecules, (a)Fab molecules, and (a)F(ab')2 molecules; in particular (a)Fab molecules. In another embodiment, the antibody fragment is (a) a bispecific antibody, (a) a triplicate antibody, or (a) a quadruplespecific antibody.

[0126] In one embodiment, the first antigen-binding domain is a Fab molecule. In a preferred embodiment, the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain, particularly the variable domains VL and VH, are substituted for each other (the first antigen-binding domain is a crossover Fab molecule).

[0127] In further embodiments, antibodies according to any of the above embodiments may incorporate any of the features individually or in combination, as described in Sections II.A.1-8 below.

[0128] In a preferred embodiment, the antibody includes an Fc domain, particularly an IgG Fc domain, more specifically an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain. In another embodiment, the Fc domain is a human IgG1 Fc domain. The Fc domain consists of first and second subunits, and with respect to Fc domain variants, any of the features described below may be incorporated individually or in combination (Section II.A.8).

[0129] In another preferred embodiment, the antibody comprises a second and optionally third antigen-binding domain that binds to a second antigen (i.e., the antibody is a multispecific antibody, as further described below in this specification (Section II.A.7)).

[0130] 1. Antibody fragment In certain embodiments, the antigen-binding domain provided herein is an antibody fragment.

[0131] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 molecule, particularly the Fab molecule described herein. A "Fab' molecule" is distinct from a Fab molecule by the addition of residues at the carboxyl terminus of one or more cysteine-containing CH1 domains from the antibody hinge region. Fab'-SH is a Fab' molecule in which the cysteine ​​residues of the constant domain have free thiol groups. Pepsin treatment yields an F(ab')2 molecule having two antigen-binding sites (two Fab molecules) and a portion of the Fc region.

[0132] In another embodiment, the antibody fragment is a bispecific antibody, a triplicate antibody, or a quadruplicate antibody. A “bispecific antibody” is an antibody fragment having two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097;WO1993 / 01161;Hudson et al., Nat. Med. 9:129-134(2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448(1993). Triplicate and quadruplicate antibodies are also described in Hudson et al., Nat. Med. 9:129-134(2003).

[0133] In a further embodiment, the antibody fragment is a single-chain Fab molecule. The "single-chain Fab molecule" or "scFab" comprises an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-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 to 50 amino acids. The single-chain Fab molecule is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab molecules can be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to Kabat numbering).

[0134] In another embodiment, the antibody fragment is a single-chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody, linked by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids, usually rich in glycine for flexibility and rich in serine or threonine for solubility, and can be linked from the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. For a review of the scFv fragment, see, for example, Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); and WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0135] In another embodiment, the antibody fragment is a single-domain antibody. A "single-domain antibody" is an antibody fragment that contains all or part of the heavy-chain variable domain or all or part of the light-chain variable domain of an antibody. In a particular embodiment, the single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).

[0136] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (e.g., Escherichia coli), as described herein.

[0137] 2. Humanized antibodies In certain embodiments, the antibodies provided herein (e.g., bispecific antibodies) are humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains in which the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies also optionally include at least a portion of the human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residue is derived) to restore or improve the specificity or affinity of the antibody, for example.

[0138] Humanized antibodies and their production methods have been reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (Specificity Determination Region (SDR) Grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (explains "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (explains "FR shuffle"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (explains the "guided selection" approach to FR shuffle).

[0139] Human framework regions that can be used for humanization include, but are not limited to, the following: framework regions selected using the “best fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of the FR library (see, e.g., Baca et al., J. Biol. Chem. See 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0140] 3. Glycosylated variants In certain embodiments, antibodies provided herein (e.g., bispecific antibodies) are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be easily achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0141] If an antibody contains an Fc region, the oligosaccharides bound to it may be modified. Native antibodies produced by mammalian cells typically contain branched or bifurcated oligosaccharides commonly bound by an N-bond to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the bifurcated oligosaccharide structure. In some embodiments, modification of oligosaccharides in the antibodies of the present invention may be performed to produce antibody variants with specific improved properties.

[0142] In one embodiment, an antibody variant is provided having an oligosaccharide structure lacking a non-fucosylated oligosaccharide, i.e., a fucose (directly or indirectly) bound to the Fc region. Such a non-fucosylated oligosaccharide (also called an "afucosylated" oligosaccharide) is in particular an N-linked oligosaccharide, which lacks a fucose residue bound to the first GlcNAc of the branched oligosaccharide structure's stem. In one embodiment, an antibody variant is provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to the native antibody or the parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The proportion of non-fucosylated oligosaccharides is the (average) amount of fucose-lacking oligosaccharides relative to the total of all oligosaccharides bound to Asn 297, as measured by MALDI-TOF mass spectrometry, as described, for example, in WO2006 / 082515 (e.g., complex, hybrid, and high-mannose structures). Asn297 refers to the asparagine residue located approximately 297th in the Fc region (EU numbering of Fc region residues); however, due to slight sequence differences in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of 297, i.e., between 294 and 300. Antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US2003 / 0157108; US2004 / 0093621.

[0143] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545(1986); US2003 / 0157108; and WO 2004 / 056312, especially at Example 11), and knockout cell lines such as those containing the α-1,6-fucosyltransferase gene, FUT8, and knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622(2004); Kanda, Y. et al., Biotechnol. Bioeng., See 94(4):680-688(2006); and WO2003 / 085107), or cells with reduced or lost cGDP-fucose synthesis or transporter protein activity (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

[0144] In a further context, antibody variants are provided, for example, with a bifid oligosaccharide in which the branched oligosaccharide bound to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced 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); WO99 / 54342; WO2004 / 065540, WO2003 / 011878.

[0145] Antibody variants having at least one galactose residue in an oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087;WO1998 / 58964; and WO1999 / 22764.

[0146] 4. Cysteine-modified antibody variants In certain embodiments, it may be desirable to produce cysteine-modified antibodies, such as THIOMAB® antibodies in which one or more residues of the antibody are substituted with cysteine ​​residues. In preferred embodiments, the substituted residues occur at accessible sites on the antibody. As further described herein, by substituting these residues with cysteine, a reactive thiol group is positioned at an accessible site on the antibody, allowing the antibody to be conjugated to a drug moiety or other moiety, such as a linker-drug moiety, for use in producing an immunocomplex. Cysteine-modified antibodies are described, for example, in U.S. Patents 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0147] 5. Antibody derivatives In certain embodiments, antibodies provided herein (e.g., bispecific antibodies) can be further modified to include additional non-proteinoid moieties known and readily available in the art. Moieties suitable for antibody derivatization 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, carboxymethylcellulose, 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 glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may offer advantages in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers that bind to an antibody varies, and if multiple polymers are bound, they may be the same molecule or different molecules. Generally, the number and / or types of polymers used in derivatization can be determined based on considerations including, but are not limited to, the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for treatment under defined conditions.

[0148] 6. Immune complex The present invention also provides an immune complex comprising the anti-CD3 / anti-FolR1 antibody of this specification, which is conjugated (chemically bonded) to one or more therapeutic agents such as cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes.

[0149] In one aspect, the immune complex is an antibody-drug conjugate (ADC) in which an antibody is bound to one or more of the above therapeutic agents. The antibody is typically bound to one or more therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents, drugs, and linkers, is described in Pharmacol Review 68:3-19 (2016).

[0150] In another aspect, the immune complex comprises an antibody of the present invention bound to an enzymatically active toxin or a fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, aleurite fordii protein, dianthin protein, phytolaca americana protein (PAPI, PAPII, and PAP-S), nigaui inhibitor, curcin, crotin, saponaria inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene.

[0151] In another aspect, the immune complex comprises an antibody of the present invention bound to a radioactive atom to form a radioactive complex. Various radioisotopes can be used for the production of the radioactive conjugate. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioisotopes of Lu. When the radioactive complex is used for detection, radioactive atoms for scintigraphic studies, such as Tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (such as I123, I131, In111, F19, C13, N15, O17, gadolinium, manganese, or iron, also known as magnetic resonance imaging, MRI) can be included.

[0152] Antibody-cytotoxic agent conjugates can be prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidylsberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bisactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for the binding of radioactive nucleotides to antibodies. See WO94 / 11026. Linkers may be "cleavable linkers" that facilitate the release of cytotoxic drugs within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0153] The immunocomplexes or ADCs used herein are expressly intended to be such complexes prepared with crosslinking reagents, but are not limited to those, and include BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are available from the market (e.g., Pierce Biotechnology, Inc., Rockford, IL., USA), but are not limited to those.

[0154] 7. Multispecific antibodies The antibodies provided herein are multispecific antibodies, particularly bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different antigenic determinants (e.g., two different proteins, or two different epitopes on the same protein). In certain embodiments, a multispecific antibody has three or more binding specificities. In certain embodiments, one binding specificity is for CD3, and the other specificity is for FolR1. In certain embodiments, a multispecific antibody may bind to two (or more) different epitopes of CD3. Multispecific (e.g., bispecific) antibodies may also be used as cytotoxic agents or to localize cells to express CD3. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0155] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168, Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies are produced by manipulating the electrostatic steering effect to create Fc heterodimer molecules of antibodies (see, e.g., WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO2011 / 034605); using common light chain techniques to avoid light chain mispairing problems (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and Tutt et al. J. Immunol. It can also be prepared by using the single-chain Fv(sFv) dimer described in 147:60(1991).

[0156] Modified antibodies having three or more antigen-binding sites, such as "octopus antibody" or DVD-Ig, are also included herein (see, for example, WO2001 / 77342 and WO2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in WO2010 / 115589, WO2010 / 112193, WO2010 / 136172, WO2010 / 145792, and WO2013 / 026831. Multispecific antibodies or their antigen-binding fragments also include "dual-acting FAb" or "DAF" containing antigen-binding sites that bind to CD3 and another different antigen, or to two different epitopes of CD3 (see, for example, US2008 / 0069820 and WO2015 / 095539).

[0157] Multispecific antibodies can also be provided in an asymmetric form where domains cross over one or more binding arms of the same antigen specificity (so-called "CrossMab" technology), i.e., by exchanging VH / VL domains (see, e.g., WO2009 / 080252 and WO2015 / 150447), CH1 / CL domains (see, e.g., WO2009 / 080253), or complete Fab arms (see, e.g., WO2009 / 080251, WO2016 / 016299; also see Schaefer et al., PNAS, 108(2011)1187-1191, and Klein et al., MAbs 8(2016)1010-20). Asymmetric Fab arms can also be manipulated by introducing charged or uncharged amino acid mutations into the domain interface to direct correct Fab pairing. For example, see WO2016 / 172485.

[0158] Multispecific antibodies are also provided in which binding arms of different specificities share a common light chain. The inventors of the present invention have generated a bispecific antibody in which the binding portion retains the specificity and efficacy of a parent monospecific antibody against CD3 and shares a common light chain that can bind to a second antigen (e.g., FolR1) using the same light chain. Generating a bispecific molecule having a common light chain that retains the binding properties of the parent antibody is not straightforward because the common CDR of the hybrid light chain must achieve binding specificity to both targets. In one embodiment, the present invention provides a T cell-activated bispecific antigen-binding molecule comprising first and second antigen-binding portions, one of which is a Fab molecule that can specifically bind to CD3 and the other is a Fab molecule that can specifically bind to CD3, and the first and second Fab molecules have the same VLCL light chain. In one embodiment, the same light chain (VLCL) includes the light chain CDRs of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. In one embodiment, the same light chain (VLCL) includes SEQ ID NO: 129.

[0159] Various further molecular forms of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67(2015)95-106).

[0160] Certain types of multispecific antibodies, also included herein, are bispecific antibodies designed to simultaneously bind to surface antigens such as FolR1, to target cells such as tumor cells, and to the activating and invariant components of T cell receptor (TCR) complexes such as CD3 for retargeting T cells to kill target cells. In a preferred embodiment, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, in which one binding specificity is to CD3 and the other is to FolR1.

[0161] Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules, in which two scFv molecules are fused by a flexible linker (e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567, Nagarsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); bispecific antibodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives such as tandem bispecific antibodies ("TandAb"; Kipriyanov et al., J Mol Biol 293, 41-56(1999)); "DART" (dual affinity retargeting) molecules based on the bispecific antibody format but featuring a C-terminal disulfide crosslink for additional stabilization (Johnson et al., J Mol Biol 399, 436-449(2010)); and triomab, the so-called all-hybrid mouse / rat IgG molecule (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467(2010)). Specific T cell bispecific antibody formats included herein are described in WO2013 / 026833, WO2013 / 026839, WO2016 / 020309; Bacac et al., Oncoimmunology 5(8)(2016)e1203498.

[0162] Preferred embodiments of the multispecific antibody of the present invention are described below.

[0163] In one embodiment, the present invention provides a CD3-binding antibody comprising a first antigen-binding domain that binds to CD3, and a second and optionally third antigen-binding domain that binds to FolR1, as described herein.

[0164] In a preferred embodiment of the present invention, the antigen-binding domain contained in the antibody is a Fab molecule (i.e., an antigen-binding domain composed of a heavy chain and a light chain, each containing a variable domain and a constant domain). In one embodiment, the first, second and / or third antigen-binding domains, if present, are Fab molecules. In one embodiment, the Fab molecule is human. In a preferred embodiment, the Fab molecule is humanized. In yet another embodiment, the Fab molecule contains human heavy chain and light chain constant domains.

[0165] In a preferred embodiment of the present invention, a (multispecific) antibody can simultaneously bind to a first antigen (i.e., CD3) and a second antigen (i.e., FolR1). In one embodiment, the (multispecific) antibody can crosslink T cells and target cells by simultaneous binding to CD3 and FolR1. In a more preferred embodiment, such simultaneous binding results in the lysis of target cells, particularly tumor cells expressing the target cell antigen (i.e., FolR1). In one embodiment, such simultaneous binding results in T cell activation. In other embodiments, such simultaneous binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one embodiment, binding of a (multispecific) antibody to CD3 without simultaneous binding to FolR1 does not result in T cell activation.

[0166] In one embodiment, a (multispecific) antibody can redirect the cytotoxic activity of T cells to target cells. In a preferred embodiment, such redirection is independent of MHC-mediated peptide antigen presentation by target cells and / or T cell specificity.

[0167] Preferably, the T cells according to any aspect of the present invention are cytotoxic T cells. In some aspects, the T cells are CD4 + or CD8 + T cells, especially CD8 + These are T cells.

[0168] 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 preferred embodiments, CD3 is human CD3 (SEQ ID NO: 112) or cynomolgus monkey CD3 (SEQ ID NO: 113), most particularly human CD3. In one embodiment, the first antigen-binding domain is cross-reactive (i.e., specifically binds) to human and cynomolgus monkey CD3. In some embodiments, CD3 is the epsilon subunit of CD3 (CD3 epsilon).

[0169] In a preferred embodiment, the (bispecific) antibody comprises one or fewer antigen-binding domains that bind to CD3. In one embodiment, the (bispecific) antibody provides monovalent binding to CD3.

[0170] In one embodiment, the antigen-binding domain that binds to CD3 is an antibody fragment selected from the group consisting of Fv molecules, scFv molecules, Fab molecules, and F(ab')2 molecules. In a preferred embodiment, the antigen-binding domain that binds to CD3 is a Fab molecule.

[0171] b) Second (and third) antigen-binding domains In certain embodiments, the (multispecific) antibody of the present invention comprises at least one antigen-binding domain, particularly a Fab molecule, that binds to a second antigen. The second antigen is preferably not CD3, i.e., different from CD3. In one embodiment, the second antigen is an antigen expressed on cells different from CD3 (e.g., expressed on cells other than T cells). In one embodiment, the second antigen is a target cell antigen, particularly a tumor cell antigen. In a preferred embodiment, the second antigen is FolR1. The second antigen-binding domain can direct the (multispecific) antibody to a target site, for example, a specific type of tumor cell expressing the second antigen.

[0172] In one embodiment, the antigen-binding domain that binds to the second antigen (i.e., FolR1) is an antibody fragment selected from the group consisting of Fv molecules, scFv molecules, Fab molecules, and F(ab')2 molecules. In a preferred embodiment, the antigen-binding domain that binds to the second antigen is a Fab molecule.

[0173] In certain embodiments, the (multispecific) antibody comprises two antigen-binding domains, particularly Fab molecules, that bind to a second antigen. In preferred embodiments, each of these antigen-binding domains binds to the same antigenic determinant. In more preferred embodiments, all of these antigen-binding domains are identical, i.e., they have the same molecular form (e.g., a conventional Fab molecule) and contain the same amino acid sequence (if any) with the same amino acid substitutions in the CH1 and CL domains as described herein. In one embodiment, the (multispecific) antibody comprises two or fewer antigen-binding domains, particularly Fab molecules, that bind to a second antigen.

[0174] In one embodiment, the second (and, if present, third) antigen-binding domain includes a human constant region. In one embodiment, the second (and, if present, third) antigen-binding domain is a Fab molecule including a human constant region, particularly the human CH1 and / or CL domain. Exemplary sequences of the human constant domain are shown in SEQ ID NOs: 120 and 121 (human kappa and lambda CL domains, respectively) and SEQ ID NOs: 122 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one embodiment, the second (and, if present, third) antigen-binding domain includes a light chain constant region, particularly the amino acid sequence of SEQ ID NOs: 120, which includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs: 120 or 121. In particular, the light chain constant region may include amino acid mutations as described herein under “charge modification” and / or, in the case of a crossover Fab molecule, one or more (particularly two) N-terminal amino acid deletions or substitutions. In some embodiments, the first (and, if present, the third) antigen-binding domain includes a heavy chain constant region comprising an amino acid sequence that 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. In particular, the heavy chain constant region (specifically the CH1 domain) may include amino acid mutations as described herein under "charge modification".

[0175] TYRP-1 In some aspects of this disclosure, the second antigen is TYRP-1, in particular human TYRP-1 (SEQ ID NO: 114).

[0176] In one embodiment, the second (and, if present, third) antigen-binding domain includes a heavy chain variable region (VH) containing the 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, and a light chain variable region (VL) containing the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 19, LCDR2 of SEQ ID NO: 20, and LCDR3 of SEQ ID NO: 21.

[0177] In one embodiment, the second (and, if present, third) antigen-binding domain is derived from a humanized antibody. In one embodiment, the second (and, if present, third) antigen-binding domain is a humanized antigen-binding domain (i.e., 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 are humanized variable regions.

[0178] In one embodiment, the VH and / or VL of the second (or third, if present) antigen-binding domains include a receptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0179] In one embodiment, the VH of the second (and, if present, third) antigen-binding domain consists of one or more heavy chain framework sequences of SEQ ID NO: 18 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, the VH includes 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: 18. In one embodiment, the VH includes 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 includes an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody containing that sequence retains the ability to bind to TYRP-1. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., FR). In one embodiment, VH contains the amino acid sequence of SEQ ID NO: 18. Optionally, VH contains the amino acid sequence of SEQ ID NO: 18, including post-translational modifications of that sequence.

[0180] In one embodiment, the VL of the second (and, if present, third) antigen-binding domain consists of one or more light chain framework sequences of SEQ ID NO: 22 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, the VL includes 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: 22. In one embodiment, the VL includes 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 includes an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 22. In certain embodiments, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody containing that sequence retains the ability to bind to TYRP-1. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., FR). In one embodiment, VL contains the amino acid sequence of SEQ ID NO: 22. Optionally, VL contains the amino acid sequence of SEQ ID NO: 22, including post-translational modifications of that sequence.

[0181] In one embodiment, the VH of the second (and, if present, third) antigen-binding domain includes 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: 18, and the VL of the second (and, if present, third) antigen-binding domain includes 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: 22. In one embodiment, VH includes the amino acid sequence of SEQ ID NO: 18, and VL includes the amino acid sequence of SEQ ID NO: 22.

[0182] In a further embodiment, the second (and third, if present) antigen-binding domain comprises a VH containing the sequence of SEQ ID NO: 18 and a VL containing the sequence of SEQ ID NO: 22.

[0183] In a further embodiment, the second (and third, if present) antigen-binding domain includes the VH sequence of SEQ ID NO: 18 and the VL sequence of SEQ ID NO: 22.

[0184] In another embodiment, the second (and third, if present) antigen-binding domain comprises VH containing the heavy chain CDR sequence of VH of SEQ ID NO: 18 and VL containing the light chain CDR sequence of VL of SEQ ID NO: 22.

[0185] In a further embodiment, the second (and third, if present) antigen-binding domain includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH in SEQ ID NO: 18 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL in SEQ ID NO: 22.

[0186] In one embodiment, the second (and, if present, third) antigen-binding domain VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 18 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 18. In one embodiment, VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 18 and a framework having at least 95% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 18. In another embodiment, VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 18 and a framework having at least 98% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 18.

[0187] In one embodiment, the second (and, if present, third) antigen-binding domain VL comprises the light chain CDR sequence of VL of SEQ ID NO: 22 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the framework sequence of VL of SEQ ID NO: 22. In one embodiment, VL comprises the light chain CDR sequence of VL of SEQ ID NO: 22 and a framework having at least 95% sequence identity with respect to the framework sequence of VL of SEQ ID NO: 22. In another embodiment, VL comprises the light chain CDR sequence of VL of SEQ ID NO: 22 and a framework having at least 98% sequence identity with respect to the framework sequence of VL of SEQ ID NO: 22.

[0188] EGFRvIII In some embodiments of this disclosure, the second antigen is EGFRvIII, in particular human EGFRvIII (SEQ ID NO: 115).

[0189] In one embodiment, the second (and, if present, third) antigen-binding domain includes a heavy chain variable region (VH) containing the 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, and a light chain variable region (VL) containing the light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 89, LCDR2 of SEQ ID NO: 90, and LCDR3 of SEQ ID NO: 91.

[0190] In one embodiment, the second (and, if present, the third) antigen-binding domain is derived from a humanized antibody. In one embodiment, the second (and, if present, the third) antigen-binding domain is a humanized antigen-binding domain (i.e., the antigen-binding domain of a humanized antibody). In one embodiment, the VH and / or VL of the second (and, if present, the third) antigen-binding domain are humanized variable regions.

[0191] In one embodiment, the VH and / or VL of the second (or third, if present) antigen-binding domains include a receptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0192] In one embodiment, the VH of the second (and, if present, third) antigen-binding domain consists of one or more heavy chain framework sequences of SEQ ID NO: 88 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, the VH includes 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: 88. In one embodiment, the VH includes 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 includes an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 88. In certain embodiments, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody containing it retains the ability of the sequence to bind to EGFRvIII. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 88. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., FR). In one embodiment, VH contains the amino acid sequence of SEQ ID NO: 88. Optionally, VH contains the amino acid sequence of SEQ ID NO: 88, including post-translational modifications of that sequence.

[0193] In one embodiment, the VL of the second (and, if present, third) antigen-binding domain consists of one or more light chain framework sequences of SEQ ID NO: 92 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, the VL includes 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: 92. In one embodiment, the VL includes 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 includes an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 92. In certain embodiments, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody containing it retains the ability of the sequence to bind to EGFRvIII. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 92. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., FR). In one embodiment, VL contains the amino acid sequence of SEQ ID NO: 92. Optionally, VL contains the amino acid sequence of SEQ ID NO: 92, including post-translational modifications of that sequence.

[0194] In one embodiment, the VH of the second (and, if present, third) antigen-binding domain includes 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: 88, and the VL of the second (and, if present, third) antigen-binding domain includes 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: 92. In one embodiment, VH includes the amino acid sequence of SEQ ID NO: 88, and VL includes the amino acid sequence of SEQ ID NO: 92.

[0195] In a further embodiment, the second (and third, if present) antigen-binding domain comprises a VH containing the sequence of SEQ ID NO: 88 and a VL containing the sequence of SEQ ID NO: 92.

[0196] In a further embodiment, the second (and third, if present) antigen-binding domain includes the VH sequence of SEQ ID NO: 88 and the VL sequence of SEQ ID NO: 92.

[0197] In another embodiment, the second (and third, if present) antigen-binding domain comprises VH containing the heavy chain CDR sequence of VH of SEQ ID NO: 88 and VL containing the light chain CDR sequence of VL of SEQ ID NO: 92.

[0198] In a further embodiment, the second (and third, if present) antigen-binding domain includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH in SEQ ID NO: 88 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL in SEQ ID NO: 92.

[0199] In one embodiment, the second (and, if present, third) antigen-binding domain VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 88 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 88. In one embodiment, VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 88 and a framework having at least 95% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 88. In another embodiment, VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 88 and a framework having at least 98% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 88.

[0200] In one embodiment, the second (and, if present, third) antigen-binding domain VL comprises the light chain CDR sequence of VL of SEQ ID NO: 92 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the framework sequence of VL of SEQ ID NO: 92. In one embodiment, VL comprises the light chain CDR sequence of VL of SEQ ID NO: 92 and a framework having at least 95% sequence identity with respect to the framework sequence of VL of SEQ ID NO: 92. In another embodiment, VL comprises the light chain CDR sequence of VL of SEQ ID NO: 92 and a framework having at least 98% sequence identity with respect to the framework sequence of VL of SEQ ID NO: 92.

[0201] In another aspect, the second (and third if present) antigen-binding domain comprises a VH sequence in any of the aspects provided in the above section with respect to EGFRvIII, and a VL sequence in any of the aspects provided in that section, but instead of SEQ ID NO: 85 (HCDR1), 86 (HCDR2), 87 (HCDR3), 88 (VH), 89 (LCDR1), 90 (LCDR2), 91 (LCDR3) and 92 (VL), is based on the following sequences (in row order): TIFF0007848142000001.tif129170

[0202] FolR1 In some aspects of the present disclosure, the second antigen is FolR1, particularly human FolR1 (SEQ ID NO: 137).

[0203] In one aspect, the second (and third if present) antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 124, HCDR2 of SEQ ID NO: 125, and HCDR3 of SEQ ID NO: 126, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9 and LCDR3 of SEQ ID NO: 10.

[0204] In one aspect, the second (and third if present) antigen-binding domain is a humanized antibody (derived). In one aspect, the second (and third if present) antigen-binding domain is a humanized antigen-binding domain (i.e., the antigen-binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second (and third if present) antigen-binding domain is a humanized variable region.

[0205] In one aspect, the VH and / or VL of the second (and third if present) antigen-binding domain comprises a receptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0206] In one embodiment, the VH of the second (and, if present, third) antigen-binding domain consists of one or more heavy chain framework sequences of SEQ ID NO: 123 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, the VH includes 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: 123. In one embodiment, the VH includes an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 123. In one embodiment, the VH includes an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 123. In certain embodiments, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody containing that sequence retains the ability to bind to FolR1. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 123. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., FR). In one embodiment, VH contains the amino acid sequence of SEQ ID NO: 123. Optionally, VH contains the amino acid sequence of SEQ ID NO: 123, including post-translational modifications of that sequence.

[0207] In one embodiment, the VL of the second (and, if present, third) antigen-binding domain consists of one or more light chain framework sequences of SEQ ID NO: 11 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, the VL includes 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 embodiment, the VL includes an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 11. In one embodiment, the VL includes 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 includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody containing that sequence retains the ability to bind to FolR1. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., FR). In one embodiment, VL contains the amino acid sequence of SEQ ID NO: 11. Optionally, VL contains the amino acid sequence of SEQ ID NO: 11, including post-translational modifications of that sequence.

[0208] In one embodiment, the VH of the second (and, if present, third) antigen-binding domain includes 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: 123, and the VL of the second (and, if present, third) antigen-binding domain includes 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 embodiment, VH includes the amino acid sequence of SEQ ID NO: 123, and VL includes the amino acid sequence of SEQ ID NO: 11.

[0209] In a further embodiment, the second (and third, if present) antigen-binding domain comprises a VH containing the sequence of SEQ ID NO: 123 and a VL containing the sequence of SEQ ID NO: 11.

[0210] In a further embodiment, the second (and third, if present) antigen-binding domain includes the VH sequence of SEQ ID NO: 123 and the VL sequence of SEQ ID NO: 11.

[0211] In another embodiment, the second (and third, if present) antigen-binding domain comprises VH containing the heavy chain CDR sequence of VH of SEQ ID NO: 123 and VL containing the light chain CDR sequence of VL of SEQ ID NO: 11.

[0212] In a further embodiment, the second (and third, if present) antigen-binding domain includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH in SEQ ID NO: 123 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL in SEQ ID NO: 11.

[0213] In one embodiment, the second (and, if present, third) antigen-binding domain VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 123 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 123. In one embodiment, VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 123 and a framework having at least 95% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 123. In another embodiment, VH comprises the heavy chain CDR sequence of VH of SEQ ID NO: 123 and a framework having at least 98% sequence identity with respect to the framework sequence of VH of SEQ ID NO: 123.

[0214] In one embodiment, the second (and, if present, third) antigen-binding domain VL comprises the light chain CDR sequence of VL of SEQ ID NO: 11 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the framework sequence of VL of SEQ ID NO: 11. In one embodiment, VL comprises the light chain CDR sequence of VL of SEQ ID NO: 11 and a framework having at least 95% sequence identity with respect to the framework sequence of VL of SEQ ID NO: 11. In another embodiment, VL comprises the light chain CDR sequence of VL of SEQ ID NO: 11 and a framework having at least 98% sequence identity with respect to the framework sequence of VL of SEQ ID NO: 11.

[0215] In one embodiment, the second (and, if present, third) antigen-binding domain comprises the VH sequence in any of the embodiments provided in this section above, and the VL sequence in any of the embodiments provided in this section above.

[0216] Anti-TYRP-1 and anti-EGFRvIII antibodies The Disclosure also provides antibodies that bind to TYRP-1, comprising a VH sequence as in any of the embodiments provided in this Section above with respect to TYRP-1, and a VL sequence as in any of the embodiments provided in this Section above with respect to TYRP-1 (for example, an antibody that binds to TYRP-1 comprising a heavy chain variable region (VH) including 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, and a light chain variable region (VL) including light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 19, LCDR2 of SEQ ID NO: 20, and LCDR3 of SEQ ID NO: 21; or an antibody that binds to TYRP-1 comprising a VH containing the sequence of SEQ ID NO: 18 and a VL containing the sequence of SEQ ID NO: 22).

[0217] The Disclosure also provides antibodies that bind to EGFRvIII, comprising a VH sequence as in any of the embodiments provided in this section above with respect to EGFRvIII, and a VL sequence as in any of the embodiments provided in this section above with respect to EGFRvIII (for example, an antibody that binds to EGFRvIII comprising a heavy chain variable region (VH) including 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, and a light chain variable region (VL) including light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 89, LCDR2 of SEQ ID NO: 90, and LCDR3 of SEQ ID NO: 91; or an antibody that binds to TYRP-1 comprising a VH containing the sequence of SEQ ID NO: 88 and a VL containing the sequence of SEQ ID NO: 92).

[0218] Anti-FolR1 antibody The present invention also provides an antibody that binds to FolR1, comprising a VH sequence as described in any of the embodiments provided in this section above with respect to FolR1, and a VL sequence as described in any of the embodiments provided in this section above with respect to FolR1 (for example, an antibody that binds to FolR1 comprising a heavy chain variable region (VH) including heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 124, HCDR2 of SEQ ID NO: 125, and HCDR3 of SEQ ID NO: 126, and a light chain variable region (VL) including light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10; or an antibody that binds to FolR1 comprising a VH including the sequence of SEQ ID NO: 123 and a VL including the sequence of SEQ ID NO: 11).

[0219] In a further aspect of the present invention, an antibody that binds to FolR1 according to any of the above embodiments may incorporate any of the features described with respect to an antibody that binds to CD3, either alone or in combination (unless such as a binding sequence is clearly specific to the anti-CD3 antibody).

[0220] c) Change of charge The (bispecific) antibodies of the present invention may contain amino acid substitutions in the Fab molecules therein, which are particularly effective in reducing mispairing with heavy chains that do not match the light chains (Bence-Jones type byproducts), which can occur in the production of Fab-based multispecific antibodies with VH / VL exchange in one of the binding arms (two or more in the case of molecules containing two or more antigen-binding Fab molecules) (see also PCT publication number WO2015 / 150447, particularly examples therein which the whole is incorporated herein by reference). The ratio of desirable (bispecific) antibodies to undesirable byproducts, particularly the Bence-Jones type byproducts that occur in multispecific antibodies with VH / VL domain exchange in one of the binding arms, can be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

[0221] Thus, in some embodiments, both the first and second (and third if present) antigen-binding domains of the (bispecific) antibody are Fab molecules, and in one of the antigen-binding domains (particularly the first antigen-binding domain), the variable domains VL and VH of the Fab light and heavy chains are replaced with each other, i) in the constant domain CL of the second (and third if present) antigen-binding domain, the amino acid at position 124 is replaced by a positively charged amino acid (numbering according to Kabat), and in the constant domain CH1 of the second (and third if present) antigen-binding domain, the amino acid at position 147 or 213 is replaced by a negatively charged amino acid (numbering according to the Kabat Eu index); or ii) in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is replaced by a positively charged amino acid (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or 213 is replaced by a negatively charged amino acid (numbering according to the Kabat EU index).

[0222] (Bispecific) antibodies do not contain both of the modifications referred to in i) and ii). The constant domains CL and CH1 of the antigen-binding domain having VH / VL exchange are not replaced with each other (i.e., remain unexchanged).

[0223] In a more specific embodiment, i) in the constant domain CL of the second (and third if present) antigen-binding domain, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (numbering according to Kabatt), and in the constant domain CH1 of the second (and third if present) antigen-binding domain, the amino acid at position 147 or 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index); or ii) In the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is substituted with lysine (K), arginine (R), or histidine (H) (as numbered by Kabatt), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or position 213 is substituted with glutamic acid (E) or aspartic acid (D) (as numbered by the Kabatt EU index).

[0224] In one embodiment, in the constant domain CL of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is lysine (K), arginine (R), or histidine (H) (numbered by Kabatt), and in the constant domain CH1 of the second (and, if present, third) antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is an acid (D) independently substituted by glutamic acid (E) or aspartic acid (numbered by Kabatt EU index).

[0225] In a further embodiment, in the constant domain CL of the second (or third, if present) antigen-binding domain, the amino acid at position 124 is substituted with lysine (K), arginine (R), or histidine (H) (as numbered by Kabatt), and in the constant domain CH1 of the second (or third, if present) antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) or aspartic acid (D) (as numbered by the Kabatt EU index).

[0226] In a preferred embodiment, in the constant domain CL of the second (or third, if present) antigen-binding domain, the amino acid at position 124 is substituted with lysine (K), arginine (R), or histidine (H) (as numbered by Kabatt), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (as numbered by Kabatt), and in the constant domain CH1 of the second (or third, if present) antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) or aspartic acid (D) (as numbered by the Kabatt EU index), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (as numbered by the Kabatt EU index).

[0227] In a more preferred embodiment, in the constant domain CL of the second (or third, if present) antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (numbered by Kabat), the amino acid at position 123 is substituted with lysine (K) (numbered by Kabat), and in the constant domain CH1 of the second (or third, if present) antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (numbered by the Kabat EU index), and the amino acid at position 213 is substituted with glutamic acid (E) (numbered by the Kabat EU index).

[0228] In a more preferred embodiment, in the constant domain CL of the second (or third, if present) antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with arginine (R) (Kabat numbering), and in the constant domain CH1 of the second (or third, if present) antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0229] In a preferred embodiment, when the amino acid substitutions in the above-described region occur in the constant domain CL and the constant domain CH1 of the second (and third, if present) antigen-binding domain, the constant domain CL of the second (and third, if present) antigen-binding domain is a kappa isotype.

[0230] Alternatively, the amino acid substitutions described above may be made in the constant domain CL and constant domain CH1 of the first antigen-binding domain, instead of the constant domain CL and the constant domain CH1 of the second (or third, if present) antigen-binding domain. In a preferred embodiment, the constant domain CL of the first antigen-binding domain is a kappa isotype.

[0231] 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) (as numbered by Kabat), and 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 is by the Kabat EU index).

[0232] In a further embodiment, in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is substituted with lysine (K), arginine (R), or histidine (H) (as numbered by Kabat), and 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) (as numbered by the Kabat EU index).

[0233] In yet another embodiment, in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is substituted with lysine (K), arginine (R), or histidine (H) (as numbered by Kabatt), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (as numbered by Kabatt), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) or aspartic acid (D) (as numbered by the Kabatt EU index), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (as numbered by the Kabatt EU index).

[0234] In one embodiment, in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (numbered by Kabat), and the amino acid at position 123 is substituted with lysine (K) (numbered by Kabat), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (numbered by Kabat EU index), and the amino acid at position 213 is substituted with glutamic acid (E) (numbered by Kabat EU index).

[0235] In another embodiment, in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), and the amino acid at position 123 is substituted with arginine (R) (Kabat numbering), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0236] In a preferred embodiment, the (bispecific) antibody of the present invention is (a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other, and the first antigen-binding domain comprises a heavy chain variable region (VH) containing heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 5, and a light chain variable region (VL) containing light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10, (b) Second and optionally third antigen-binding domains that bind to the target antigen; In the constant domain CL of the second (and third, if present) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (in a preferred embodiment, independently substituted with lysine (K) or arginine (R)) (Kabatt numbering), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (in a preferred embodiment, independently substituted with lysine (K) or arginine (R)) (Kabatt numbering), and in the constant domain CH1 of the second (and third, if present) antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabatt EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabatt EU index numbering).

[0237] d) Multispecific antibody format The (bispecific and / or multispecific) antibody according to the present invention can have various configurations. An exemplary configuration is shown in Figure 1.

[0238] 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, etc. antigen-binding domains may be referred to herein as the first, second, third, etc. Fab molecules, respectively.

[0239] In one embodiment, the first and second antigen-binding domains of a (bispecific) antibody are optionally fused to each other via a peptide linker. In a preferred embodiment, the first and second antigen-binding domains are each a Fab molecule. In one 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. In embodiments where (i) 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, or (ii) 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 Fab light chain of the first antigen-binding domain and the Fab light chain of the second antigen-binding domain may optionally be fused to each other via a peptide linker.

[0240] Antibodies possessing a single antigen-binding domain (such as a Fab molecule) (bispecific antibodies) can specifically bind to a second antigen, such as a target cell antigen like FolR1 (e.g., A, D, G, H, K, L in Figure 1), which is particularly useful when internalization of the second antigen is expected after binding of a high-affinity antigen-binding domain. In such cases, the presence of two or more antigen-binding domains specific to the second antigen may enhance the internalization of the second antigen, thereby reducing its availability.

[0241] However, in other cases, it is advantageous to have a second antigen, such as a bispecific antibody containing two or more antigen-binding domains (e.g., Fab molecules) specific to the target cell antigen (see examples shown in Figures 1B, 1C, 1E, 1F, 1I, 1J, 1M, 1N, 33A, 33B), which can, for example, optimize targeting to the target site or enable cross-linking of the target cell antigen.

[0242] Therefore, in a preferred embodiment, the (multispecific, e.g., bispecific) antibody according to the present invention includes a third antigen-binding domain.

[0243] In one embodiment, the third antigen-binding domain binds to a second antigen, such as a target cell antigen like FolR1. In another embodiment, the third antigen-binding domain is a Fab molecule.

[0244] In one embodiment, the third antigen domain is identical to the second antigen-binding domain.

[0245] In some embodiments, the third and second antigen-binding domains are each Fab molecules, 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 configuration (i.e., conventional or crossover). Furthermore, in these embodiments, the third antigen-binding domain contains the same amino acid substitutions as the second antigen-binding domain, if any. For example, the amino acid substitutions described herein as "charge modifications" occur in the constant domains CL and CH1 of the second and third antigen-binding domains, respectively. Alternatively, the amino acid substitutions may occur in the constant domain CL and CH1 of the first antigen-binding domain (which is also a Fab molecule in a preferred embodiment), but not in the constant domains CL and CH1 of the second and third antigen-binding domains.

[0246] Similar to the second antigen-binding domain, the third antigen-binding domain is preferably a conventional Fab molecule. All Fab molecules may share a common light chain. However, embodiments in which the second and third antigen-binding domains are crossover Fab molecules (and the first antigen-binding domain is a conventional Fab molecule) are also intended. Thus, in some embodiments, the second and third antigen-binding domains are each conventional Fab molecules, and the first antigen-binding domain is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL, or the constant domains CL and CH1 of the Fab heavy and light chains, are exchanged / substituted for each other. In other embodiments, the second and third antigen-binding domains are each crossover Fab molecules, and the first antigen-binding domain is a conventional Fab molecule.

[0247] If 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 bind to a second antigen, particularly a target cell antigen such as FolR1.

[0248] As shown above and in Figures 33A to 33E, in one embodiment, the T cell activating bispecific antigen-binding molecule comprises at least two Fab fragments having the same light chain (VLCL) and different heavy chains (VHCL) to confer specificity to two different antigens, namely, one Fab fragment that can specifically bind to the T cell activating antigen CD3 and the other Fab fragment that can specifically bind to the target cell antigen FolR1.

[0249] In a preferred embodiment, the (multispecific) antibody of the present invention comprises an Fc domain composed of first and second subunits. The first and second subunits of the Fc domain are capable of stable association.

[0250] The (multispecific, e.g., bispecific) antibodies according to the present invention may have different configurations. That is, the first, second (and optionally third) antigen-binding domains may be fused to each other and to the Fc domain in different ways. The components may be fused to each other directly, or preferably via one or more suitable peptide linkers. When the fusion of the Fab molecule is at the N-terminus of a subunit of the Fc domain, it is typically via an immunoglobulin hinge region.

[0251] In some embodiments, the first and second antigen-binding domains are each a Fab molecule, and the first antigen-binding domain 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 embodiments, the second antigen-binding domain may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding domain of the Fab heavy chain, or to the other N-terminus of the subunit of the Fc domain. In preferred embodiments, the second antigen-binding domain is a conventional Fab molecule, and the first antigen-binding domain is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL, or the constant domains CL and CH1 of the Fab heavy and light chains, are exchanged / substituted for each other. In other embodiments, the second antigen-binding domain is a crossover Fab molecule, and the first antigen-binding domain is a conventional Fab molecule.

[0252] In one embodiment, the first and second antigen-binding domains are each a Fab molecule, the first antigen-binding domain 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, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding domain. In a particular embodiment, a (multispecific, e.g., bispecific) antibody consists essentially of first and second Fab molecules, the Fc domain is composed of first and second subunits and optionally one or more peptide linkers, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first Fab molecule, and the first 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. Such configurations are schematically shown in Figures 1G and 1K (in these examples, the first antigen-binding domain is a 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 can be further fused with each other.

[0253] In another embodiment, the first and second antigen-binding domains are each Fab molecules, and the first and second antigen-binding domains are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In a particular embodiment, a (multispecific, e.g., bispecific) antibody consists essentially of first and second Fab molecules, and the Fc domain is composed of first and second subunits and optionally one or more peptide linkers, and the first and second Fab molecules are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. Such configurations are schematically shown in Figures 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 a conventional Fab molecule) and Figure 33E (in this example, the light chains of the first and second antigen-binding domains are identical). The first and second Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a preferred embodiment, the first and second Fab molecules are each fused to an Fc domain via an immunoglobulin hinge region. In a particular embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, in particular when the Fc domain is an IgG1Fc domain.

[0254] In some embodiments, the first and second antigen-binding domains are each a Fab molecule, and the second antigen-binding domain 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 embodiments, the first antigen-binding domain may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding domain of the Fab heavy chain, or (as described above) to the other N-terminus of the subunit of the Fc domain. In preferred embodiments, the second antigen-binding domain is a conventional Fab molecule, and the first antigen-binding domain is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL, or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged / substituted for each other. In other embodiments, the second antigen-binding domain is a crossover Fab molecule, and the first antigen-binding domain is a conventional Fab molecule.

[0255] In one embodiment, the first and second antigen-binding domains are each a Fab molecule, the second antigen-binding domain 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, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding domain. In a particular embodiment, a (multispecific, e.g., bispecific) antibody consists essentially of a first and a second Fab molecule, the Fc domain being composed of a first and a second subunit and optionally one or more peptide linkers, the first Fab molecule being 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 second Fab molecule being fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such configurations are schematically shown in Figures 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 a conventional Fab molecule) and Figures 33C and 3D (in these examples, the light chains of the first and second antigen-binding domains are identical). Optionally, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule can be further fused with each other.

[0256] In a preferred embodiment, the first and third antigen-binding domains are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, 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 Fab molecule. In a particular embodiment, a (multispecific, e.g., bispecific) antibody consists essentially of first, second, and third Fab molecules, where the Fc domain is composed of first and second subunits and optionally one or more peptide linkers, 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, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The first and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a preferred embodiment, the first and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a particular embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, especially when the Fc domain is an IgG1Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

[0257] In another such embodiment, the second and third antigen-binding domains are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding domain. In a particular embodiment, a (multispecific, e.g., bispecific) antibody consists essentially of first, second, and third Fab molecules, where the Fc domain is composed of first and second subunits and optionally one or more peptide linkers, 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, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The second and third Fab molecules may be fused to the Fc domain directly or via peptide linkers. In a preferred embodiment, the second and third Fab molecules are each fused to an Fc domain via an immunoglobulin hinge region. In a particular embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, in particular when the Fc domain is an IgG1Fc domain. Optionally, the Fab light chains of the first Fab molecule and the second Fab molecule may be further fused to each other.

[0258] In the composition of a (multispecific) antibody in which a Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each subunit of the Fc domain via the immunoglobulin hinge region, the two Fab molecules, with their hinge regions and Fc domains, essentially form an immunoglobulin molecule. In a preferred embodiment, the immunoglobulin molecule is an IgG-class immunoglobulin. In a more preferred embodiment, the immunoglobulin is an IgG1 subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4 subclass immunoglobulin. In a further preferred embodiment, the immunoglobulin is a human immunoglobulin. In yet another embodiment, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the immunoglobulin includes a human constant region, particularly a human Fc region.

[0259] In some of the (multispecific) antibodies of the present invention, the Fab light chain of a first Fab molecule and the Fab light chain of a second Fab molecule are optionally fused to each other via a peptide linker. Depending on the configuration of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. The fusion of the Fab light chains of the first and second Fab molecules further reduces mispairing of mismatched Fab heavy and light chains and also reduces the number of plasmids required for the expression of some of the (multispecific) antibodies of the present invention.

[0260] The antigen-binding domain may be fused to the Fc domain, directly to each other, or via a peptide linker, and may contain one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n (SG4) n (G4S) n Or G4 (SG4) nA peptide linker is included. "n" is generally an integer from 1 to 10, typically from 2 to 4. In one embodiment, the peptide linker has at least 5 amino acid lengths, in one embodiment 5 to 100 amino acid lengths, and in further embodiments 10 to 50 amino acid lengths. In one embodiment, the peptide linker is (GxS) n or (GxS) n G m (wherein G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3), or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3), in one embodiment x=4, and n=2 or 3, in further embodiments 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. An exemplary peptide linker suitable for linking the Fab heavy chains of the first and second Fab fragments includes sequence (D)-(G4S)2 (SEQ ID NOs: 118 and 119). Another suitable such linker includes sequence (G4S)4. Furthermore, the linker may include (part of) an immunoglobulin hinge region. In particular, when a Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused via the immunoglobulin hinge region or a portion thereof, with or without the presence of an additional peptide linker.

[0261] In some embodiments, the bispecific antigen-binding molecule includes a common light chain. In one embodiment, the present invention provides a bispecific antigen-binding molecule comprising a first and a second antigen-binding moiety, one of which is a Fab molecule capable of specifically binding to CD3, and the other being a Fab molecule capable of specifically binding to CD3, wherein the first and second Fab molecules have the same VLCL light chain. In one embodiment, the same light chain (VLCL) includes the light chain CDR of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. In one embodiment, the same light chain (VLCL) includes SEQ ID NO: 133.

[0262] In one embodiment, the present invention provides a T cell-activating bispecific antigen-binding molecule, the T cell-activating bispecific antigen-binding molecule comprising: (i) a first antigen-binding moiety, which is a Fab molecule that can specifically bind to CD3, and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs. 2, SEQ ID NOs. 3 and SEQ ID NOs. 5, and at least one light chain CDR selected from the group consisting of SEQ ID NOs. 8, SEQ ID NOs. 9 and SEQ ID NOs. 10; and (ii) a second antigen-binding moiety, which comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs. 124, SEQ ID NOs. 125 and SEQ ID NOs. 126, and at least one light chain CDR selected from the group consisting of SEQ ID NOs. 8, SEQ ID NOs. 9 and SEQ ID NOs. 10.

[0263] In one such embodiment, the CD3 antigen-binding portion includes the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 3, the heavy chain CDR3 of SEQ ID NO: 5, the light chain CDR1 of SEQ ID NO: 8, the light chain CDR2 of SEQ ID NO: 9, and the light chain CDR3 of SEQ ID NO: 10, and the FolR1 antigen-binding portion 5 includes the heavy chain CDR1 of SEQ ID NO: 124, the heavy chain CDR2 of SEQ ID NO: 125, the heavy chain CDR3 of SEQ ID NO: 126, the light chain CDR1 of SEQ ID NO: 8, the light chain CDR2 of SEQ ID NO: 9, and the light chain CDR3 of SEQ ID NO: 10.

[0264] In one embodiment, the present invention provides a T cell-activating bispecific antigen-binding molecule comprising: (i) a first antigen-binding moiety which is a Fab molecule capable of specifically binding to CD3, comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO: 7 and a variable light chain containing the amino acid sequence of SEQ ID NO: 11; and (ii) a second antigen-binding moiety which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO: 123 and a variable light chain containing the amino acid sequence of SEQ ID NO: 11.

[0265] In one embodiment, the present invention provides a T cell-activating bispecific antigen-binding molecule comprising: (i) a first antigen-binding moiety which is a Fab molecule capable of specifically binding to CD3, comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO: 7 and a variable light chain containing the amino acid sequence of SEQ ID NO: 11; and (ii) a second antigen-binding moiety which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO: 123 and a variable light chain containing the amino acid sequence of SEQ ID NO: 11.

[0266] In one embodiment, the T cell-activating bispecific antigen-binding molecule further comprises (iii) a third antigen-binding moiety (which is a Fab molecule) that can specifically bind to FolR1. In one such embodiment, the second and third antigen-binding moieties, which can specifically bind to FolR1, include the same heavy chain complementarity-determining region (CDR) and light chain CDR sequence. In one such embodiment, the third antigen-binding moiety is identical to the second antigen-binding moiety.

[0267] Therefore, in one embodiment, the present invention provides a T cell-activating bispecific antigen-binding molecule comprising the following: (i) A first antigen-binding moiety, which is a Fab molecule capable of specifically binding to CD3, comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs. 37, SEQ ID NOs. 38, and SEQ ID NOs. 39, and at least one light chain CDR selected from the group consisting of SEQ ID NOs. 32, SEQ ID NOs. 33, and SEQ ID NOs. 34; and (ii) A second antigen-binding moiety, which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs. 16, SEQ ID NOs. 17, and SEQ ID NOs. 18, and at least one light chain CDR selected from the group consisting of SEQ ID NOs. 32, SEQ ID NOs. 33, and SEQ ID NOs. 34; (iii) A third antigen-binding moiety, which is a Fab molecule that can specifically bind to folate receptor 1 (FolR1), and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs. 16, SEQ ID NOs. 17, and SEQ ID NOs. 18, and at least one light chain CDR selected from the group consisting of SEQ ID NOs. 32, SEQ ID NOs. 5, 33, and SEQ ID NOs. 34.

[0268] In one such embodiment, the CD3 antigen-binding portion includes the heavy chain CDR1 of SEQ ID NO: 37, the heavy chain CDR2 of SEQ ID NO: 38, the heavy chain CDR3 of SEQ ID NO: 39, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO: 34. The FolR1 antigen-binding portion includes the heavy chain CDR1 of SEQ ID NO: 16, the heavy chain CDR2 of SEQ ID NO: 17, the heavy chain CDR3 of SEQ ID NO: 18, the light chain CDR1 of SEQ ID NO: 32, the light chain CDR2 of SEQ ID NO: 33, and the light chain CDR3 of SEQ ID NO: 34.

[0269] In one embodiment, the present invention provides a T cell-activating bispecific antigen-binding molecule comprising the following: (i) A first antigen-binding moiety, which is a Fab molecule that can specifically bind to CD3, comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO: 36 and a variable light chain containing the amino acid sequence of SEQ ID NO: 31, (ii) A second antigen-binding moiety which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain containing the amino acid sequence of SEQ ID NO: 31. (iii) A third antigen-binding moiety which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), comprising a variable heavy chain containing the amino acid sequence of SEQ ID NO: 15 and a variable light chain containing the amino acid sequence of SEQ ID NO: 31.

[0270] Therefore, in one embodiment, the present invention relates to a bispecific molecule having an identical light chain and a corresponding modified heavy chain, each having at least two binding sites that confer specific binding to the T cell activating antigen CD3 and the target cell antigen FolR1, respectively. This so-called "common light chain" principle, that is, combining two binders that share one light chain but have individual specificities, prevents light chain mispairing. Consequently, there are fewer byproducts during production, and it becomes easier to prepare homogeneous T cells that activate the bispecific antigen-binding molecule.

[0271] In some embodiments, the T cell-activating bispecific antigen-binding molecule includes an Fc domain composed of first and second subunits capable of stable association. Exemplary embodiments of T cell-activating bispecific antigen-binding molecules including the Fc domain are described below.

[0272] In one embodiment, the present invention relates to an antibody (multispecific, e.g., bispecific), a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule and comprises a heavy chain variable region (VH) including the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 2, the HCDR2 of SEQ ID NO: 3, and the HCDR3 of SEQ ID NO: 5, and a light chain variable region (VL) including the light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, the LCDR2 of SEQ ID NO: 9, and the LCDR3 of SEQ ID NO: 10; b) A second antigen-binding domain, which binds to a second antigen, particularly a target cell antigen, more specifically FolR1, and is a Fab molecule containing a light chain variable region (VL) including the light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10; c) An Fc domain consisting of a first and a second subunit; We provide antibodies that include, (i) The first antigen-binding domain under a) is fused to the C-terminus of the Fab heavy chain, and the second antigen-binding domain under b) is fused to the N-terminus of one of the subunits of the Fc domain under c), or (ii) The second antigen-binding domain under (b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding domain under (a) of the Fab heavy chain, and the first antigen-binding domain under (a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under (c).

[0273] In a preferred scenario, the present invention relates to a (multispecific) antibody, a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule and comprises a heavy chain variable region (VH) including the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 2, the HCDR2 of SEQ ID NO: 3, and the HCDR3 of SEQ ID NO: 5, and a light chain variable region (VL) including the light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, the LCDR2 of SEQ ID NO: 9, and the LCDR3 of SEQ ID NO: 10; b) Second and third antigen-binding domains, each comprising a Fab molecule that binds to the second and third antigens, particularly target cell antigens, more specifically FolR1, and includes a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10; c) An Fc domain consisting of a first and a second subunit; We provide antibodies that include, (i) The first antigen-binding domain under a) 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 under b), and the second antigen-binding domain under b) and the third antigen-binding domain under 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 under c), respectively, or (ii) The second antigen-binding domain under (b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding domain under (a) of the Fab heavy chain, and the first antigen-binding domain under (a) and the third antigen-binding domain under (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 under (c), respectively.

[0274] In another embodiment, the present invention relates to a (multispecific) antibody, a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule and comprises a heavy chain variable region (VH) including the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 2, the HCDR2 of SEQ ID NO: 3, and the HCDR3 of SEQ ID NO: 5, and a light chain variable region (VL) including the light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, the LCDR2 of SEQ ID NO: 9, and the LCDR3 of SEQ ID NO: 10; b) A second antigen-binding domain, which binds to a second antigen, particularly a target cell antigen, more specifically FolR1, and is a Fab molecule containing a light chain variable region (VL) including the light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10; c) An Fc domain consisting of a first and a second subunit; We provide antibodies that include, (i) The first antigen-binding domain under a) and the second antigen-binding domain under 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 under c), respectively.

[0275] According to any of the above embodiments, components of an antibody (e.g., a multispecific, e.g., bispecific) (e.g., a Fab molecule, Fc domain) can be fused directly or via various linkers, particularly peptide linkers containing one or more amino acids, typically about 2 to 20 amino acids, which are described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n (SG4) n (G4S)n Or G4 (SG4) n It contains a peptide linker, where n is generally an integer between 1 and 10, typically between 2 and 4.

[0276] In a preferred scenario, the present invention relates to a (bispecific) antibody, a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is Fab and comprises the heavy chain variable region (VH) including the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 5; b) Second and third antigen-binding domains that bind to FolR1, wherein the second and third antigen-binding domains are each Fab molecules and include heavy chain variable regions (VH) comprising the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 124, HCDR2 of SEQ ID NO: 125, and HCDR3 of SEQ ID NO: 126; c) Fc domains consisting of first and second subunits; and We provide antibodies that include, The first antigen-binding domain and the second and third antigen-binding domains include light chain variable regions (VLs) containing the light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10.

[0277] In one aspect of these embodiments of the present invention, 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), 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) (numbered according to the Kabat EU index).

[0278] In further embodiments of these aspects of the present invention, the first subunit of the Fc domain is further modified by substituting the serine residue at position 354 with a cysteine ​​residue (S354C), the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is substituted with a cysteine ​​residue), and the second subunit of the Fc domain is further modified by substituting the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index).

[0279] In yet another aspect of these embodiments of the present invention, in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (numbered according to the Kabat EU index).

[0280] In yet another aspect of these aspects of the present invention, the Fc domain is a human IgG1Fc domain.

[0281] In a preferred specific embodiment, the bispecific antibody comprises 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: 127, 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: 128, and a polypeptide (particularly three polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In a further preferred specific embodiment, the (multispecific, e.g., bispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 127, a polypeptide comprising the amino acid sequence of SEQ ID NO: 128, and a polypeptide (particularly three polypeptides) comprising the amino acid sequence of SEQ ID NO: 129.

[0282] In a preferred embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 127, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 128, and a polypeptide (particularly three polypeptides) comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129.

[0283] In a specific embodiment, the bispecific antibody comprises 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: 130, 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: 128, and a polypeptide (in particular three polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In a further specific embodiment, the (multispecific, e.g., bispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 130, a polypeptide comprising the amino acid sequence of SEQ ID NO: 128, and a polypeptide (in particular three polypeptides) comprising the amino acid sequence of SEQ ID NO: 129.

[0284] In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a polypeptide containing an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 130, a polypeptide containing an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 128, and a polypeptide (particularly three polypeptides) containing an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a polypeptide containing an amino acid sequence of SEQ ID NO: 130, a polypeptide containing an amino acid sequence of SEQ ID NO: 128, and a polypeptide (particularly three polypeptides) containing an amino acid sequence of SEQ ID NO: 129.

[0285] In a specific embodiment, the bispecific antibody comprises 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: 131, 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: 132, and a polypeptide (in particular three polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In a further specific embodiment, the (multispecific, e.g., bispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 131, a polypeptide comprising the amino acid sequence of SEQ ID NO: 132, and a polypeptide (in particular three polypeptides) comprising the amino acid sequence of SEQ ID NO: 129.

[0286] In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a polypeptide containing an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 131, a polypeptide containing an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 132, and a polypeptide (particularly three polypeptides) containing an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1-1, the antibody comprising a polypeptide containing an amino acid sequence of SEQ ID NO: 131, a polypeptide containing an amino acid sequence of SEQ ID NO: 132, and a polypeptide (particularly three polypeptides) containing an amino acid sequence of SEQ ID NO: 129.

[0287] In a specific embodiment, the bispecific antibody comprises 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: 133, 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: 134, and a polypeptide (in particular three polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In a further specific embodiment, the (multispecific, e.g., bispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 133, a polypeptide comprising the amino acid sequence of SEQ ID NO: 134, and a polypeptide (in particular three polypeptides) comprising the amino acid sequence of SEQ ID NO: 129.

[0288] In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a polypeptide containing an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 133, a polypeptide containing an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 134, and a polypeptide (particularly three polypeptides) containing an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a polypeptide containing an amino acid sequence of SEQ ID NO: 133, a polypeptide containing an amino acid sequence of SEQ ID NO: 134, and a polypeptide (particularly three polypeptides) containing an amino acid sequence of SEQ ID NO: 129.

[0289] In a specific embodiment, the bispecific antibody comprises 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: 135, 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: 136, and a polypeptide (in particular two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In a further specific embodiment, the (multispecific, e.g., bispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 135, a polypeptide comprising the amino acid sequence of SEQ ID NO: 136, and a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 129.

[0290] In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 135, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 136, and a polypeptide (particularly two polypeptides) comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 129. In one embodiment, the present invention provides a bispecific antibody that binds to CD3 and FolR1, the antibody comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 135, a polypeptide comprising an amino acid sequence of SEQ ID NO: 136, and a polypeptide (particularly two polypeptides) comprising an amino acid sequence of SEQ ID NO: 129.

[0291] e) Fc domain variant In a preferred embodiment, the antibody of the present invention (multispecific, e.g., bispecific) comprises an Fc domain composed of a first and a second subunit.

[0292] The Fc domain of a (multispecific, e.g., bispecific) antibody consists of a pair of polypeptide chains containing the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each subunit contains the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably bind to each other. In one embodiment, the (multispecific, e.g., bispecific) antibody of the present invention comprises one or fewer Fc domains.

[0293] In one embodiment, the Fc domain of the antibody (multispecific, e.g., bispecific) is an IgG Fc domain. In a preferred embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing an amino acid substitution, particularly the amino acid substitution S228P, at position S228 (Kabat EU index numbering). This amino acid substitution reduces Fab arm exchange in vivo of the IgG4 antibody (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In yet another embodiment, the Fc domain is a human Fc domain. In an even more preferred embodiment, the Fc domain is a human IgG1 Fc domain. An exemplary sequence of the human IgG1 Fc region is given in Sequence ID No. 117.

[0294] f) Modification of the Fc domain that promotes heterodimerization The (multispecific, e.g., bispecific) antibody according to the present invention comprises different antigen-binding domains that can be fused to one or the other of two subunits of the Fc domain, and therefore the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization result in several possible combinations of the two polypeptides. Therefore, to improve the yield and purity of the (multispecific, e.g., bispecific) antibody in recombinant production, it is advantageous to introduce modifications to the Fc domain of the (multispecific, e.g., bispecific) antibody that promote the binding of the desired polypeptide.

[0295] Therefore, in a preferred embodiment, the Fc domain of the (multispecific, e.g., bispecific) antibody according to the present invention includes modifications that promote the association of the first and second subunits of the Fc domain. The site where the most extensive protein-protein interaction occurs between the two subunits of the human IgG Fc domain is the CH3 domain of the Fc domain. Therefore, in one embodiment, the modifications are located in the CH3 domain of the Fc domain.

[0296] Several approaches exist for modifying the CH3 domain of the Fc domain to force heterodimerization, which are well described in, for example, WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, and WO 2013096291. Typically, in all such approaches, the CH3 domains of the first subunit of the Fc domain and the second subunit of the Fc domain are both designed complementaryly, so that each CH3 domain (or the heavy chain comprising it) cannot homodimerize on its own, but is forced to heterodimerize with other complementaryly engineered CH3 domains (so that the first and second CH3 domains heterodimerize and homodimers are not formed between two first or two second CH3 domains). These different approaches to improving heavy chain heterodimerization are intended as alternatives in combination with heavy-light chain modifications in antibodies that reduce heavy / light chain mispairing and Bence Jones-type byproducts (multispecific, e.g., bispecific) (e.g., exchange / substitution of VH and VL in one binding arm, and introduction of substitution of charged amino acids with opposite charges at the CH1 / CL interface).

[0297] In certain embodiments, the modification that facilitates the binding of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0298] The knob-into-hole technique is described, for example, in US5,731,168; US7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a bump ("knob") at the interface of a first polypeptide so that the bump can be positioned within a cavity to promote heterodimer formation and prevent homodimer formation, and introducing a corresponding cavity ("hole") at the interface of a second polypeptide. The bump is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the bump is created at the interface of the second polypeptide by replacing the large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine).

[0299] Therefore, in a preferred embodiment, the CH3 domain of the first subunit of the Fc domain of the antibody (multispecific, e.g., bispecific) is substituted with an amino acid residue having a larger side chain volume, thereby creating a bulge within the CH3 domain of the first subunit that can be placed in a cavity within the CH3 domain of the second subunit, and the CH3 domain of the second subunit of the Fc domain is substituted with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit in which the bulge within the CH3 domain of the first subunit can be placed.

[0300] Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0301] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0302] The ridges and cavities can be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0303] In certain embodiments, the first subunit ("knob" subunit) of the Fc domain (the CH3 domain) is modified by substituting the threonine residue at position 366 with a tryptophan residue (T366W), and the second subunit ("whole" subunit) of the Fc domain (the CH3 domain) is modified by substituting the tyrosine residue at position 407 with a valine residue (Y407V). In one embodiment, the second subunit of the Fc domain is further modified by substituting the threonine residue at position 366 with a serine residue (T366S) and the leucine residue at position 368 with an alanine residue (L368A) (numbered by Kabat EU).

[0304] In a further embodiment, the first subunit of the Fc domain is further modified by substituting the serine residue at position 354 with a cysteine ​​residue (S354C), the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is substituting with a cysteine ​​residue), and the second subunit of the Fc domain is further modified by substituting the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). The introduction of these two cysteine ​​residues forms a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0305] In a preferred embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the Kabat EU index).

[0306] In a preferred embodiment, the antigen-binding domain that binds to CD3 is fused (including the "knob" modification) to the first subunit of the Fc domain (optionally, via a second antigen-binding domain that binds to a second antigen (i.e., FolR1), and / or via a peptide linker). While we do not wish to be bound by theory, fusion of the CD3-binding antigen-binding domain to the knob-containing subunit of the Fc domain further minimizes the production of antibodies containing two antigen-binding domains that bind to CD3 (steric collision of the two knob-containing polypeptides).

[0307] Other techniques for CH3 modification to carry out heterodimerization are intended as alternatives according to the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, and WO 2013 / 096291.

[0308] In one embodiment, the heterodimerization approach described in EP 1870459 is used instead. This approach is based on introducing charged amino acids with opposite charges at specific amino acid positions at the CH3 / CH3 domain interface between two subunits of the Fc domain. A particular embodiment of the (multispecific) antibody of the present invention is an amino acid mutation R409D;K370E in one of the two CH3 domains (of the Fc domain), and an amino acid mutation D399K;E357K in the other CH3 domain of the Fc domain (numbered by the Kabat EU index).

[0309] In another embodiment, the (multispecific, e.g., bispecific) antibodies of the present invention include the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, as well as the amino acid mutations R409D; K370E and D399K; E357K within the CH3 domain of the first subunit of the Fc domain (numbered by the Kabat EU index).

[0310] In another embodiment, the (multispecific, e.g., bispecific) antibody of the present invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the (multispecific, e.g., bispecific) antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and further comprises 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 (all numbering is by Kabat EU index).

[0311] In one embodiment, the heterodimerization approach described in WO2013 / 157953 is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366K, and the second CH3 domain contains the amino acid mutation L351D (numbered according to the Kabat EU index). In a further embodiment, the first CH3 domain contains a further amino acid mutation L351K. In a further embodiment, the second CH3 domain further contains amino acid mutations selected from Y349E, Y349D, and L368E (especially L368E) (numbered according to the Kabat EU index).

[0312] In one embodiment, the heterodimerization approach described in WO2012 / 058768 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises further amino acid mutations at positions T411, D399, S400, F405, N390, or K392, for example 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, K392L, K392F, or K392E (Kabat Selected from (numbered according to the EU index). In a further embodiment, the first CH3 domain contains amino acid mutations L351Y and Y407A, and the second CH3 domain contains amino acid mutations T366V and K409F. In a further embodiment, the first CH3 domain contains amino acid mutation Y407A, and the second CH3 domain contains amino acid mutations T366A and K409F. In a further embodiment, the second CH3 domain further contains amino acid mutations K392E, T411E, D399R and S400R (numbered according to the Kabat EU index).

[0313] In one embodiment, the heterodimerization approach described in WO2011 / 143545 is used as an alternative, along with amino acid modification at a position selected from the group consisting of 368 and 409 (numbered according to the Kabat EU index).

[0314] In one embodiment, the heterodimerization approach described in WO2011 / 090762, which also uses the knob-in-to-hole technique described above, is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366W and the second CH3 domain contains the amino acid mutation Y407A. In another embodiment, the first CH3 domain contains the amino acid mutation T366Y and the second CH3 domain contains the amino acid mutation Y407T (numbered according to the Kabat EU index).

[0315] In one embodiment, the antibody (multispecific, e.g., bispecific) or its Fc domain is of the IgG2 subclass, and the heterodimerization approach described in WO2010 / 129304 is used instead.

[0316] In another embodiment, modifications that promote the association of the first and second subunits of the Fc domain include modifications that mediate an electrostatic steering effect, as described, for example, in PCT Publication WO2009 / 089004. Generally, this method involves substituting one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues so that homodimerization is electrostatically unfavorable, while heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain includes an amino acid substitution of K392 or N392 with a charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), particularly K392D or N392D), and the second CH3 domain includes an amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), particularly D399K, E356K, D356K, or E357K, more specifically D399K and E356K). In a further embodiment, the first CH3 domain further includes an amino acid substitution of K409 or R409 with a charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), particularly K409D or R409D). In a further embodiment, the first CH3 domain further or alternatively comprises an amino acid substitution of negatively charged amino acids (e.g., glutamic acid (E) or aspartic acid (D)) at K439 and / or K370 (all numbering is according to the Kabat EU index).

[0317] In a further embodiment, the heterodimerization approach described in WO2007 / 147901 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations K253E, D282K, and K322D, and the second CH3 domain contains amino acid mutations D239K, E240K, and K292D (numbered according to the Kabat EU index).

[0318] In yet another embodiment, the heterodimerization approach described in WO2007 / 110205 can be used instead.

[0319] In one embodiment, the first subunit of the Fc domain includes amino acid substitutions K392D and K409D, and the second subunit of the Fc domain includes amino acid substitutions D356K and D399K (numbered according to the Kabat EU index).

[0320] g) Fc domain modification that reduces Fc receptor binding and / or effector function The Fc domain confers desirable pharmacokinetic properties to (multispecific) antibodies, including a long serum half-life that contributes to good accumulation in target tissues and a favorable tissue-to-blood distribution ratio. However, at the same time, it can lead to undesirable targeting of (multispecific, e.g., bispecific) antibodies against cells expressing the Fc receptor, rather than cells that possess the desired antigen. Furthermore, co-activation of the Fc receptor signaling pathway can lead to cytokine release, and combined with T cell activation properties and the long half-life of (multispecific) antibodies, this can result in excessive activation of cytokine receptors and serious side effects from systemic administration. Activation of immune cells other than T cells (that possess the Fc receptor) can even reduce the effectiveness of (multispecific) antibodies, for example, by the potential destruction of T cells by NK cells.

[0321] Therefore, in a preferred embodiment, the Fc domain of the (multispecific, e.g., bispecific) antibody according to the present invention exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the natural IgG1Fc domain. In one such embodiment, the Fc domain (or the (multispecific) antibody containing the Fc domain) exhibits less than 50%, particularly less than 20%, more particularly less than 10%, and most particularly less than 5% binding affinity to the Fc receptor compared to the natural IgG1Fc domain (or the (multispecific, e.g., bispecific) antibody containing the natural IgG1Fc domain), and / or exhibits less than 50%, particularly less than 20%, more particularly less than 10%, and most particularly less than 5% effector function compared to the natural IgG1Fc domain (or the (multispecific, e.g., bispecific) antibody containing the natural IgG1Fc domain). In one embodiment, the Fc domain (or the (multispecific, e.g., bispecific) antibody containing the Fc domain) substantially does not bind to the Fc receptor and / or does not 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, most specifically 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 embodiment, the Fc domain domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG1 Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or an antibody containing the Fc domain (multispecific, e.g., bispecific)) has a binding affinity to FcRn of the natural IgG1Fc domain (or an antibody containing the natural IgG1Fc domain (multispecific, e.g., bispecific)) that is greater than about 70%, particularly greater than about 80%, and even more particularly greater than about 90%.

[0322] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to the Fc receptor and / or reduced effector function compared to an unengineered Fc domain. In preferred embodiments, the Fc domain of a (multispecific, e.g., bispecific) antibody contains one or more amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptor and / or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to 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 twofold, at least fivefold, or at least tenfold. In embodiments where there are two or more amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to the Fc receptor by at least tenfold, at least twentyfold, or even more than fiftyfold. In one embodiment, an antibody containing a modified Fc domain (multispecific, e.g., bispecific) exhibits a binding affinity to the Fc receptor of less than 20%, particularly less than 10%, and more particularly less than 5%, compared to an antibody containing an unmodified Fc domain (multispecific, e.g., bispecific). 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, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly to C1q, is also reduced. In one embodiment, binding affinity to the neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e., maintenance of the binding affinity of the Fc domain to the receptor, is achieved when the Fc domain (or an antibody containing the Fc domain (multispecific, e.g., bispecific)) has a binding affinity to FcRn that exceeds about 70% of that of the unmanipulated form of the Fc domain (or an antibody containing the unmanipulated form of the Fc domain (multispecific, e.g., bispecific)). The Fc domain, or the (multispecific) antibody of the present invention containing the Fc domain, may have such affinity exceeding about 80%, and even exceeding about 90%. In certain embodiments, the Fc domain of the (multispecific, e.g., bispecific) antibody is manipulated to have reduced effector function compared to the unmanipulated Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cell-mediated cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling that induces apoptosis, reduced crosslinking of target-binding antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group consisting of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a preferred embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% ADCC induced by an unmodified Fc domain (or an antibody containing an unmodified Fc domain (multispecific, e.g., bispecific)).

[0323] 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 contains an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331, and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain contains an amino acid substitution at a position selected from the group L234, L235, and P329 (numbered according to the Kabat EU index). In some embodiments, the Fc domain contains amino acid substitutions L234A and L235A (numbered according to the Kabat EU index). In one such embodiment, the Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. In one embodiment, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbered according to the Kabat EU index). In one embodiment, the Fc domain includes an amino acid substitution at position P329 and further amino acid substitutions (numbered according to the Kabat EU index) at positions selected from E233, L234, L235, N297, and P331. In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a preferred embodiment, the Fc domain includes amino acid substitutions at positions P329, L234, and L235 (numbered according to the Kabat EU index). In a more preferred embodiment, the Fc domain includes amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA", or "LALAPG").Specifically, in a preferred embodiment, each subunit of the Fc domain includes amino acid substitutions L234A, L235A, and P329G (numbered according to the Kabat EU index), that is, in the first and second subunits of the Fc domain, the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A), and the proline residue at position 329 is substituted with a glycine residue (P329G) (numbered according to the Kabat EU index).

[0324] In one such embodiment, the Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. The amino acid substitution combination "P329G LALA" almost completely inactivates the Fcγ receptor (and complement) binding of the human IgG1Fc domain, as described in PCT Publication WO2012 / 130831, which is incorporated herein by reference in its entirety. WO2012 / 130831 also describes methods for preparing such mutant Fc domains and for determining their properties, such as Fc receptor binding or effector function.

[0325] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to IgG1 antibodies. Therefore, in some embodiments, the Fc domain of the (multispecific) antibody of the present invention is an IgG4Fc domain, particularly a human IgG4Fc domain. In one embodiment, the IgG4Fc domain includes an amino acid substitution at position S228, specifically amino acid substitution S228P (numbered according to the Kabat EU index). To further reduce its binding affinity to Fc receptors and / or its effector function, in one embodiment, the IgG4Fc domain includes an amino acid substitution at position L235, specifically amino acid substitution L235E (numbered according to the Kabat EU index). In another embodiment, the IgG4Fc domain includes an amino acid substitution at position P329, specifically amino acid substitution P329G (numbered according to the Kabat EU index). In a preferred embodiment, the IgG4Fc domain includes amino acid substitutions at positions S228, L235, and P329, specifically amino acid substitutions S228P, L235E, and P329G (numbered according to the Kabat EU index). Such IgG4Fc domain variants and their Fcγ receptor binding properties are described in PCT publication number WO2012 / 130831, which is incorporated herein by reference in its entirety.

[0326] In a preferred embodiment, the Fc domain exhibiting reduced binding affinity to the Fc receptor and / or reduced effector function is either a human IgG1 Fc domain containing the amino acid substitutions L234A, L235A, and optionally P329G, or a human IgG4 Fc domain (numbered according to the Kabat EU index) containing the amino acid substitutions S228P, L235E, and optionally P329G, compared to a native IgG1 Fc domain.

[0327] In certain embodiments, N-glycosylation of the Fc domain is excluded. In one such embodiment, the Fc domain contains an amino acid mutation at position N297, particularly an amino acid substitution (numbered according to the Kabat EU index) that replaces asparagine with alanine (N297A) or aspartic acid (N297D).

[0328] In addition to the Fc domains described above and in PCT Publication No. WO2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those in which one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent No. 6,737,056) (numbered by the Kabat EU Index). Such Fc variants include those with two or more substitutions of amino acids at positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0329] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of encoding DNA sequences, PCR, and gene synthesis. Precise nucleotide changes can be confirmed, for example, by sequencing.

[0330] Binding to Fc receptors can be readily determined, for example, by ELISA or surface plasmon resonance (SPR) using standard instruments such as the BIAcore instrument (GE Healthcare), and using Fc receptors obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain or an antibody containing an Fc domain (multispecific) to Fc receptors can be evaluated using cell lines known to express specific Fc receptors, such as human NK cells expressing the FcγIIIa receptor.

[0331] The effector function of an Fc domain, or an antibody containing an Fc domain (multispecific, e.g., bispecific), can be measured by methods known in the art. Examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent 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); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, for example, as disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0332] In some embodiments, the binding of the Fc domain to complement components, particularly to C1q, is reduced. Therefore, in some embodiments where the Fc domain is manipulated to have reduced effector function, this reduced effector function includes reduced CDC. A C1q binding assay can be performed to determine whether an antibody containing the Fc domain, or an antibody containing the Fc domain (multispecific, e.g., bispecific), can bind to C1q and thus possess CDC activity. See, for example, the C1q and C3c binding ELISAs of WO2006 / 029879 and WO2005 / 100402. CDC assays can be performed to evaluate complement activation (e.g., Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0333] The determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769(2006); WO2013 / 120929).

[0334] B. Polynucleotides The present invention further provides isolated polynucleotides encoding the antibodies of the present invention. The isolated polynucleotides may be a single polynucleotide or a plurality of polynucleotides.

[0335] The polynucleotide encoding the (multispecific, e.g., bispecific) antibody of the present invention may be expressed as a single polynucleotide encoding the entire antibody, or as multiple (e.g., two or more) polynucleotides co-expressed. Polypeptides encoded by co-expressed polynucleotides may associate, for example, via disulfide bonds or other means, to form a functional antibody. For example, the light chain portion of an antibody may be encoded by a polynucleotide separate from the antibody portion containing the heavy chain. When co-expressed, the heavy chain polypeptide associates with the light chain polypeptide to form the antibody. In another example, an antibody portion containing one of two Fc domain subunits and optionally one or more Fab molecules (or parts thereof) may be encoded by separate polynucleotides from the antibody portion containing the other of the two Fc domain subunits and optionally Fab molecules (or parts thereof). When co-expressed, the Fc domain subunits associate to form an Fc domain.

[0336] In some embodiments, the isolated polynucleotide encodes the entire antibody molecule according to the present invention as described herein. In other embodiments, the isolated polynucleotide encodes the polypeptide contained in the antibody according to the present invention as described herein.

[0337] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

[0338] C. Recombination The antibodies of the present invention can be obtained, for example, by solid peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the antibody are isolated, for example, as described above, and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In one embodiment, a vector, particularly an expression vector, containing the polynucleotide of the present invention (i.e., a single or multiple polynucleotides) is provided. An expression vector containing the encoding sequence of the antibody, along with appropriate transcription / translation control signals, can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY (1989). An expression vector may be a plasmid, part of a virus, or a nucleic acid fragment. An expression vector comprises an expression cassette, which is cloned with a polynucleotide encoding an antibody (i.e., a coding region) operably bound to a promoter and / or other transcriptional or translational regulatory elements. As used herein, “coding region” is a portion of a nucleic acid consisting of codons translated into amino acids. “Stop codons” (TAG, TGA, or TAA) are not translated into amino acids but may be considered part of the coding region if present; however, promoters, adjacent sequence transcriptional terminators such as ribosome binding sites, introns, and 5' and 3' untranslated regions are not part of the coding region.Two or more coding regions may be present, for example, in a single polynucleotide construct on a single vector, or in separate polynucleotide constructs on separate (different) vectors. Furthermore, any vector may contain a single coding region or two or more coding regions; for example, the vector of the present invention may encode one or more polypeptides that are separated post-translation or concurrently with translation to a final protein via proteolytic cleavage. Furthermore, the vector, polynucleotide, or nucleic acid of the present invention may encode heterogeneous coding regions fused or unfused to the polynucleotide encoding the antibody or its variant or derivative of the present invention. Heterogeneous coding regions may include, but are not limited to, special elements or motifs such as secretory signal peptides or heterogeneous functional domains. Manipulable binding is when the coding region of a gene product, for example, a polypeptide, associates with one or more regulatory sequences in such a way that the expression of the gene product is influenced by or controlled by the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "functionally associated" if the promoter's function induces the transcription of mRNA encoding the gene product of interest, and the nature of the binding between the two DNA fragments does not interfere with the ability of an expression regulatory sequence to direct gene product expression or the transcriptional ability of a DNA template. Therefore, if a promoter can transcribe a polypeptide-encoding nucleic acid, the promoter region is operably associated with the polypeptide-encoding nucleic acid. A promoter may be a cell-specific promoter that directs substantial transcription of DNA only in a given cell. Other transcriptional regulatory elements besides promoters, such as enhancers, operators, repressors, and transcription termination signals, can be operably conjugated to polynucleotides to direct cell-specific transcription. Suitable promoters and other transcriptional regulatory regions are disclosed herein. Various transcriptional regulatory regions are known to those skilled in the art.These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, such as promoter and enhancer segments from cytomegalovirus (e.g., pre-early promoter in combination with intron A), Simianvirus 40 (e.g., early promoter), and retroviruses (e.g., Roussarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences that can regulate gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., tetracycline-induced promoters). Similarly, various translational regulatory elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation start and termination codons, and viral elements (in particular, internal ribosome entry sites or IRESs, also known as CITE sequences). Expression cassettes may also include other features such as origins of replication and / or chromosomal integration elements, such as retroviral long-terminal repeats (LTRs) or adeno-associated virus (AAV) reverse-terminal repeats (ITRs).

[0339] The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretory peptides or signal peptides that direct the secretion of polypeptides encoded by the polynucleotides of the present invention. For example, if antibody secretion is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid encoding the antibody or a fragment thereof of the present invention. According to the signaling hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein when transport of the growing protein chain across the coarse endoplasmic reticulum begins. Those skilled in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secretory or “mature” form of the polypeptide. In certain embodiments, natural signal peptides, such as immunoglobulin heavy chain or light chain signal peptides, or functional derivatives of their sequences that retain the ability to direct the secretion of polypeptides operably associated therewith, are used. Alternatively, heterologous mammalian signal peptides or their functional derivatives can be used. For example, the wild-type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0340] DNA encoding short protein sequences (e.g., histidine tags) or DNA that assists in antibody labeling, which can be used to facilitate subsequent purification, may be contained within or at the ends of an antibody (fragment) encoding a polynucleotide.

[0341] In further embodiments, host cells comprising the polynucleotides of the present invention (i.e., a single polynucleotide or multiple polynucleotides) are provided. In certain embodiments, host cells comprising vectors of the present invention are provided. The polynucleotides and vectors may each incorporate, individually or in combination, any of the features described herein in relation to the polynucleotides and vectors, respectively. In such embodiments, the host cell comprises (e.g., transformed or transfected) one or more vectors comprising one or more polynucleotides encoding (part of) the antibody of the present invention. As used herein, the term “host cell” refers to any type of cell line that can be manipulated to produce the antibody or fragment thereof of the present invention. Host cells suitable for antibody replication and supportive expression are well known in the art. Such cells can be appropriately transfected or transduced with a particular expression vector, and large quantities of the vector containing the cells can be grown and seeded in a large fermenter to obtain sufficient quantities of antibody for clinical application. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides can be produced in bacteria, especially when glycosylation is not required. After expression, polypeptides can be isolated from bacterial cell paste into a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi and yeasts are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in polypeptides with partially or completely human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Host cells suitable for the expression of (glycosylated) polypeptides also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda cells.Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell lines (e.g., 293 or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad Sci 383). These include MRC 5 cells and FS4 cells (as described in 44-68 (1982)). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. 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. 255-268 (2003).Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, but also, to name just a few, cells contained within transgenic animals, transgenic plants, or cultured plants or animal tissues. In one embodiment, the host cell is a eukaryotic cell, in particular a mammalian cell such as Chinese hamster ovary (CHO) cells, human fetal kidney (HEK) cells, or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In another embodiment, the host cell is not a cell from the human body.

[0342] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing polypeptides containing either the heavy or light chain of an antigen-binding domain, such as antibodies, can be genetically engineered to also express other antibody chains, such that the expression product is an antibody having both heavy and light chains.

[0343] In one embodiment, a method for producing an antibody according to the present invention is provided, comprising culturing host cells containing a polynucleotide encoding an antibody provided herein under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0344] The components of the (multispecific, e.g., bispecific) antibody of the present invention can be genetically fused with one another. The (multispecific, e.g., bispecific) antibody can be designed so that its components are fused with one another directly or indirectly via linker sequences. The composition and length of the linkers can be determined according to methods well known in the art and tested for efficacy. Examples of linker sequences between different components of a (multispecific) antibody are provided here. If necessary, additional sequences, e.g., endopeptidase recognition sequences, can be included to incorporate cleavage sites for separating the individual components of the fusion.

[0345] Antibodies prepared as described herein can be purified by known techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a particular protein depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. In affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the antibody binds can be used. For example, in affinity chromatography purification of the antibody of the present invention, a matrix containing protein A or protein G can be used. Antibodies can be isolated using sequential protein A or G affinity chromatography and size exclusion chromatography, essentially as described in the examples. The purity of the antibody can be determined by any of the various well-known analytical methods, including gel electrophoresis and high-pressure liquid chromatography.

[0346] D. assay The antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0347] 1. Binding assay The binding (affinity) of an antibody to an Fc receptor or target antigen can be determined, for example, by surface plasmon resonance (SPR) using standard instruments such as the BIAcore instrument (GE Healthcare), and by the receptor or target protein obtained by recombinant expression. Alternatively, the binding of antibodies to different receptors or target antigens can be evaluated, for example, by flow cytometry (FACS) using cell lines expressing a specific receptor or target antigen. Specific illustrative and exemplary embodiments for measuring binding activity to CD3 are described below. The illustrated assay can be readily adapted to measure binding activity to FolR1 by using the FolR1 antigen instead of the CD3 antigen and making minor adjustments readily recognizable to those skilled in the art.

[0348] In one embodiment, the binding activity to CD3 is determined by SPR as follows: SPR is performed using a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibody (GE Healthcare, #28958325) is immobilized on a Series S sensor chip CM5 (GE Healthcare) with a surface density of 4000-6000 resonance units (RUs) using standard amine-bonded chemistry. HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) is used as the run and dilution buffer. CD3 antibody at a concentration of 2 μg / ml (in 20 mM His, 140 mM NaCl, pH 6.0) is injected at a flow rate of 5 μl / min for approximately 60 seconds. The CD3 antigen used is a heterodimer of CD3 delta and CD3 epsilon ectodomain fused to a human Fc domain with knob-in-two-hole modification and a C-terminal Avi tag (see SEQ ID NOs. 28 and 29). CD3 antigen is injected at a concentration of 10 μg / ml for 120 seconds, and dissociation is monitored at a flow rate of 5 μl / min for approximately 120 seconds. The tip surface is regenerated by injecting 10 mM glycine pH 2.1 twice consecutively for approximately 60 seconds each. Differences in bulk refractive index are corrected by subtracting the blank injection and the response obtained from the blank control flow cell. For evaluation, the binding response is acquired 5 seconds after the end of injection. To normalize the binding signal, CD3 binding is divided by the anti-Fab response (the signal (RU) obtained when the CD3 antibody is captured on the immobilized anti-Fab antibody). The binding activity of an antibody after one treatment relative to the binding activity of antibodies after different treatments (also called the relative activity concentration (RAC)) is calculated by referencing the binding activity of one antibody sample after one treatment to the binding activity of the corresponding antibody sample after another treatment.

[0349] 2. Activity assay The biological activity of the (multispecific, e.g., bispecific) antibodies of the present invention can be measured by various assays 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, induction of lysis of target cells such as tumor cells, and induction of tumor regression and / or improvement of survival rates.

[0350] E. Composition, formulation, and route of administration In further embodiments, the present invention provides a pharmaceutical composition comprising any of the (multispecific, e.g., bispecific) antibodies provided herein for use in, for example, any of the following therapeutic methods. In one embodiment, the pharmaceutical composition comprises the antibody according to the present invention and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises the (multispecific, e.g., bispecific) antibody according to the present invention and at least one additional therapeutic agent, as described below, for example.

[0351] Also provided is a method for producing the antibody of the present invention in a form suitable for in vivo administration, the method comprising (a) obtaining the antibody according to the present invention, and (b) compounding the antibody using at least one pharmaceutically acceptable carrier, thereby compounding the antibody preparation for in vivo administration.

[0352] The pharmaceutical compositions of the present invention comprise an effective amount of antibody dissolved or dispersed in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means molecular entities and compositions that are generally nontoxic to recipients at the doses and concentrations used; that is, they do not cause adverse reactions, allergic reactions, or other harmful reactions when administered to animals, such as humans, as required. The preparation of pharmaceutical compositions comprising antibodies and optionally additional active ingredients is known to those skilled in the art, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference. Furthermore, for administration to animals (e.g., humans), it is understood that the formulations should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Biotechnology Bureau or the corresponding authorities in other countries. Preferred compositions are lyophilized preparations or aqueous solutions. As used herein, “pharmaceutically acceptable carriers” include, as known to those skilled in the art, all kinds of materials and combinations thereof, such as solvents, buffers, dispersions, coatings, surfactants, antioxidants, preservatives (antimicrobials, antifungals, etc.), isotonic agents, absorption retarders, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, vehicles, disintegrants, lubricants, sweeteners, flavorings, dyes, etc. (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289–1329). Their use in pharmaceutical compositions is intended unless conventional carriers are incompatible with the active ingredient.

[0353] The (multispecific, e.g., bispecific) antibody (and any additional therapeutic agent) of the present invention may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if local treatment is desired, intrafocal administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, such as intravenous or subcutaneous injection, depending on whether the administration is short-term or long-term.

[0354] Parenteral compositions include those designed for administration by injection, such as subcutaneous, intradermal, intrafocal, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the antibodies of the present invention may be prepared in aqueous solutions, particularly physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically suitable saline buffer. The solution may contain preparations such as suspensions, stabilizers, and / or dispersants. Alternatively, the antibodies may be in powder form for preparation with a suitable vehicle, such as sterile water free of pyrogens, before use. Sterile injectable solutions are prepared by incorporating the required amount of the antibodies of the present invention into a suitable solvent, along with various other components listed below, as needed. Sterility can be easily achieved, for example, by filtration with 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 components. For sterile powders used to prepare sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, which yields any additional desired components from the liquid medium, in addition to the active ingredient powder, from the liquid medium, which has been previously sterile filtered. If necessary, the liquid medium should be properly buffered, and the liquid diluent should be made isotonic before injecting sufficient saline or glucose. The composition must be stable under manufacturing and storage conditions and must be preserved against microbial contamination such as bacteria and fungi. It will be understood that endotoxin contamination should be kept to a minimum, e.g., less than 0.5 ng / mg of protein.Suitable pharmaceutically acceptable carriers include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; serum albumin, gelatin, immunoassay This includes, but is not limited to, proteins such as globulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., 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, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or reagents to increase the solubility of the compound and enable the preparation of high-concentration solutions. Furthermore, the suspension of the active compound can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethylcrete or triglycerides, or liposomes.

[0355] The active ingredient may be encapsulated in microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules), or macroemulsions, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, prepared by coacervation technology or interfacial polymerization, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations can be prepared. A suitable example of a sustained-release preparation involves a semipermeable matrix of a solid hydrophobic polymer containing a polypeptide, the matrix in the form of a molded article, e.g., a film or microcapsules. In certain embodiments, prolonged absorption of an injectable composition can be achieved by using an absorption-delaying reagent in the composition, for example, aluminum monostearate, gelatin, or a combination thereof.

[0356] In addition to the compositions described above, antibodies can also be prepared as depot preparations. Such sustained-release preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Therefore, for example, antibodies can be prepared using suitable polymer or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, such as sparingly soluble salts.

[0357] The pharmaceutical compositions comprising the (multispecific, e.g., bispecific) antibodies of the present invention can be prepared by conventional mixing, dissolution, emulsification, encapsulation, capture, or lyophilization processes. The pharmaceutical compositions can be prepared conventionally using one or more physiologically acceptable carriers, diluents, vehicles, or adjuvants that facilitate the processing of proteins into pharmaceutically usable preparations. The appropriate formulation depends on the chosen route of administration.

[0358] Antibodies can be formulated into compositions of free acids or free bases, neutral or salt forms. A pharmaceutically acceptable salt is one that substantially retains the biological activity of the free acid or base. These include acid addition salts, for example, those formed from free amino groups of proteinaceous compositions, or those formed from inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed from free carboxyl groups can be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or from organic salts such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than their corresponding free base forms.

[0359] F. Treatment methods and compositions Any of the antibodies (multispecific, e.g., bispecific) provided herein can be used in therapeutic methods. The antibodies of the present invention can be used, for example, as immunotherapeutic agents in the treatment of cancer.

[0360] For use in therapeutic applications, the (multispecific, e.g., bispecific) antibodies of the present invention are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals.

[0361] In one embodiment, the present invention provides an antibody (multispecific, e.g., bispecific) for use as a pharmaceutical. In a further embodiment, the present invention provides an antibody (multispecific, e.g., bispecific) for use in the treatment of a disease. In a particular embodiment, the present invention provides an antibody (multispecific, e.g., bispecific) for use in a therapeutic method. In one embodiment, the present invention provides an antibody (multispecific, e.g., bispecific) for use in the treatment of a disease in an individual that requires it. In a particular embodiment, the present invention provides an antibody (multispecific, e.g., bispecific) for use in a method of treating an individual having a disease, comprising administering an effective amount of the antibody to the individual. In a particular embodiment, the disease to be treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In a particular embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, e.g., an anticancer agent, to the individual if the disease to be treated is cancer. In a further embodiment, the present invention provides an antibody for use in inducing the lysis of target cells, particularly tumor cells. In certain embodiments, the present invention provides (multispecific, e.g., bispecific) antibodies for use in a method of inducing the lysis of target cells, particularly tumor cells, in an organism, which comprises administering to the organism an amount of the antibody effective in inducing the lysis of target cells. The "organism" in any of the above embodiments is a mammal, preferably a human.

[0362] In further embodiments, the present invention provides the use of the (multispecific, e.g., bispecific) antibody of the present invention in the manufacture or preparation of pharmaceuticals. In one embodiment, the drug is for the treatment of a disease in an individual that requires it. In further embodiments, the drug is for use in a method of treating a disease, comprising administering an effective amount of the drug to an individual having the disease. In a particular embodiment, the disease to be treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In one embodiment, the method, if the disease to be treated is cancer, further comprises administering an effective amount of at least one additional therapeutic agent, e.g., an anticancer agent, to the individual. In further embodiments, the drug is for inducing the lysis of target cells, particularly tumor cells. In further embodiments, the drug is for use in a method of inducing the lysis of target cells, particularly tumor cells, in an individual, comprising administering an effective amount of the drug to the individual to induce the lysis of target cells. The “individual” in any of the above embodiments may be a mammal, preferably a human.

[0363] Further, the present invention provides a method for treating a disease. In one embodiment, the method comprises administering an effective amount of the antibody of the present invention to an individual having such a disease. In one embodiment, a composition comprising the antibody of the present invention in a pharmaceutically acceptable form is administered to the individual. In a particular embodiment, the disease to be treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In a particular embodiment, if the disease to be treated is cancer, the method further comprises administering an effective amount of at least one additional therapeutic agent, such as an anticancer agent, to the individual. The “individual” in any of the above embodiments may be a mammal, preferably a human.

[0364] In further embodiments, the present invention provides a method for inducing the lysis of target cells, particularly tumor cells. In one embodiment, the method comprises contacting target cells with the antibody of the present invention in the presence of T cells, particularly cytotoxic T cells. In further embodiments, a method is provided for inducing the lysis of target cells, particularly tumor cells, in an individual. In one such embodiment, the method comprises administering an effective amount of the antibody of the present invention to an individual to induce the lysis of target cells. In one embodiment, “individual” is a human.

[0365] In certain embodiments, the diseases treated are proliferative disorders, particularly cancer. Non-limited examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, hematological cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other proliferative disorders that can be treated with the antibodies of the present invention include, but are not limited to, tumors located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system. Precancerous conditions or precancerous lesions and cancer metastases are also included. In certain embodiments, 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 one embodiment, the cancer is a cancer that expresses (or overexpresses) FolR1, in particular when the antibody is a bispecific antibody that binds to FolR1 as a second antigen. In one embodiment, the cancer is ovarian cancer, lung cancer, breast cancer, or kidney cancer, in particular when the antibody is a bispecific antibody that binds to FolR1 as a second antigen. Those skilled in the art will readily recognize that in many cases antibodies may not result in a cure and may only provide partial benefit. In some embodiments, any physiological change that provides some benefit is also considered therapeutically beneficial. Therefore, in some embodiments, the amount of antibody that produces a physiological change is considered an "effective dose". The subject, patient, or individual requiring treatment is typically a mammal, and more specifically, a human.

[0366] In some embodiments, an effective amount of the antibody of the present invention is administered to an individual for the treatment of a disease.

[0367] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the route of administration, the patient's weight, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's medical history and response to the antibody, and the discretion of the attending physician. In any case, the practicing physician administering the drug will determine the concentration of the active ingredient in the composition and the appropriate dose for each individual. Various dosing schedules are envisioned herein, including but not limited to single or multiple doses, bolus administration, and pulse infusion at various time points.

[0368] Antibodies are administered to the patient appropriately, either in a single dose or over a series of treatments. Depending on the type and severity of the disease, for example, an initial candidate dose of antibody of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be administered to the patient, either in a single dose, multiple separate doses, or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may range from approximately 1 μg / kg to 100 mg / kg or more. Depending on the condition, in cases of repeated administration over several days or more, treatment is generally continued until the desired suppression of disease symptoms occurs. An example of antibody dosage ranges from approximately 0.005 mg / kg to approximately 10 mg / kg. In other non-limiting examples, doses may also include approximately 1 microgram / kg body weight, approximately 5 micrograms / kg body weight, approximately 10 micrograms / kg body weight, approximately 50 micrograms / kg body weight, approximately 100 micrograms / kg body weight, approximately 200 micrograms / kg body weight, approximately 350 micrograms / kg body weight, approximately 500 micrograms / kg body weight, approximately 1 milligram / kg body weight, approximately 5 milligrams / kg body weight, approximately 10 milligrams / kg body weight, approximately 50 milligrams / kg body weight, approximately 100 milligrams / kg body weight, approximately 200 milligrams / kg body weight, approximately 350 milligrams / kg body weight, approximately 500 milligrams / kg body weight, approximately 1000 milligrams / kg body weight, or more, and any range derived therefrom. In non-limiting examples of ranges deriveable from the numbers listed herein, doses may be administered based on the above numbers, such as from approximately 5 mg / kg body weight to approximately 100 mg / kg body weight, from approximately 5 micrograms / kg body weight to approximately 500 milligrams / kg body weight, etc. Therefore, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives approximately 2 to 20 antibody doses, or for example, approximately 6 doses). A high loading dose may be administered first, followed by one or more low doses. However, other drug regimens may be useful. The progress of this treatment can be easily monitored by conventional techniques and assays.

[0369] The antibodies of the present invention are generally used in an effective amount to achieve the intended purpose. For use in treating or preventing a disease condition, the antibodies or their pharmaceutical compositions of the present invention are administered or applied in an effective amount.

[0370] For systemic administration, the effective dose can first be estimated from in vitro assays such as cell culture assays. Subsequently, the IC is determined by cell culture. 50 Doses can be prescribed in animal models to achieve a circulating concentration range that includes [specific concentration range]. Such information can be used to more accurately determine useful doses in humans.

[0371] The initial dose can also be estimated from in vivo data, such as animal models, using techniques well known in the art.

[0372] The dosage and administration interval can be individually adjusted to provide sufficient plasma antibody levels to maintain therapeutic effect. Typical patient doses for administration by injection range from approximately 0.1 to 50 mg / kg / day, typically from approximately 0.5 to 1 mg / kg / day. Therapeutably effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by HPLC.

[0373] The effective dose of the antibody of the present invention generally provides therapeutic benefits without causing substantial toxicity. The toxicity and therapeutic effect of the antibody can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Using cell culture assays and animal studies, LD 50 (Lethal dose for 50% of the population) and ED 50 (The therapeutically effective dose for 50% of the population) can be determined. The dose-to-toxicity ratio is the therapeutic index, and the LD50 is the therapeutic index. 50 / ED 50It can be expressed as a ratio. Antibodies exhibiting a large therapeutic index are preferred. In one embodiment, the antibody according to the present invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a dose range suitable for human use. The dosage is determined to have little to no toxicity. 50 It is preferable that the circulating concentration is within the range including [specific component]. The dosage may vary within this range depending on various factors, such as the dosage form used, the route of administration used, and the patient's condition. The exact formulation, route of administration, and dosage can be selected by the individual physician in consideration of the patient's condition (see, for example, Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, which is incorporated herein by reference in its entirety).

[0374] The attending physician of a patient treated with the antibody of the present invention knows how and when to discontinue, interrupt, or adjust the administration in the event of toxicity, organ dysfunction, etc. Conversely, the attending physician also knows how to adjust the treatment to a higher level if the clinical response is inadequate (i.e., to eliminate toxicity). The size of the dose administered in the management of the disorder of interest varies depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition can be partially assessed, for example, by standard prognostic assessment methods. Furthermore, the dose and possibly the frequency of administration also vary depending on the age, weight, and response of the individual patient.

[0375] The (multispecific, e.g., bispecific) antibodies of the present invention can be administered in combination with one or more other agents in treatment. For example, the antibodies of the present invention may be administered concurrently with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent administered to treat a symptom or disease of an individual requiring treatment. Such additional therapeutic agents may include any active ingredient suitable for the specific disease being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, a cell proliferation inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis-inducing factors. In preferred embodiments, the additional therapeutic agent is an anticancer agent, e.g., a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

[0376] Such other agents are appropriately present in combination in amounts 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 other factors discussed above. Antibodies are generally used in the same doses and routes of administration as described herein, or at about 1–99% of the doses described herein, or in any dose and route that is determined to be empirically / clinically appropriate.

[0377] Such combination therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate compositions) and separate administration, in which case the administration of the (multispecific, e.g., bispecific) antibody of the present invention may be performed before administration, simultaneously with and / or after administration of additional therapeutic agents and / or adjuvants. The antibody of the present invention may also be used in combination with radiotherapy.

[0378] G. Manufactured products In another aspect of the present invention, a product is provided comprising a material useful for the treatment, prevention and / or diagnosis of the above-mentioned disorders. The product comprises a container and a label or accompanying documentation on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition, either by itself or in combination with another composition effective for treating, preventing and / or diagnosing the condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a pierceable stopper by a subcutaneous injection needle). At least one activator in the composition is the antibody of the present invention. The label or accompanying documentation indicates that the composition is used to treat a selected condition. Furthermore, the product comprises (a) a first container containing a composition comprising the antibody of the present invention, and (b) a second container containing the composition, the composition comprising further cytotoxic agents or other therapeutic agents. The product in this aspect of the present invention may further include accompanying documentation indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0379] H. Methods and compositions for diagnosis and detection In certain embodiments, any of the antibodies provided herein are useful for detecting the presence of their target (e.g., CD3, FolR1) in a biological sample. The term “detect” as used herein encompasses quantitative or qualitative detection. In certain contexts, the biological sample includes cells or tissues, such as prostate tissue.

[0380] In one embodiment, an antibody according to the present invention is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of CD3 and FolR1 in a biological sample is provided. In a particular embodiment, the method includes contacting a biological sample with the antibody of the present invention under conditions that allow the binding of the antibody to CD3 and FolR1, and detecting whether a complex is formed between the antibody and CD3 and FolR1. Such a method may be in vitro or in vivo. In one embodiment, the antibody of the present invention is used to select subjects eligible for treatment with an antibody that binds to CD3 and FolR1, for example, when CD3 and FolR1 are biomarkers for patient selection.

[0381] Exemplary disorders that can be diagnosed using the antibodies of the present invention include cancer, particularly skin cancer or brain cancer.

[0382] In certain embodiments, antibodies according to the present invention are provided, wherein the antibody is labeled. Labeling includes, but is not limited to, directly detectable labels or moieties (such as fluorescence, chromophores, high electron density, chemiluminescence, and radioactive labeling), as well as indirectly detectable moieties, such as enzymes or ligands mediated by enzymatic reactions or molecular interactions. Exemplary labels include radioactive isotopes. 32 P, 14 C, 125 I, 3 H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases, such as glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, conjugated with enzymes that use hydrogen peroxide to oxidize pigment precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin-labeled, bacteriophage-labeled, and stable free radicals.

[0383] III.array TIFF0007848142000002.tif252170TIFF0007848142000003.tif255170TIFF0007848142000004.t if255170TIFF0007848142000005.tif252170TIFF0007848142000006.tif247170TIFF0007848142 000007.tif248170TIFF0007848142000008.tif248170TIFF0007848142000009.tif253170TIFF00 07848142000010.tif255170TIFF0007848142000011.tif246170TIFF0007848142000012.tif37170

[0384] Bispecific CD3 / FolR1 antibody sequence: JPEG0007848142000013.jpg255170JPEG0007848142000014.jpg255170JPEG0007848142000015.jpg225170

[0385] IV. Examples The following are examples of the methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be implemented.

[0386] Example 1 - Generation of an optimized CD3 binder This specification refers to "CD3 orig Starting from a previously described CD3 binder (see, for example, WO2014 / 131712, incorporated herein by reference), which is called and contains the VH and VL sequences of sequence numbers 6 and 11, respectively, we conjugate this binder by removing two asparagine deamidation sequence motifs located at Kabat positions 97 and 100 of the heavy chain CDR3.

[0387] To this end, we created an antibody library suitable for heavy chain phage display by removing asparagine at both Kabat positions 97 and 100 and further randomizing CDR H1, H2, and H3 to compensate for the affinity loss caused by substituting Asn97 and Asn100 through the affinity maturation process.

[0388] This library was loaded onto linear phages via fusion with the minor coat protein p3 (Marks et al. (1991) J Mol Biol 222, 581-597), and recombinant CD3 ε I selected the combination with [this].

[0389] Ten candidate clones were identified in the initial screening, and they showed acceptable binding to recombinant antigens as measured by SPR, as Fab fragments (generated in E. coli).

[0390] However, only one of these clones showed acceptable binding activity to CD3-expressing cells when measured by flow cytometry after conversion to IgG format.

[0391] In this specification, "CD3 optSelected clones, referred to as "[...]", containing the VH and VL sequences of sequence numbers 7 and 11 respectively, were further evaluated and converted to a bispecific format as described below.

[0392] Example 2 - Optimized CD3 Binder Bonding to CD3 Binding to recombinant CD3 The binding to recombinant CD3 is done using the optimized CD3 binder "CD3 opt " and the original CD3 binder "CD3 orig Determined by surface plasmon resonance (SPR), both are human IgG1 formats with P329G L234A L235A ("PGLALA", EU numbering) mutations in the Fc region (SEQ ID NOs. 12 and 14 (CD3). orig ) and sequence numbers 13 and 14 (CD3 opt ))

[0393] To evaluate the effect of deamide removal and its impact on antibody stability, the binding of original and optimized CD3 binders to recombinant CD3 was tested after 14 days of temperature stress at 37°C or 40°C. Samples stored at -80°C were used as references. The reference samples and samples stressed at 40°C were in 20 mM His, 140 mM NaCl, pH 6.0, while the samples stressed at 37°C were in PBS, pH 7.4, all at concentrations of 1.2–1.3 mg / ml. After the stress period (14 days), samples in PBS were dialyzed to 20 mM His, 140 mM NaCl, pH 6.0 for further analysis.

[0394] The relative activity concentration (RAC) of the sample was determined by SPR as follows:

[0395] SPR was performed using a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibody (GE Healthcare, #28958325) was immobilized on a Series S sensor tip CM5 (GE Healthcare) using standard amine-bonding chemistry to obtain a surface density of 4000–6000 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used as the run and dilution buffer. CD3 antibody at a concentration of 2 μg / ml was injected at a flow rate of 5 μl / min for 60 seconds. CD3 antigen (see below) at a concentration of 10 μg / ml was injected for 120 seconds, and dissociation was monitored at a flow rate of 5 μl / min for 120 seconds. The tip surface was regenerated by injecting 10 mM glycine pH 2.1 twice consecutively for 60 seconds each. The difference in bulk refractive index was corrected by subtracting the blank injection and the response obtained from the blank control flow cell. For evaluation, the binding response was acquired 5 seconds after the end of injection. To normalize the binding signal, CD3 binding was divided by the anti-Fab response (signal (RU) obtained when CD3 antibody is captured on immobilized anti-Fab antibody). Relative activity concentrations were calculated by referencing each temperature-stressed sample with the corresponding non-stressed sample.

[0396] The CD3 antigen used was a heterodimer of a CD3 delta and a CD3 epsilon ectodomain fused to a human Fc domain with a knob-in-two-hole modification and a C-terminal Avi tag (see SEQ ID NOs. 28 and 29).

[0397] The results of this experiment are shown in Figure 2. As can be seen from the figure, the optimized CD3 binder CD3 opt This is the original CD3 binder CD3 orig In comparison, binding to CD3 was significantly improved after temperature stress (37°C, pH 7.4 for 2 weeks). This result indicates that the deamide site was successfully removed, resulting in an antibody with excellent stability related to in vivo half-life, and an antibody formulation at neutral pH.

[0398] Binding of CD3 to Jurkat cells The binding of CD3 to the human reporter T cell line Jurkat NFAT is achieved by the optimized CD3 binder "CD3 opt " and the original CD3 binder "CD3 orig It was determined by FACS that both are human IgG1 formats with the P329G L234A L235A ("PGLALA", EU numbering) mutation in the Fc region (SEQ ID NOs. 12 and 14 (CD3) orig ) and sequence numbers 13 and 14 (CD3 opt ))

[0399] Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphoblastic leukemia reporter cell line possessing an NFAT promoter expressing human CD3. Cells were cultured at 0.1–0.5 mio cells / ml in RPMI1640, 2 g / l glucose, 2 g / l NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1 × NEAA, and 1 × sodium pyruvate. A final concentration of 200 μg of hygromycin B per 1 ml was added after each cell passage.

[0400] 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, 100,000–200,000 cells were seeded per well. The plate was centrifuged at 400×g for 4 minutes, and the supernatant was removed. The test antibody was diluted in FACS buffer, and 20 μl of the antibody solution was added to the cells at 4°C for 30 minutes. To remove unbound antibody, the cells were washed twice with FACS buffer before adding the diluted secondary antibody (PE-conjugated AffiniPure F(ab')2 Fragment goat anti-human IgG Fcg Fragment Specific; Jackson ImmunoResearch #109-116-170). After incubation at 4°C for 30 minutes, the unbound secondary antibody was washed away. Prior to measurement, cells were resuspended in 200 μl of FACS buffer and analyzed by flow cytometry using a BD Canto II device.

[0401] As shown in Figure 3, the optimized CD3 binder "CD3 opt " and the original CD3 binder "CD3 orig It bound relatively well to CD3 on Jurkat cells.

[0402] Example 3 - Functional activation of optimized CD3 binder Optimized CD3 Binder "CD3 opt The functional activity of the original CD3 binder "CD3" was tested using the Jurkat reporter cell assay. orig The activity was compared with that of IgG. To test the functional activity of IgG, anti-PGLALA expressing CHO cells were subjected to increasing concentrations of CD3. opt Human IgG1 PGLALA or CD3 orig Jurkat NFAT reporter cells were co-incubated in the presence of human IgG1 PGLALA. Activation of CD3 on Jurkat NFAT reporter cells during T cell crosslinking induced luciferase production, and luminescence can be measured as an activation marker. origHuman IgG1 wt was included as a negative control because it could not bind to anti-PGLALA expressing CHO cells and therefore could not crosslink on Jurkat NFAT cells. A schematic diagram of the assay is shown in Figure 4.

[0403] Anti-PGLALA expressing CHO cells are CHO-K1 cells engineered to express an antibody that specifically binds to human IgG1Fc (PGLALA) on their surface (see WO2017 / 072210, incorporated herein by reference). These cells were cultured in DMEM / F12 medium containing 5% FCS + 1% GluMax. Jarcut NFAT reporter cells were as described in Example 2.

[0404] When CD3 huIgG1 PGLALA simultaneously binds to anti-PGLALA expressed in CHO cells and CD3 expressed in Jurkat-NFAT reporter cells, the NFAT promoter is activated, and active firefly luciferase is expressed. The intensity of the luminescence signal (obtained by adding a luciferase substrate) is proportional to the activation of CD3 and the intensity of signal transduction. Jurkat-NFAT reporter cells were grown in suspension and cultured on RPMI1640, 2 g / l glucose, 2 g / l NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1 × NEAA, 1 × sodium pyruvate (0.1-0.5 mio cells / ml), and 200 μg / ml hygromycin. For the assay, CHO cells were harvested and viability was measured using ViCell. 30,000 target cells / well were plated into a flat-bottomed, white-walled 96-well plate (Greiner bio-one #655098) with 100 μl of medium and 50 μl / well of diluted antibody, or medium (control) was added to the CHO cells. Jurkat-NFAT reporter cells were then harvested and their viability was assessed using ViCell. The cells were resuspended in hygromycin B-free cell culture medium at 1.2 mio cells / ml and added to the CHO cells at 60,000 cells / well (50 μl / well) to obtain a final effector-to-target (E:T) ratio of 2:1 and a final volume of 200 μl per well. Next, 4 μl of GloSensor (Promega #E1291) was added to each well (2% of the final volume). The cells were incubated in a humidified incubator at 37°C for 24 hours. At the end of the incubation period, luminescence was detected using a TECAN Spark 10M.

[0405] As shown in Figure 5, the optimized CD3 binder CD3 opt CD3 is used against Jurkat NFAT cells during cross-linking. orig It showed similar activity to [another product].

[0406] Example 4 - Generation of T cell bispecific antibodies containing an optimized CD3 binder TYRP1 TCB The optimized CD3 binder identified in Example 1 ("CD3 opt Using sequence numbers 7(VH) and 11(VL), we generated a T cell bispecific antibody (TCB) targeting CD3 and TYRP1 ("TYRP1 TCB").

[0407] The TYRP1 binder contained in this TCB is generated by humanization of the TYRP1 binder "TA99" (see GenBank entries AXQ57811 and AXQ57813 for the heavy and light chains, respectively), and includes the heavy and light chain variable region sequences shown in sequence numbers 18 and 22, respectively.

[0408] A schematic diagram of the TCB molecule is shown in Figure 6, and its complete sequence is shown in sequence numbers 23, 24, 25, and 27.

[0409] Similar molecules with the original CD3 binding sequence were also prepared (SEQ ID NOs: 23, 24, 25, and 26).

[0410] The bispecific molecule was generated by transient transfection of HEK293EBNA cells. Cells were transfected with the corresponding expression vector in a 1:2:1:1 ratio ("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)"), centrifuged, and the medium was replaced with preheated CDCHO medium (Thermo Fisher, #10743029). The expression vector was mixed with CDCHO medium, PEI (polyethyleneimine, Polysciences, #23966-1) was added, the solution was vortexed, and incubated at room temperature for 10 minutes. Subsequently, cells (2mio / ml) were mixed with the vector / PEI solution, transferred to a flask, and incubated at 37°C for 3 hours in a shaking incubator under a 5% CO2 atmosphere. After incubation, Excel medium containing supplements (80% of the total volume) was added. One day after transfection, supplements (feed, 12% of the total volume) were added. Cell supernatant was collected after 7 days by centrifugation and subsequent filtration (0.2 μm filter).

[0411] Proteins were purified from filtered cell culture supernatant using standard methods. Briefly, Fc-containing proteins were purified from cell culture supernatant by protein A affinity chromatography (MabSelect Sure, 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 immediately by neutralization of the sample pH. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (#UFC903096)), and aggregated proteins were separated from monomeric proteins by size exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0412] The concentration of purified protein was determined by measuring the absorption at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence, according to Pace et al. (1995), Protein Science 4, 2411-23. Protein purity and molecular weight were analyzed by CE-SDS using LabChipGXII (Perkin Elmer) in and out of the presence of a reducing agent (Table 1). Aggregate content was measured using running buffer (25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7 or 200 mM KH2PO4, 250 mM KCl, pH 6.2, respectively) (Table 2).

[0413] Table 1. CE-SDS analysis (non-reducible) of TYRP1 TCB. TIFF0007848142000016.tif27170

[0414] Table 2. Overview of TYRP1 TCB production and purification. TIFF0007848142000017.tif33170

[0415] Example 5 - Binding of T cell bispecific antibody containing an optimized CD3 binder to CD3 and TYRP1 Binding to recombinant CD3 The binding of TYRP1 TCB to recombinant CD3 is optimized (TYRP1 TCB CD3 opt ) or the original (TYRP1 TCB CD3 orig Evaluated by SPR using TCBs containing a CD3 binding sequence.

[0416] The SPR experiment was performed on a Biacore T200 using HBS-EP as the run buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 0.05% (v / v) Surfactant P20 (GE Healthcare)).

[0417] TYRP1 TCBs were captured on the surface of the CM5 sensor chip using an immobilized antibody that specifically binds to human IgG1Fc(PGLALA) (see WO2017 / 072210, as incorporated herein by reference). The capture antibody was conjugated to the sensor chip surface by directly immobilizing approximately 8700 resonance units (RUs) at pH 5.0 using a standard amine conjugation kit (GE Healthcare). The TCB molecules were captured at a flow rate of 10 μl / min at 5 nM for 30 seconds.

[0418] Human and cynomolgus monkey antigens (see below) were passed through a flow cell at a flow rate of 30 μl / min for 240 seconds at concentrations ranging from 12.35 to 3000 nM. The dissociation phase was monitored for 240 seconds and triggered by switching from the sample solution to HBS-EP. The tip surface was regenerated in each cycle by injecting 10 mM glycine pH 2.0 once for 30 seconds.

[0419] The antigens used were heterodimers of either human or cynomolgus monkey CD3 delta and CD3 epsilon ectodomain, fused to the human Fc domain using knob-to-hole modification and a C-terminal Avi tag (see SEQ ID NOs. 28 and 29 (human CD3) and SEQ ID NOs. 30) and 31 (cynomolgus monkey CD3).

[0420] The difference in bulk refractive index was corrected by subtracting the response obtained from the reference flow cell (where the TCB was not captured). The affinity constant was derived from the rate constant by fitting it to a 1:1 Langmuir coupling using BIAeval software (GE Healthcare).

[0421] The KD values ​​for binding to human and cynomolgus monkey CD3 were determined to be 50 nM and 20 nM, respectively, for TYRP1 TCB CD3opt, which were similar to those for TYRP1 TCB CD3orig (50 nM and 40 nM, respectively).

[0422] This is because, in a stress-free state, CD3 opt or CD3 orig This indicates that both TCBs containing either of these ingredients bound relatively well to recombinant CD3.

[0423] The binding of TYRP1 TCBs to recombinant human CD3 was also evaluated after 14 days of temperature stress at 37°C or...

Claims

1. A bispecific antibody that binds to CD3 and FolR1, wherein the bispecific antibody is (i) A first antigen-binding domain that can specifically bind to CD3, comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11, (ii) A second antigen-binding domain that can specifically bind to FolR1 and A bispecific antibody containing [specific antibody].

2. The bispecific antibody according to claim 1, wherein the first antigen-binding domain is a Fab molecule.

3. A bispecific antibody according to claim 1 or 2, comprising an Fc domain composed of a first and a second subunit.

4. A bispecific antibody according to any one of claims 1 to 3, comprising a third antigen-binding domain capable of specifically binding to FolR1.

5. The bispecific antibody according to claim 4, wherein the second and / or third antigen-binding domain is a Fab molecule.

6. A bispecific antibody according to any one of claims 1 to 5, wherein the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH or the constant domain CL and CH1 of the Fab light chain and Fab heavy chain are substituted for each other.

7. The bispecific antibody according to claim 6, wherein the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other.

8. A bispecific antibody according to any one of claims 4 to 7, wherein the second and, if present, third antigen-binding domains are conventional Fab molecules.

9. A bispecific antibody according to any one of claims 4 to 8, wherein the second and, if present, third antigen-binding domains are Fab molecules, and in the constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat), and in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat EU index), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat EU index).

10. A bispecific antibody according to any one of claims 4 to 9, wherein the first and second antigen-binding domains are fused to each other.

11. The bispecific antibody according to claim 10, wherein the first and second antigen-binding domains are fused to each other via a peptide linker.

12. A bispecific antibody according to any one of claims 4 to 11, wherein the first and second antigen-binding domains are each Fab molecules, and (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain of the first antigen-binding domain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, or (ii) 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.

13. The bispecific antibody according to any one of claims 4 to 12, wherein the first, second, and third antigen-binding domains, if present, are each Fab molecules, and the bispecific antibody comprises an Fc domain composed of first and second subunits, wherein (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain of the first antigen-binding domain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding domain, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain, if present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

14. A bispecific antibody according to any one of claims 3 to 13, wherein the Fc domain is an IgG Fc domain.

15. The bispecific antibody according to claim 14, wherein the Fc domain is an IgG1 Fc domain.

16. A bispecific antibody according to any one of claims 3 to 15, wherein the Fc domain is a human Fc domain.

17. A bispecific antibody according to any one of claims 3 to 16, wherein Fc comprises a modification that promotes the association of the first and second subunits of the Fc domain.

18. A bispecific antibody according to any one of claims 3 to 17, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

19. A bispecific antibody according to any one of claims 4 to 18, wherein the second and, if present, third antigen-binding domains comprise a VH containing HCDR1 of SEQ ID NO: 124, HCDR2 of SEQ ID NO: 125, and HCDR3 of SEQ ID NO: 126, and a VL containing LCDR1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO:

10.

20. A bispecific antibody according to any one of claims 4 to 19, wherein the second and, if present, third antigen-binding domains comprise a VH containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 123, and / or a VL containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

11.

21. An isolated polynucleotide encoding a bispecific antibody according to any one of claims 1 to 20.

22. A host cell containing an isolated polynucleotide as described in claim 21.

23. A method for producing a bispecific antibody that binds to CD3 and FolR1, comprising (a) culturing the host cells described in claim 22 under conditions suitable for the expression of a bispecific antibody, and (b) optionally recovering the bispecific antibody.

24. A bispecific antibody that binds to CD3 and FolR1, produced by the method described in claim 23.

25. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 20 or 24 and a pharmaceutically acceptable carrier.

26. A bispecific antibody according to any one of claims 1 to 20 or 24, or a pharmaceutical composition according to claim 25, for use as a pharmaceutical.

27. A bispecific antibody according to any one of claims 1 to 20 or 24, or a pharmaceutical composition according to claim 25, for use in the treatment of cancer.

28. Use in the manufacture of a pharmaceutical product of the bispecific antibody according to any one of claims 1 to 20 or 24 or the pharmaceutical composition according to claim 25.

29. Use of the bispecific antibody according to any one of claims 1 to 20 or 24 or the pharmaceutical composition according to claim 25 in the manufacture of a pharmaceutical for the treatment of cancer.

30. A pharmaceutical for treating a disease of an individual, comprising a bispecific antibody according to any one of claims 1 to 20 or 24, or a pharmaceutical composition according to claim 25.

31. The pharmaceutical product according to claim 30, wherein the disease is cancer.

Images