RU2832669C2 - Antibodies binding to cd3 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an antibody which binds to CD3 and to TYRP-1, containing a first antigen-binding domain, which binds CD3 and a second and optionally a third antigen-binding domain, which binds TYRP-1, a method for production thereof, as well as a composition containing same. Also disclosed is an isolated polynucleotide coding said antibody, as well as a host cell containing said antibody.

EFFECT: invention is effective for treating cancer in an individual.

35 cl, 32 dwg, 7 tbl, 13 ex

Description

AREA OF TECHNOLOGY

The present invention generally relates to antibodies that bind to CD3, including multispecific antibodies, for example, for T cell activation. The present invention further relates to polynucleotides encoding such antibodies and to vectors and host cells containing such polynucleotides. The invention further relates to methods for producing antibodies and to methods for using them in the treatment of disease.

LEVEL OF TECHNOLOGY

CD3 (cluster of differentiation 3) is a protein complex consisting of four subunits - a CD3γ chain, a CD3δ chain, and two CD3ε chains. CD3 binds to the T-cell receptor and the ζ chain, generating an activation signal in T lymphocytes.

CD3 has been extensively studied as a drug target. Monoclonal antibodies targeting CD3 have been used as immunosuppressive therapy in autoimmune diseases such as type 1 diabetes or in the treatment of transplant rejection. The CD3 antibody, muromonab-CD3 (OKT3), was the first monoclonal antibody approved for clinical use in humans in 1985.

A more recent application of CD3 antibodies is in the form of bispecific antibodies that bind CD3 on one side and a tumor cell antigen on the other. The simultaneous binding of such an antibody to both of its targets will promote a transient interaction between the target cell and the T cell, leading to activation of any cytotoxic T cell and subsequent lysis of the target cell.

For therapeutic purposes, an important requirement that antibodies must satisfy is sufficient stability both in vitro (for storing the drug) and in vivo (after administration to the patient).

Modifications such as asparagine deamidation are typical degradation pathways for recombinant antibodies and can affect both in vitro stability and in vivo biological functions.

Given the enormous therapeutic potential of antibodies, particularly bispecific antibodies, for T cell activation, there is a need for anti-CD3 antibodies with optimized properties.

ESSENCE OF THE INVENTION

The present invention provides antibodies, including multispecific (e.g. bispecific) antibodies, that bind to CD3 and are resistant to degradation by asparagine deamidation and thus exceptionally stable, which is required for therapeutic purposes. The proposed (multispecific) antibodies are further characterized by a combination of good efficacy and manufacturability with low toxicity and favorable pharmacokinetic properties.

As shown herein, antibodies, such as the multispecific antibodies that bind to CD3, provided in the present invention, retain greater than about 90% of the binding activity to CD3 after 2 weeks at pH 7.4, 37°C, compared to the binding activity after 2 weeks at pH 6, -80°C, as determined by surface plasmon resonance (SPR).

In one aspect, the present invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10. In one aspect, the VH 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 a sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11.

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

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

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

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

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

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

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

In one aspect, the second and, if present, the third antigen-binding domain is a Fab molecule, wherein in the CL constant domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the CH1 constant domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the EU index according to Kabat).

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

In one aspect, each of the first and second antigen-binding domains is a Fab molecule and either (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, 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.

In one aspect, each of the first, second and, if present, third antigen-binding domains is a Fab molecule, and the antibody comprises an Fc domain consisting of a first and a second subunit; and wherein either (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and the first antigen-binding domain is 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 Fab heavy chain 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.

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

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

In one aspect, the second antigen is TYRP-1. In one aspect, the second and, if present, the third antigen-binding domain comprises a VH comprising HCDR 1 of SEQ ID NO: 15, HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17, and a VL comprising LCDR 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21. In one aspect, the second and, if present, the third antigen-binding domain comprises a VH comprising 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: 18 and/or a VL comprising 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: 18 100% identical to the amino acid sequence of SEQ ID NO: 22.

In one aspect, the second antigen is EGFRvIII. In one aspect, the second and, if present, the third antigen-binding domain comprises a VH comprising HCDR 1 of SEQ ID NO: 85, HCDR 2 of SEQ ID NO: 86 and HCDR 3 of SEQ ID NO: 87, and a VL comprising LCDR 1 of SEQ ID NO: 89, LCDR 2 of SEQ ID NO: 90 and LCDR 3 of SEQ ID NO: 91. In one aspect, the second and, if present, the third antigen-binding domain comprises a VH comprising 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: 88 and/or a VL comprising 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: 88 100% identical to the amino acid sequence of SEQ ID NO: 92.

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

In another aspect, a method for producing an antibody that binds to CD3 is provided, comprising the steps of (a) culturing a host cell of the invention under conditions suitable for expressing the antibody, and optionally (b) isolating the antibody. The invention also includes an antibody that binds to CD3 obtained by the method of the invention.

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

Also included in the invention are methods of using the antibody and pharmaceutical composition of the invention. In one aspect, the invention provides an antibody or pharmaceutical composition according to the invention for use as a medicament. In one aspect, the antibody or pharmaceutical composition according to the invention is provided for use in the treatment of a disease. In a particular aspect, the disease is cancer.

Also proposed is the use of an antibody or a pharmaceutical composition according to the invention in the production of a medicinal product, the use of an antibody or a pharmaceutical composition according to the invention in the production of a medicinal product for the treatment of a disease, in particular cancer. The invention also proposes a method for treating a disease in an individual, comprising administering to said individual an effective amount of an antibody or a pharmaceutical composition according to the invention.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1. Exemplary configurations of (multispecific) antibodies according to the invention. (A, G) Illustration of a “1+1 CrossMab” molecule. (B, E) Illustration of a “2+1 IgG Crossfab” molecule with an alternative order of the Crossfab and Fab components (“inverted molecule”). (C, E) Illustration of a “2+1 IgG Crossfab” molecule. (G, K) Illustration of a “1+1 IgG Crossfab” molecule with an alternative order of the Crossfab and Fab components (“inverted molecule”). (I, M) Illustration of a “1+1 IgG Crossfab” molecule. (I, I) Illustration of a “2+1 IgG Crossfab” molecule with two Cross Fabs. (J, O) Illustration of a “2+1 IgG Crossfab” molecule with two CrossFabs and an alternative order of the Crossfab and Fab components (“inverted molecule”). (U, F) Illustration of a “Fab-Crossfab” molecule. (R, F) Illustration of the Crossfab-Fab molecule. (C, X) Illustration of the (Fab)2-Crossfab molecule. (T, C) Illustration of the Crossfab-(Fab)2 molecule. (H, Щ) Illustration of the Fab-(Crossfab)2 molecule. (SH, Ы) Illustration of the (Crossfab)2-Fab molecule. Black dot: optional modification in the Fc domain that promotes heterodimerization. ++, --: amino acids of opposite charges optionally introduced into the CH1 and CL domains. The Crossfab molecules depicted contain an interchange of the VH and VL regions, but may, in aspects in which the charge modifications are introduced into the CH1 and CL domains, alternatively contain an interchange of the CH1 and CL domains.

Fig. 2. Relative binding activity of original and optimized CD3 binding fragments CD3 _orig and CD3 _opt to recombinant CD3 according to SPR measurements under stress-free conditions after 14 days at 40°C, pH 6, or after 14 days at 37°C, pH 7.4 (IgG format).

Fig. 3. Binding of original and optimized CD3 binding fragments CD3 _orig and CD3 _opt to Jurkat NFAT cells as measured by flow cytometry (IgG format). Detection of antibodies bound to Jurkat NFAT cells was performed using a fluorescently labeled anti-human Fc-specific secondary antibody.

Fig. 4. Schematic illustration of the CD3 activation assay used in Example 3.

Fig. 5. Activation of Jurkat NFAT by original and optimized CD3 binding fragments CD3 _orig and CD3 _opt (IgG format). Jurkat NFAT reporter cells were co-incubated with anti-PGLALA expressing CHO cells (CHO-PGLALA) in the presence of CD3 _orig or CD3 _opt IgG PGLALA or CD3 _opt IgG wt as a negative control. Quantification of CD3 activation was performed by measuring luminescence after 24 h.

Fig. 6. Schematic illustration of the T-cell bispecific antibody (TCB) molecules obtained in the examples. All tested TCB antibody molecules were obtained as “2+1 IgG CrossFab, inverted” with charge modifications (VH/VL interchange in the CD3 binding molecule, charge modifications in the target antigen binding molecules, EE=147E, 213E; RK=123R, 124K).

Fig. 7. Relative binding activity of TYRP1 TCBs containing original or optimized CD3 binding fragments CD3 _orig or CD3 _opt with recombinant CD3 as 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.

Fig. 8. Relative binding activity of TYRP1 TCBs containing original or optimized CD3 binding fragments CD3 _orig or CD3 _opt , or the corresponding TYRP1 IgG with recombinant TYRP1 according to measurements by the SPR method under stress-free conditions after 14 days at 40°C, pH 6, or after 14 days at 37°C, pH 7.4.

Fig. 9. Binding of TYRP1 TCBs containing the original or optimized CD3 binding fragments CD3 _orig or CD3 _opt to Jurkat NFAT cells as measured by flow cytometry. Detection of TCBs bound to Jurkat NFAT cells was performed using a fluorescently labeled anti-human Fc-specific secondary antibody.

Fig. 10. Jurkat NFAT TYRP1 activation by TCB containing original or optimized CD3 binding molecules. Jurkat NFAT reporter cells were co-incubated with the melanoma cell line M150543 in the presence of TYRP1 TCB CD3 _orig or TYRP1 TCB CD3 _opt . CD3 activation in the presence of TCB was quantified by measuring luminescence after 24 h.

Fig. 11. Tumor cell killing and T cell activation by TYRP1 TCB containing native or optimized CD3 binding fragments. Killing of the melanoma cell line M150543 after treatment with TYRP1 TCB CD3 _orig and TYRP1 TCB CD3 _opt PBMCs from three different healthy donors (A-E: donor 1, F-M: donor 2, H-T: donor 3) was determined by LDH release after 24 h (A, F, H) and 48 h (B, H, O). In parallel, as a marker of T cell activation, upregulation of CD25 (B, D, I, L, P, C) and CD69 (G, E, K, M, R, T) on CD8 (D, E, L, M, C, T) and CD4 (B, G, I, L, P, R) T cells in PBMCs was measured by flow cytometry after 48 h.

Fig. 12. Specific binding of EGFRvIII IgG PGLALA. Specific binding of EGFRvIII IgG PGLALA antibodies to EGFRvIII without cross-reactivity with EGFRwt was studied by flow cytometry in CHO-EGFRvIII (A), EGFRvIII-positive DK-MG (B), and EGFRwt-expressing MKN-45 (C). Cetuximab was included as a positive control for EGFRwt expression.

Fig. 13. CAR J activation by EGFRvIII IgG PGLALA. Jurkat NFAT reporter cells expressing anti-PGLALA CAR were co-incubated with EGFRvIII-expressing DK-MG cells and EGFRvIII IgG PGLALA or DP47 IgG PGLALA antibodies as a negative control. Jurkat NFAT cells were quantified by measuring luminescence after 22 h.

Fig. 14. Binding of EGFRvIII IgG PGLALA and corresponding TCBs to EGFRvIII. Specific binding of EGFRvIII binding molecules, in the form of IgG PGLALA and converted to TCB, to CHO-EGFRvIII (A) and MKN-45 (B) cells was measured by flow cytometry.

Fig. 15. Jurkat NFAT activation by EGFRvIII TSV. Jurkat NFAT activation was determined as a marker of CD3 interaction with EGFRvIII TSV in the presence of EGFRvIII-positive DK-MG cells. DP47 TSV was included as a negative control.

Fig. 16. Lysis of tumor cells by EGFRvIII TSV. Induction of specific lysis of tumor cells by EGFRvIII TSV was determined after co-cultivation with freshly isolated PBMCs and EGFRvIII-positive DK-MG cells (A, B) or EGFRwt-positive MKN-45 cells (C, D) for 24 h (A, C) or 48 h (B, D).

Fig. 17. EGFRvIII T cell activation by TSV. Induction of EGFRvIII T cell activation by TSV was determined after co-culture with freshly isolated PBMCs and EGFRvIII-positive DK-MG cells (A, B, E, F) or EGFRwt-positive MKN-45 cells (C, D, G, H) 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 (D-3).

Fig. 18. Cytokine release from EGFRvIII TSV. Induction of IFNy (A, D), TNFa (B, E), and granzyme B (C, E) release by EGFRvIII TSV was determined after co-culture with freshly isolated PBMCs and EGFRvIII-positive DK-MG cells (A, B) or EGFRwt-positive MKN-45 cells (D-E).

Fig. 19. Specific binding of EGFRvIII affinity-matured IgG PGLALA. Specific binding of affinity-matured anti-EGFRvIII antibodies to EGFRvIII was compared with the parental EGFRvIII binding molecule on U87MG-EGFRvIII cells (A) and on the EGFRwt-positive MKN-45 cell line (B).

Fig. 20. Activation of Jurkat NFAT by EGFRvIII TSV. Jurkat NFAT activation was determined as a marker of CD3 interaction with EGFRvIII TSV in the presence of EGFRvIII-positive DK-MG cells (A), U87MG-EGFRvIII cells (B), and MKN-45 cells (C). DP47 TSV was included as a negative control.

Fig. 21. Relative binding activity of EGFRvIII TCBs containing original or optimized CD3 binding fragments CD3 _orig or CD3 _opt with recombinant CD3 as 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.

Fig. 22. Relative binding activity of EGFRvIII TCBs containing original or optimized CD3 binding fragments CD3 _orig or CD3 _opt with recombinant EGFRvIII according to SPR measurements under stress-free conditions after 14 days at 40°C, pH 6, or after 14 days at 37°C, pH 7.4.

Fig. 23. Binding of EGFRvIII TCBs containing the original or optimized CD3 binding fragments CD3 _orig or CD3 _opt to Jurkat NFAT cells as measured by flow cytometry. Detection of TCBs bound to Jurkat NFAT cells was performed using a fluorescently labeled anti-human Fc-specific secondary antibody.

Fig. 24. Binding of EGFRvIII TCBs containing the EGFRvIII binding fragment P063.056 or P056.021 to U87MG-EGFRvIII cells as measured by flow cytometry. Detection of TCBs bound to U87MG-EGFRvIII cells was performed using a fluorescently labeled anti-human Fc-specific secondary antibody.

Fig. 25. Tumor cell lysis and T cell activation by EGFRvIII TSV. Induction of specific tumor cell lysis (A, B) and T cell activation (C, D) by EGFRvIII TSV was determined after co-culture with freshly isolated PBMCs and U87MG-EGFRvIII cells for 24 h (A, C) or 48 h (B, D). DP47 TSV was included as a negative control.

Fig. 26. Jurkat NFAT activation comparing 2+1 and 1+1 format EGFRvIII TCB. Jurkat NFAT activation was determined as a marker of CD3 interaction with EGFRvIII TCB in the inverted 2+1 format and head-to-tail 1+1 format in the presence of EGFRvIII-positive U87MG-EGFRvIII cells.

Fig. 27. Tumor cell lysis and T cell activation comparing 2+1 and 1+1 formats of EGFRvIII TSV. Induction of specific tumor cell lysis (A, B) and T cell activation (C, D) by EGFRvIII TSV in the inverted 2+1 format and head-to-tail 1+1 format was determined after co-culture with freshly isolated PBMCs and U87MG-EGFRvIII cells for 24 h (A, C) or 48 h (B, D).

Fig. 28. Activation and proliferation of T cells by EGFRvIII TSV. Induction of T cell proliferation (A, B) and activation of CD4 T cells (A, B) and CD8 T cells (C, D) by TSV to EGFRvIII was determined after co-cultivation of U87MG-EGFRvIII and PBMC isolated from healthy donors.

Fig. 29. Tumor cell lysis, T cell activation, and cytokine release by EGFRvIII TSV. Induction of tumor cell lysis (A, B), T cell activation (C, D), and IFNγ and TNFα release (D, E) by EGFRvIII TSV was determined after co-culture of U87MG-EGFRvIII cells with PBMCs. Tumor cell lysis was determined at 24 h and 48 h after treatment, T cell activation and cytokine release were determined at 48 h.

Fig. 30. Tumor cell lysis, T cell activation, and cytokine release by TYRP-1 TSV. Induction of tumor cell lysis (A, B), T cell activation (C, D), and IFNγ and TNFα release (D, E) by TYRP-1 TSV was determined after co-culture of the patient-derived melanoma cell line M150543 with PBMC. Tumor cell lysis was determined at 24 h and 48 h after treatment, and T cell activation and cytokine release were determined at 48 h.

Fig. 31. In vivo efficacy of TYRP-1 TCB. Human melanoma cell line IGR-1 was subcutaneously injected into humanized NSG mice to investigate tumor growth inhibition in a subcutaneous melanoma xenograft model. Significant tumor growth inhibition (TGI) was observed in the TYRP-1 TCB group (68% TGI, p=0.0058*) compared with the vehicle group.

Fig. 32. In vivo efficacy of EGFRvIII TSV. Human glioblastoma cell line U87-huEGFRvIII was subcutaneously injected into humanized NSG mice to investigate tumor growth inhibition in a subcutaneous glioblastoma xenograft model. Significant tumor control was observed in the EGFRvIII TSV group, in which all mice achieved complete remission.

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

I. DEFINITIONS

In this document, terms are used as commonly understood in the art unless otherwise defined below.

In the context of this document, the terms "first", "second" or "third" with respect to antigen-binding domains, etc. are used to facilitate distinction when more than one of each type of fragment is present. The use of these terms does not imply a particular order or orientation of the fragment unless explicitly stated otherwise.

The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to an antibody that is capable of binding CD3 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting CD3. In one aspect, the extent of binding of an anti-CD3 antibody to an unrelated protein other than CD3 is less than about 10% of the binding of the antibody to CD3, as measured, for example, by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to CD3 has a dissociation constant (K _D ) of ≤ 1 μM, ≤ 500 nM, ≤ 200 nM, or ≤ 100 nM. An antibody is said to "specifically bind" to CD3 if the antibody has a K _{D of} 1 μM or less, as measured, for example, by SPR. In certain aspects, the anti-CD3 antibody binds to an epitope of CD3 that is conserved between CD3 from different species.

The term "antibody" as used herein is used in the broadest sense and includes various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the necessary antigen-binding activity.

"Antibody fragment" refers to a molecule, other than an intact antibody, that contains the portion of an intact antibody that binds 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 (e.g., scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments. For a review of some antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

In the context of this document, the terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably to refer to an antibody having a structure substantially similar to the structure of a native antibody.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, with the exception of possible variant antibodies, such as those containing natural mutations or arising during the production of the monoclonal antibody preparation, such variants generally being present in small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of methods, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and methods using transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for producing monoclonal antibodies are described herein.

An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some aspects, the antibody is purified to greater than 95% or 99% purity by determination, such as by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reversed phase HPLC, affinity chromatography, size exclusion chromatography) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some aspects, the antibodies provided herein are isolated antibodies.

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

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody comprises substantially all of at least one, and typically two, variable domains in which 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 a "humanized variable region." A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. In some aspects, some of the FR residues in a humanized antibody are substituted with corresponding residues of a non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized.

"A human antibody" is an antibody that has an amino acid sequence corresponding to an antibody sequence produced by a human or human cell, or that is obtained from a source other than a human that utilizes human antibody repertoires or other sequences encoding human antibodies. This definition of a human antibody explicitly excludes a humanized antibody that contains non-human antigen-binding residues. In certain aspects, a human antibody is obtained from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain aspects, a human antibody is obtained from a hybridoma cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered human antibodies or human antibody fragments herein.

The term "antigen-binding domain" refers to a portion of an antibody that contains a region that binds to and is complementary to a portion of an antigen or to the entire antigen. The antigen-binding domain can be formed, for example, by one or more variable domains of an antibody (also called variable regions of an antibody). In preferred aspects, the antigen-binding domain comprises a variable domain of an antibody light chain (VL) and a variable domain of an antibody heavy chain (VH).

The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that participates in the binding of the antibody to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain containing four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, ^6th ed., W H Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated by using the VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880–887 (1993); Clarkson et al., Nature 352:624–628 (1991). In the context of this document, in connection with variable region sequences, “Kabat numbering” refers to the numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

In the context of this document, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to herein as “Kabat numbering” or “Kabat numbering.” In particular, the Kabat numbering system (see pages 647-660 in 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 the kappa or lambda isotype, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge region, CH2, and CH3), which is then further emphasized in this document by the designation "Kabat EU index numbering" or "Kabat EU index numbering".

As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of the variable domain of an antibody that are hypervariable in sequence and that determine antigen-binding specificity, such as the "complementarity determining regions" ("CDRs"). Antibodies generally contain six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs as used herein include:

(a) hypervariable loops located 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 located at amino acid residues 24-34 (LI), 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

(c) antigenic contacts located 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)).

Unless otherwise noted, CDRs are defined in accordance with Kabat et al., supra. One skilled in the art will recognize that assignment of CDRs may also be defined in accordance with Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

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

Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

For the purposes of this document, a "human acceptor framework" is a framework comprising an amino acid sequence of a variable light chain (VL) framework or a variable heavy chain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A human acceptor framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may comprise changes in the amino acid sequence. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, a human VL acceptor framework is identical in sequence to a human immunoglobulin VL framework sequence or a human consensus framework sequence.

A "human consensus framework" is a framework region that represents the most frequently occurring amino acid residues in a set of human immunoglobulin VL or VH framework sequences. In general, a set of human immunoglobulin VL or VH sequences is derived from a subset of variable domain sequences. In general, the subset of sequences is the subset according to Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3.

As used herein, the term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons, consisting of two light chains and two heavy chains that are linked by disulfide bonds. In the direction from the N-terminus to the C-terminus, each heavy chain contains a variable domain (VH), also called the heavy chain variable domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, in the direction from the N-terminus to the C-terminus, each light chain contains a variable domain (VL), also called the light chain variable domain or light chain variable region, followed by a light chain constant domain (CL), also called the light chain constant region. The immunoglobulin heavy chain can be classified into one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, such as γ ₁ (IgG ₁ ), γ ₂ (IgG ₂ ), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ), and α ₂ (IgA ₂ ). The immunoglobulin light chain can be classified into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. Immunoglobulin consists primarily of two Fab molecules and an Fc domain linked by the immunoglobulin hinge region.

The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region contained in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

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

By "crossover" Fab molecule (also called "Crossfab") is meant a Fab molecule in which the variable domains or constant domains of the heavy and light chains of the Fab are exchanged (i.e. substituted for each other), i.e. a crossover Fab molecule comprises a peptide chain consisting of the light chain variable domain VL and the heavy chain constant domain 1 CHI (VL-CH1, in the N- to C-terminal direction), and a peptide chain consisting of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in the N- to C-terminal direction). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. At the same time, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

In contrast, a "standard" Fab molecule means that the Fab molecule has its natural format, i.e. it contains a heavy chain consisting of the variable and constant domains of the heavy chain (VH-CH1, in the direction from N- to C-terminus), and a light chain consisting of the variable and constant domains of the light chain (VL-CL, in the direction from N- to C-terminus).

As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. In one aspect, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expressing a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or may comprise a cleaved version of a full-length heavy chain. This may occur when the last two C-terminal amino acids of the heavy chain are glycine (G ₄ 46) and lysine (K447, numbering according to the EU index of Kabat). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. The amino acid sequences of the heavy chains, including the Fc region (or Fc domain subunit as defined herein) are designated herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one aspect, a heavy chain, including an Fc region (subunit), as defined herein, contained in an antibody of the invention comprises an additional C-terminal glycine-lysine dipeptide (G ₄ 46 and K447, numbering according to the EU index of Kabat). In one aspect, a heavy chain, including an Fc region (subunit), as defined herein, contained in an antibody of the invention comprises an additional C-terminal glycine residue (G ₄ 46, numbering according to the EU index of Kabat). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also referred to as the EU index, 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, an Fc domain "subunit" refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide containing the C-terminal constant regions of an immunoglobulin heavy chain capable of stable self-association. For example, the Fc domain subunit of IgG contains the CH2 and CH3 constant domains of IgG.

By "fused" is meant that the components (e.g., the Fab molecule and the Fc domain subunit) are linked by peptide bonds, either directly or through one or more peptide linkers.

The term "multispecific" means that the antibody is capable of specifically binding to at least two distinct antigenic determinants. A multispecific antibody may, for example, be a bispecific antibody. Typically, a bispecific antibody comprises two antigen-binding sites, each of which is specific for a distinct antigenic determinant. In certain aspects, a multispecific (e.g., bispecific) antibody is capable of simultaneously binding two antigenic determinants, in particular two antigenic determinants expressed on two different cells.

In the context of this document, the term "valent" refers to the presence of a specified number of antigen-binding sites in an antigen-binding molecule. Therefore, the term "monovalent antigen binding" refers to the presence of one (and no more than one) antigen-binding site specific for an antigen in an antigen-binding molecule.

"Antigen-binding site" refers to the site, i.e., one or more amino acid residues, of an antigen-binding molecule that mediates interaction with an antigen. For example, the antigen-binding site of an antibody contains amino acid residues from the complementarity-determining regions (CDRs). A native immunoglobulin molecule typically has two antigen-binding sites; a Fab molecule typically has one antigen-binding site.

As used herein, the term "antigenic determinant" or "antigen" refers to a site (e.g., a continuous sequence of amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids) in a polypeptide macromolecule to which an antigen-binding domain binds to form an antigen-binding domain-antigen complex. Suitable antigenic determinants may be found, for example, on the surface of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, in free serum, and/or in the extracellular matrix (ECM). In a preferred aspect, the antigen is a human protein.

"CD3" refers to any native CD3 from any vertebrate source, 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 encompasses "full-length" unprocessed CD3 as well as any form of CD3 produced by cellular processing. The term also includes naturally occurring CD3 variants, such as splice variants or allelic variants. In one aspect, the CD3 is human CD3, in particular human CD3 epsilon (CD3ε). The amino acid sequence of human CD3ε is set forth in SEQ ID NO: 112 (without signal peptide). See also UniProt (www.uniprot.org) Accession No. P07766 (version 189) or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. In another aspect, the CD3 is cynomolgus monkey (Macaca fascicularis) CD3, particularly cynomolgus monkey CD3ε. The amino acid sequence of cynomolgus monkey CD3ε is set forth in SEQ ID NO: 113 (without signal peptide). See also NCBI GenBank No. BAB71849.1. In certain aspects, an antibody of the invention binds to an epitope of CD3 that is conserved between CD3 antigens from different species, particularly human and cynomolgus monkey CD3. In preferred aspects, the antibody binds to human CD3.

In the context of this document, "target cell antigen" refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor, such as a cancer cell or a tumor stromal cell (in this case, "tumor cell antigen"). Preferably, the target cell antigen is not CD3 and/or is expressed on a cell other than CD3. In a preferred aspect, the target cell antigen is TYRP-1, in particular human TYRP-1. In another preferred aspect, the target cell antigen is EGFRvIII, in particular human EGFRvIII.

"TYRP1" or "TYRP-1" refers to tyrosine-related protein 1, which is an enzyme involved in the synthesis of melanin. The mature form of TYRP1, also originally named gp75, is a 75 kDa transmembrane glycoprotein. The sequence of human TYRP1 is set forth in SEQ ID NO: 114 (excluding signal peptide). See also UniProt entry #P17643 (version 185). As used herein, "TYRP1" refers to any native TYRP1 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed TYRP1 as well as any form of TYRP1 produced by in-cell processing. The term also includes naturally occurring TYRP1 variants, such as splice variants or allelic variants. In one aspect, TYRP1 is human TYRP1.

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

"Affinity" refers to the strength of the net non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K _D ). Affinity can be measured by well-established methods known in the art, including the methods described herein. A preferred method for measuring affinity is surface plasmon resonance (SPR).

An "affinity matured" antibody refers to an antibody with one or more alterations in one or more complementarity determining regions (CDRs) compared to a parent antibody that does not contain such alterations, wherein such alterations result in an improvement in the affinity of the antibody for the antigen.

"Decreased binding", such as decreased binding to an Fc receptor, refers to a decrease in the affinity for the relevant interaction as defined by, for example, the SPR. For clarity, this term also includes a decrease in affinity to zero (or below the detection limit of the assay), i.e., complete elimination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the relevant interaction.

In the context of this document, "T cell activation" refers to one or more cellular responses of a lymphocyte, in particular a cytotoxic T lymphocyte, selected from: proliferation, differentiation, secretion of cytokines, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers. Suitable assays for determining T cell activation are known in the art and are described herein.

"A modification that promotes association of the first and second Fc domain subunits" is a manipulation of the peptide backbone or post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. As used herein, an association-promoting modification preferably includes separate modifications made to each of the Fc domain subunits that are to associate (i.e., the first and second Fc domain subunits), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, an association-promoting modification may alter the structure or charge of one or both Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may be non-identical in the sense that additional components fused to each of the subunits (e.g., antigen-binding domains) are not identical. In some aspects, a modification that promotes association of the first and second Fc domain subunits comprises an amino acid mutation in the Fc domain, in particular an amino acid substitution. In a preferred aspect, a modification that promotes association of the first and second Fc domain subunits comprises a separate amino acid mutation, in particular an amino acid substitution, in each of the two Fc domain subunits.

The term "effector functions" refers to the biological activities mediated by the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated uptake of antigen by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

An "activating Fc receptor" is an Fc receptor that, upon interaction with the Fc domain of an antibody, triggers signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcaRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which antibodies or their derivatives containing an Fc region specifically bind, generally via the protein portion N-terminal to the Fc region. As used herein, the term "reduced ADCC" is defined as a decrease in the number of target cells lysed in a given time at a given concentration of antibody in the medium surrounding the target cells via the ADCC mechanism defined above, and/or as an increase in the concentration of antibody in the medium surrounding the target cells required to ensure lysis of a given number of target cells in a given time via the ADCC mechanism. The reduction in ADCC is determined relative to the ADCC mediated by the same antibody produced in the same host cell type using the same standard production, purification, formulation, and storage methods (which are well known to those skilled in the art), but which has not been engineered. For example, the reduction in ADCC mediated by an antibody containing an amino acid substitution in the Fc domain that reduces ADCC is determined relative to the ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT Publication No. WO 2006/082515 or PCT Publication No. WO 2012/130831).

In the context of this document, the terms "design, designed, designing" are considered to include any manipulation of the peptide backbone or post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof.

Design involves modifications of the amino acid sequence, glycosylation profile, or side chain groups of individual amino acids, as well as combinations of these approaches.

In the context of this document, the term "amino acid mutation" is intended to include amino acid substitutions, deletions, insertions and modifications. Any combination of substitution, deletion, insertion and modification may be made to obtain the final construct, provided that the final construct has the desired characteristics, such as reduced binding to an Fc receptor or increased association with another peptide. Deletions and insertions in the amino acid sequence include amino- and/or carboxy-terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. In order to alter, for example, the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., substitution of one amino acid by another amino acid having different structural and/or chemical properties, are particularly preferred. Amino acid substitutions include substitution with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be created using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is understood that methods of altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, can also be used. Different designations can be used herein to indicate the same amino acid mutation. For example, a substitution of proline at position 329 of the Fc domain with glycine can be designated as 329G, G329, G329, P329G, or Pro329Gly.

"Percent (%) amino acid sequence identity" relative 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 a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways that are known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA software package. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. Alternatively, percent identity values can be obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code was filed with user documentation in the United States Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration Number TXU510087, and is described in WO 2001/007611.

Unless otherwise stated, for the purposes of this document % amino acid sequence identity values are obtained using the ggsearch program from the FASTA package version 36.3.8c or later with the BLOSUM50 comparison matrix. The FASTA package was written by W. R. Pearson and D. J. Lipman ("Improved Tools for Biological Sequence Analysis", PNAS 85 (1988) 2444–2448), W. R. Pearson ("Effective protein sequence comparison" Meth. Enzymol. 266 (1996) 227–258), and Pearson et al. (Genomics 46 (1997) 24–36) and is freely available at www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/fasta. Alternatively, the open server available at fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare sequences, using the ggsearch program (global protein:protein) and default options (BLOSUM50; open: -10; extend: -2; Ktup = 2) to ensure that a global rather than a local alignment is performed. Percent amino acid identity is reported in the alignment output header file.

The term "polynucleotide" or "nucleic acid molecule" includes any compound and/or substance that comprises a polymer of nucleotides. Each nucleotide consists of a base, particularly 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, a nucleic acid molecule is described by a sequence of bases, wherein said bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically presented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. A nucleic acid molecule may be linear or circular. Furthermore, the term "nucleic acid molecule" includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, a nucleic acid molecule described herein may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as a vector for direct expression of an antibody of the invention in vitro and/or in vivo, such as in a host organism or a patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule so that the mRNA can be administered to a subject to generate antibodies in vivo (see, e.g., Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2101823 B1).

An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid molecule includes a nucleic acid molecule that is contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

"Isolated polynucleotide (or nucleic acid) encoding an antibody" refers to one or more polynucleotide molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such polynucleotide molecules in a single vector or separate vectors and such polynucleotide molecules present at one or more locations in a host cell.

As used herein, the term "vector" refers to a nucleic acid molecule capable of increasing the copy number of another nucleic acid to which it is linked. This term includes a vector in the form of a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of a host cell into which it is introduced. Some vectors are capable of directing the expression of nucleic acids to which they are functionally linked. Such vectors are referred to herein as "expression vectors."

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformed" and "transformed cells," which include initially transformed cells and progeny derived therefrom, regardless of passage number. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Included herein are mutant progeny that have the same function or biological activity for which the initially transformed cells are screened or selected. A host cell is any type of cellular system that can be used to generate the antibodies of the present invention. Host cells include cultured cells, such as cultured mammalian 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 and plant cells, to name just a few, but also cells present in the body of a transgenic animal, a transgenic plant or a cultured plant or animal tissue. In one aspect, the host cell of the invention is a eukaryotic cell, in particular a mammalian cell. In one aspect, the host cell is not a cell in the human body.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that provides the effectiveness of the biological activity of the active ingredient it contains and that does not contain additional components that are unacceptably toxic to the subject to which the composition is to be administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or pharmaceutical formulation other than the active ingredient that is nontoxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, the term "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to a clinical intervention with the aim of altering the natural course of a disease in an individual being treated, and may be performed either prophylactically or during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease, and causing remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain respects, an individual or subject is a human.

An "effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

II. COMPOSITIONS AND METHODS

The invention provides antibodies that bind CD3, including multispecific antibodies that bind CD3 and a second antigen. These antibodies exhibit excellent stability in combination with other favorable properties for therapeutic use, such as in terms of efficacy and safety, pharmacokinetics, and manufacturing capability. The antibodies of the invention are useful, for example, for the treatment of diseases such as cancer.

A. Anti-CD3 antibodies

In one aspect, the invention provides antibodies that bind to CD3. In one aspect, the invention provides isolated antibodies that bind to CD3. In one aspect, the invention provides antibodies that specifically bind to CD3. In certain aspects, the anti-CD3 antibodies retain greater than about 90% of the binding activity to CD3 after 2 weeks at pH 7.4, 37°C, compared to the binding activity after 2 weeks at pH 6, -80°C, as determined by surface plasmon resonance (SPR).

In one aspect, the present invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10.

In one aspect, the antibody is a humanized antibody. In one aspect, the antigen-binding domain is a humanized antigen-binding domain (i.e., an antigen-binding domain of a humanized antibody). In one aspect, the VH and/or VL is a humanized variable region.

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

In one aspect, the VH comprises one or more heavy chain framework sequences (i.e., a FR1, FR2, FR3, and/or FR4 sequence) of the heavy chain variable region sequence of SEQ ID NO: 7. In one aspect, the VH comprises a sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In one aspect, the VH comprises a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. In one aspect, the VH comprises a sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 7. In certain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions at compared to a reference sequence, but an antibody comprising the sequence retains the ability to bind to CD3. In certain aspects, a total of 1-10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO: 7. In certain aspects, the substitutions, insertions or deletions are in regions outside the CDRs (i.e., in the FRs).

In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 7. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 7, including post-translational modifications of that sequence.

In one aspect, the VL comprises one or more light chain framework sequences (i.e., a FR1, FR2, FR3, and/or FR4 sequence) of the light chain variable region sequence of SEQ ID NO: 11. In one aspect, the VL comprises a sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In one aspect, the VL comprises a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 11. In one aspect, the VL comprises a sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 11. In certain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions compared to a reference sequence, but the antibody comprising the sequence retains the ability to bind to CD3. In certain aspects, a total of 1-10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO: 11. In certain aspects, the substitutions, insertions or deletions are in regions outside the CDRs (i.e., in the FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 11. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 11, including post-translational modifications of that sequence.

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

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

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

In another aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen-binding domain comprising a VH comprising the VH heavy chain CDR sequences of SEQ ID NO: 7 and a VL comprising the VL light chain CDR sequences of SEQ ID NO: 11.

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

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

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

In one aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen-binding domain comprising a VH sequence corresponding to any of the above aspects and a VL sequence corresponding to any of the above aspects.

In one aspect, the antibody comprises a human constant region. In one aspect, the antibody is an immunoglobulin molecule comprising a human constant region, in particular an IgG immunoglobulin molecule comprising a human CH1, CH2, CH3 and/or CL domain. Exemplary human constant domain sequences are set forth in SEQ ID NOs: 120 and 121 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 122 (human IgG1 heavy chain CH1-CH2-CH3 constant domains). In one aspect, the antibody comprises a light chain constant region comprising 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 amino acid sequence of SEQ ID NO: 120. In one aspect, the antibody comprises 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 amino acid sequence of SEQ ID NO: 122. In particular, the heavy chain constant region may comprise amino acid mutations in the Fc domain as described herein.

In one aspect, the first antigen-binding domain comprises a human constant region. In one aspect, the first antigen-binding fragment is a Fab molecule comprising a human constant region, in particular a human CH1 and/or CL domain. In one aspect, the first antigen-binding domain comprises a light chain constant region comprising 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 amino acid sequence of SEQ ID NO: 120. In particular, the light chain constant region may comprise amino acid mutations described herein as "charge modifications" and/or may comprise a deletion or substitution of one or more (in particular two) N-terminal amino acids if it belongs to a Fab crossover molecule. In some aspects, the first antigen-binding domain comprises 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 sequence of the CH1 domain contained in the amino acid sequence of SEQ ID NO: 122. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations described herein as "charge modifications."

In one aspect, the antibody is a monoclonal antibody.

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

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

In one aspect, the first antigen-binding domain is a Fab molecule. In a preferred aspect, the first antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other (i.e., the first antigen-binding domain is a crossover Fab molecule).

In a further aspect, the antibody of any of the above aspects may have any of the features, individually or in combination, described in Sections II.A.1-8. below.

In a preferred aspect, the antibody comprises an Fc domain, in particular an IgG Fc domain, more particularly an IgG1 Fc domain. In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc domain is a human IgG ₁ Fc domain. The Fc domain is comprised of a first and a second subunit and may have any of the features, individually or in combination, described below herein with respect to variants of Fc domains (Section II. A. 8).

In another preferred aspect, the antibody comprises a second and, optionally, a third antigen-binding domain that binds to a second antigen (i.e., the antibody is a multispecific antibody, as further described below herein (Section II.A. 7).

1. Antibody fragments

In certain aspects, the antibody provided herein is an antibody fragment.

In one aspect, the antibody fragment is a Fab, Fab', Fab'-SH or F(ab')2 molecule, in particular a Fab molecule as described herein. A "Fab'molecule" differs from a Fab molecule by the addition of residues at the carboxy terminus of the CH1 domain, including one or more cysteine residues from the hinge region of the antibody. Fab'-SH are Fab' molecules in which the cysteine residue(s) of the constant domains bear a free thiol group. Treatment with pepsin produces an F(ab') ₂ molecule that contains two antigen-binding sites (two Fab molecules) and a portion of the Fc region.

In another aspect, the antibody fragment is a diabody, triabody, or tetrabody. "Diabodies" are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

In a further aspect, the antibody fragment is a single-chain Fab molecule. A "single-chain Fab molecule" or "scFab" is a polypeptide consisting of 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 said antibody domains and said linker have one of the following orders in the direction from N-terminus to 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, said linker is a polypeptide of at least 30 amino acids, preferably from 32 to 50 amino acids. Said single-chain Fab molecules are stabilized by a natural disulfide bond between the CL domain and the CH1 domain. In addition, the single-chain Fab molecules can be further stabilized by creating interchain disulfide bonds by inserting cysteine residues (e.g. at position 44 of the variable domain of the heavy chain and position 100 of the variable domain of the light chain according to Kabat numbering).

In another aspect, the antibody fragment is a single-chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy (VH) and light chains (VL) of an antibody connected by a linker. In particular, the linker is a short polypeptide of 10-25 amino acids and is typically enriched in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL or vice versa. The protein retains the specificity of the parent antibody despite the removal of the constant regions and the introduction of a linker. For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269–315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.

In another aspect, the antibody fragment is a single-domain antibody. "Single-domain antibodies" are antibody fragments that comprise all or a portion of a heavy chain variable domain of an antibody, or all or a portion of a light chain variable domain of an antibody. In certain aspects, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

Antibody fragments can be produced by a variety of methods, including, but not limited to, proteolytic cleavage of an intact antibody, as well as recombinant production by recombinant host cells (e.g., E. coli), as described herein.

2. Humanized antibodies

In certain aspects, the antibody provided herein is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody optionally also comprises at least a portion of a human constant region. In some aspects, some FR residues in the humanized antibody are replaced with corresponding residues of a non-human antibody (e.g., the antibody from which the CDR residues are derived), such as to restore or improve the specificity or affinity of the antibody.

Humanized antibodies and methods for producing them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619–1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323–329 (1988); Queen et al., Proc. Nat 4 Acad. Sci. USA 86:10029–10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25–34 (2005) (which describes grafting of a specificity determining region (SDR)); Padlan, Mol. Immunol. 28:489–498 (1991) (described “surface modification”); Dall'Acqua et al., Methods 36:43–60 (2005) (described “FR shuffling”); and Osbourn et al., Methods 36:61–68 (2005) and Klimka et al., Br. J. Cancer, 83:252–260 (2000) (described a “directed selection” approach to FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected by the "best match" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light 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 mature (somatically mutated) or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678–10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611–22618 (1996)).

3. Glycosylation variants

In certain aspects, the antibody provided herein is altered to increase or decrease the degree of glycosylation of the antibody. Addition or removal of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

If the antibody comprises an Fc region, the oligosaccharide attached thereto can be altered. Native antibodies produced by mammalian cells typically comprise a branched biantennary oligosaccharide that is typically attached via an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some aspects, modifications to the oligosaccharide in an antibody of the invention can be made to create antibody variants with certain improved properties.

In one aspect, antibody variants are provided that comprise a non-fucosylated oligosaccharide, i.e., an oligosaccharide structure that lacks fucose attached (directly or indirectly) to the Fc region. In particular, such a non-fucosylated oligosaccharide (also referred to as an "afucosylated" oligosaccharide) is an N-linked oligosaccharide that lacks a fucose residue attached to the first GlcNAc in the stem of a biantennary oligosaccharide structure. In one aspect, antibody variants are provided that have an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a native or 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 percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues relative to the sum of all oligosaccharides attached to Asn 297 (e.g., complex, hybrid, and high-mannose structures), as measured by TOF-MALDI mass spectrometry, e.g., as described in WO 2006/082515. Asn297 refers to the asparagine residue located at position 297 in the Fc region (according to the EU numbering of the Fc region residues); wherein Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence variations in the antibodies. Such antibodies having an increased percentage of non-fucosylated oligosaccharides in the Fc region may exhibit improved FcγRIIIa receptor binding and/or improved effector function, in particular improved ADCC function. See, for example, US 2003/0157108; US 2004/0093621.

Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially Example 11), and knockout cell lines such as CHO cells with a knockout of the alpha-1,6-fucosyltransferase gene, FUT8 (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003/085107), or cells with reduced or eliminated activity of GDP-fucose synthesis or carrier protein (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

In a further aspect, antibody variants are provided with bisected oligosaccharides, for example, wherein a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function, as described above. Examples of such antibody variants are described in, for example, Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO 2003/011878.

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

4. Cysteine-engineered antibody variants

In certain aspects, there may be a need to create cysteine engineered antibodies, such as THIOMAB™ antibodies, in which one or more residues are substituted with cysteine residues. In preferred aspects, the substituted residues are at accessible sites on the antibody. By substituting these residues with cysteine, reactive thiol groups are located at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or drug-linker moieties, to create an immunoconjugate, as further described herein. Cysteine engineered antibodies can be created as described, for example, in U.S. Patent Nos. 7,521,541, 830,930, 7,855,275, 9000130, or WO 2016040856.

5. Antibody derivatives

In certain aspects, the antibody provided herein can be further modified to contain additional non-protein moieties that are known in the art and readily available. Moieties suitable for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers, oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymer molecules attached to the antibody may vary and, if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations that include, but are not limited to, the specific properties or functions of the antibody that are to be improved, whether the antibody derivative will be used in therapy under certain conditions, etc.

6. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CD3 antibody according to this document conjugated (chemically linked) 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 radioactive isotopes.

In one aspect, the immunoconjugate is an antibody drug conjugate (ADC), in which the antibody is conjugated to one or more therapeutic agents mentioned above. Typically, the antibody is linked to one or more therapeutic agents via linkers. A review of ADC technology, including examples of therapeutic agents, drugs, and linkers, is provided in Pharmacol Review 68:3-19 (2016).

In another aspect, the immunoconjugate comprises an antibody of the invention conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.

In another aspect, an immunoconjugate comprises an antibody of the invention conjugated to a radioactive atom to form a radioconjugate. A number of radioactive isotopes are available for the preparation of radioconjugates. Examples include At ²¹¹ , I ¹³¹ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes of Lu. If the radioconjugate is used for detection purposes, it may contain a radioactive atom for scintigraphic studies, such as Tc ^99m or I ¹²³ , or a spin label for nuclear magnetic resonance (NMR) (also known as magnetic resonance imaging, MRI) such as I ¹²¹ , I ¹³¹ , In ¹¹¹ , F ¹⁹ , C ¹³ , N ¹⁵ , O ¹⁷ , gadolinium, manganese or iron.

Conjugates of the antibody and the cytotoxic agent can be generated using a number of bifunctional protein coupling agents such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis(p-diazonium benzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin-based immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelating agent for conjugating a radionuclide to an antibody. See WO 94/11026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

As used herein, immunoconjugates or ADCs expressly include, but are not limited to, conjugates prepared using cross-linking reagents including, but not limited to, 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, and sulfo-SMPB, as well as SVSB (succinimidyl (4-vinyl sulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

7. Multispecific antibodies

In certain aspects, the antibody provided herein is a multispecific antibody, in particular a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two antigenic determinants (e.g., two different proteins or two different epitopes on a single protein). In certain aspects, a multispecific antibody has three or more binding specificities. In certain aspects, one binding specificity is directed to CD3 and the other is directed to any other antigen. In certain aspects, multispecific antibodies can bind to two (or more) different epitopes of CD3. Multispecific (e.g., bispecific) antibodies can also be used to localize cytotoxic agents or cells to cells that express CD3. Multispecific antibodies can be produced as full-length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having 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 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be generated by engineering using the electrostatic interaction effect to create Fc heterodimeric antibody molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); the use of leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547–1553 (1992) and WO 2011/034605); the use of common light chain technology to overcome the problem of light chain mispairing (see, e.g., WO 98/50431); the use of diabody technology to produce bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444–6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and the production of trispecific antibodies, as described in, e.g., Tutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more antigen-binding sites, including, for example, "octopus antibodies" or DVD-Ig, are also included herein (see, for example, WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen-binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. Multispecific antibodies or antigen-binding fragments thereof also include “dual-acting FAbs” or “DAFs” that contain an antigen-binding site that binds to CD3 as well as another different antigen or to two different epitopes of CD3 (see, e.g., US 2008/0069820 and WO 2015/095539).

Multispecific antibodies may be provided in an asymmetric form with a crossover of domains in one or more binding arms with the same antigen specificity (the so-called "CrossMab" technology), i.e. by exchanging VH/VL domains (see, e.g., WO 2009/080252 and WO 2015/150447), CH1/CL domains (see, e.g., WO 2009/080253) or complete Fab arms (see, e.g., WO 2009/080251, WO 2016/016299, see also Schaefer et al, PNAS, 108 (2011) 1187-1191 and Klein at al., MAbs 8 (2016) 1010-20). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interfaces to ensure proper Fab pairing. See, for example, WO 2016/172485.

Various other molecular formats for multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

A particular type of multispecific antibody also included herein are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, such as a tumor cell, and to an activating invariant component of the T cell receptor (TCR) complex, such as CD3, to redirect T cells to kill the target cells. Therefore, in preferred aspects, the antibody provided herein is a multispecific antibody, in particular a bispecific antibody, wherein one of the binding specificities is directed to CD3 and the other to an antigen on the target cell.

Examples of bispecific antibody formats that can be used for these purposes include, but are not limited to, so-called "BiTE" (bispecific T-cell engaging activator) molecules, in which two scFv molecules are linked by a flexible linker (see, e.g., WO 2004/106381, WO 2005/061547, WO 2007/042261 and WO 2008/119567, Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299–305 (1996)) and their derivatives, such as tandem diabodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41–56 (1999)); “DART” (dual affinity retargeting) molecules, which are based on the diabody format but have a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436–449 (2010)), and the so-called triomabs, which are fully hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458–467 (2010)). Specific T-cell bispecific antibody formats included herein are described in WO 2013/026833, WO 2013/026839, WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

Preferred aspects of the multispecific antibodies of the present invention are described below.

In one aspect, the invention provides an antibody that binds to CD3, comprising a first antigen-binding domain that binds to CD3 as described herein, and comprising a second and, optionally, a third antigen-binding domain that binds to a second antigen.

According to preferred aspects of the invention, the antigen-binding domains contained in the antibody are Fab molecules (i.e. antigen-binding domains consisting of a heavy and a light chain, each of which comprises a variable and a constant domain). In one aspect, the first, second and/or, if present, the third antigen-binding domain is a Fab molecule. In one aspect, said Fab molecule is human. In a preferred aspect, said Fab molecule is humanized. In another aspect, said Fab molecule comprises constant domains of a human heavy and light chain.

Preferably, at least one of the antigen-binding domains is a crossover Fab molecule. Such a modification reduces mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the (multispecific) antibody of the invention when produced recombinantly. In a preferred crossover Fab molecule useful for the (multispecific) antibody of the invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are exchanged. However, even with such an exchange of domains, the (multispecific) antibody preparation may contain some by-products due to the so-called Bence-Jones interaction between the mispaired heavy and light chains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To further reduce mispairing of heavy and light chains from different Fab molecules and thus improve the purity and yield of the desired (multispecific) antibody, charged amino acids of opposite charges can be introduced at specific amino acid positions in the CH1 and CL domains of the Fab molecule binding to the first antigen (CD3) or the Fab molecule(s) binding to the second antigen (e.g., a target cell antigen such as TYRP-1 or EGFRvIII), as further described herein. Charge modifications are made in standard Fab molecules contained in the (multispecific) antibody (as shown, e.g., in Fig. 1A-C, G-J) or in VH/VL crossover Fab molecules contained in the (multispecific) antibody (as shown, e.g., in Fig. 1D-E, K-O) (but not in both). In preferred aspects, charge modifications are made to standard Fab molecules contained in a (multispecific) antibody (which in preferred aspects binds to a second antigen, e.g. a target cell antigen such as TYRP-1 or EGFRvIII).

In a preferred aspect according to the invention, the (multispecific) antibody is capable of simultaneously binding to a first antigen (i.e. CD3) and a second antigen (e.g. a target antigen such as TYRP-1 or EGFRvIII). In one aspect, the (multispecific) antibody is capable of cross-linking a T cell and a target cell by simultaneously binding to CD3 and the target cell antigen. In an even more preferred aspect, such simultaneous binding results in lysis of the target cell, in particular a tumor cell expressing the target antigen (e.g. TYRP-1 or EGFRvIII). In one aspect, such simultaneous binding results in activation of T cells. In other aspects, such simultaneous binding results in a cellular response of a T lymphocyte, in particular a cytotoxic T lymphocyte, selected from the group consisting of proliferation, differentiation, secretion of cytokines, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one aspect, binding of a (multispecific) antibody to CD3 without simultaneous binding to a target cell antigen does not result in T cell activation.

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

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

a) First antigen-binding domain

The (multispecific) antibody of the invention comprises at least one antigen-binding domain (a first antigen-binding domain) that binds to CD3. In preferred aspects, the CD3 is human CD3 (SEQ ID NO: 112) or cynomolgus CD3 (SEQ ID NO: 113), in particular human CD3. In one aspect, the first antigen-binding domain is cross-reactive with (i.e. specifically binds to) human and cynomolgus CD3. In some aspects, the CD3 is the epsilon subunit of CD3 (CD3 epsilon).

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

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

In preferred aspects, the antigen-binding domain that binds to CD3 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 CH1 and CL of the heavy and light chains of the Fab are replaced/substituted with each other. In such aspects, the antigen-binding domain that binds to a second antigen (e.g. a target cell antigen such as TYRP-1 or EGFRvIII) is preferably a standard Fab molecule. In aspects in which more than one antigen-binding domain, in particular a Fab molecule that binds to a second antigen, is present in a (multispecific) antibody, the antigen-binding domain that binds to CD3 is preferably a crossover Fab molecule and the antigen-binding domains that bind to the second antigen are standard Fab molecules.

In alternative aspects, the antigen-binding domain that binds to CD3 is a standard Fab molecule. In such aspects, the antigen-binding domain that binds to a second antigen (e.g., a target cell antigen such as TYRP-1 or EGFRvIII) 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 CH1 and CL of the heavy and light chains of the Fab are replaced/substituted with each other. In aspects in which more than one antigen-binding domain, in particular a Fab molecule that binds to CD3, is present in a (multispecific) antibody, the antigen-binding domain that binds to the second antigen is preferably a crossover Fab molecule and the antigen-binding domains that bind to CD3 are standard Fab molecules.

In preferred aspects, the first antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH of the Fab light chain and the Fab heavy chain are exchanged with each other (i.e., according to such an aspect, the first antigen-binding domain is a crossover Fab molecule, wherein the variable or constant domains of the Fab light chain and the Fab heavy chain are exchanged). In one such aspect, the second (and, if present, the third) antigen-binding domain is a standard Fab molecule.

In one aspect, the (multispecific) antibody has no more than one antigen-binding domain that binds to CD3 (i.e., the antibody provides monovalent binding to CD3).

b) The second (and third) antigen-binding domain

In certain aspects, the (multispecific) antibody of the invention comprises at least one antigen-binding domain, in particular a Fab molecule, which binds to a second antigen. The second antigen is preferably not CD3, i.e. different from CD3. In one aspect, the second antigen is an antigen expressed on a cell other than CD3 (i.e. expressed on a cell other than a T cell). In one aspect, the second antigen is a target cell antigen, in particular a tumor cell antigen. In a particular aspect, the second antigen is TYRP-1. In another particular aspect, the second antigen is EGFRvIII. The second antigen-binding domain is capable of directing the (multispecific) antibody to a target site, for example to a particular tumor cell type, that expresses the second antigen.

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

In certain aspects, the (multispecific) antibody comprises two antigen-binding domains, in particular two Fab molecules, which bind to a second antigen. In such a preferred aspect, each of these antigen-binding domains binds to the same antigenic determinant. In an even more preferred aspect, all of these antigen-binding domains are identical, i.e. they have the same molecular format (e.g. a standard or crossover Fab molecule) and comprise the same amino acid sequences, comprising the same amino acid substitutions in the CH1 and CL domains, as described herein (if any). In one aspect, the (multispecific) antibody comprises no more than two antigen-binding domains, in particular two Fab molecules, which bind to a second antigen.

In preferred aspects, the antigen binding domain(s) that binds to the second antigen is/are a standard Fab molecule. In such aspects, the antigen binding domain(s) that binds to CD3 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 CH1 and CL of the heavy and light chains of the Fab are/are replaced by each other.

In alternative aspects, the antigen-binding domain(s) that binds to the second antigen 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 CH1 and CL of the heavy and light chains of the Fab are exchanged/substituted with each other. In such aspects, the antigen-binding domain(s) that binds to CD3 is a standard Fab molecule.

In one aspect, the second (and, if present, the third) antigen-binding domain comprises a human constant region. In one aspect, the second (and, if present, the third) antigen-binding domain is a Fab molecule comprising a human constant region, in particular a human CH1 and/or CL domain. Exemplary human constant domain sequences are set forth in SEQ ID NOs: 120 and 121 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 122 (human IgG1 heavy chain CH1-CH2-CH3 constant domains). In one aspect, the second (and, if present, the third) antigen-binding domain comprises a light chain constant region comprising 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 amino acid sequence of SEQ ID NO: 120. In particular, the light chain constant region may comprise amino acid mutations described herein as "charge modifications" and/or may comprise a deletion or substitution of one or more (in particular two) N-terminal amino acids if it belongs to a Fab crossover molecule. In some aspects, the second (and, if present, the third) antigen-binding domain comprises 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 sequence of the CH1 domain contained in the amino acid sequence of SEQ ID NO: 122. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations, described herein as "charge modifications."

TYRP-1

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

In one aspect, the second (and, if present, the third) antigen-binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 15, HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21.

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

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

In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3 and/or FR4 sequences) of SEQ ID NO: 18. In one aspect, the VH comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18. In one aspect, the VH comprises a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 18. In one aspect, the VH comprises a sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 18. In certain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions compared to a reference sequence, but the antibody comprising the sequence retains the ability to bind to TYRP-1. In certain aspects, a total of 110 amino acids in the amino acid sequence of SEQ ID NO: 18 have been substituted, inserted and/or deleted. In certain aspects, the substitutions, insertions or deletions are in regions outside the CDRs (i.e., in the FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 18. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 18, including post-translational modifications of that sequence.

In one aspect, the VL of the second (and, if present, third) antigen-binding domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3 and/or FR4 sequences) of SEQ ID NO: 22. In one aspect, the VL comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 22. In one aspect, the VL comprises a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 22. In one aspect, the VL comprises a sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 22. In certain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions compared to a reference sequence, but an antibody comprising the sequence retains the ability to bind to TYRP-1. In certain aspects, a total of 1-10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO: 22. In certain aspects, the substitutions, insertions or deletions are in regions outside the CDRs (i.e., in the FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 22. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 22, including post-translational modifications of that sequence.

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

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

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

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

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

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

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

EGFRvIII

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

In one aspect, the second (and, if present, the third) antigen-binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 85, HCDR 2 of SEQ ID NO: 86 and HCDR 3 of SEQ ID NO: 87, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 89, LCDR 2 of SEQ ID NO: 90 and LCDR 3 of SEQ ID NO: 91.

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

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

In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3 and/or FR4 sequences) of SEQ ID NO: 88. In one aspect, the VH comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88. In one aspect, the VH comprises a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 88. In one aspect, the VH comprises a sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 88. In certain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions compared to a reference sequence, but the antibody comprising the sequence retains the ability to bind to EGFRvIII. In certain aspects, a total of 110 amino acids in the amino acid sequence of SEQ ID NO: 88 have been substituted, inserted and/or deleted. In certain aspects, the substitutions, insertions or deletions are in regions outside the CDRs (i.e., in the FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 88. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 88, including post-translational modifications of that sequence.

In one aspect, the VL of the second (and, if present, third) antigen-binding domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3 and/or FR4 sequences) of SEQ ID NO: 92. In one aspect, the VL comprises a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 92. In one aspect, the VL comprises a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 92. In one aspect, the VL comprises a sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 92. In certain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions compared to a reference sequence, but the antibody comprising the sequence retains the ability to bind to EGFRvIII. In certain aspects, a total of 1-10 amino acids have been substituted, inserted and/or deleted in the amino acid sequence of SEQ ID NO: 92. In certain aspects, the substitutions, insertions or deletions are in regions outside the CDRs (i.e., in the FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 92. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 92, including post-translational modifications of that sequence.

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

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

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

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

In a further aspect, the second (and, if present, third) antigen binding domain comprises the amino acid sequences HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 88 and the amino acid sequences LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 92.

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

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

In alternative aspects, the second (and, if present, the third) antigen binding domain comprises a VH sequence according to any of the aspects set out above in this section in relation to EGFRvIII, and a VL sequence according to any of the aspects set out above in this section in relation to EGFRvIII, but based on the following sequences (in row order) instead of SEQ ID NO 85 (HCDR1), 86 (HCDR2), 87 (HCDR3), 88 (VH), 89 (LCDR1), 90 (LCDR2), 91 (LCDR3) and 92 (VL):

In one aspect, the second (and, if present, third) antigen binding domain comprises a VH sequence according to any of the aspects set out above in this section and a VL sequence according to any of the aspects set out above in this section.

Anti-TYRP-l and anti-EGFRvIII antibodies

The invention also provides an antibody that binds to TYRP-1, comprising a VH sequence according to any of the aspects given above in this section in connection with TYRP-1, and a VL sequence according to any of the aspects given above in this section in connection with TYRP-1 (e.g., an antibody that binds to TYRP-1, comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 15, HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21; or an antibody that binds to TYRP-1 comprising a VH comprising the sequence of SEQ ID NO: 18 and a VL comprising the sequence of SEQ ID NO: 22).

The invention also provides an antibody that binds EGFRvIII, comprising a VH sequence according to any of the aspects set out above in this section in connection with EGFRvIII, and a VL sequence according to any of the aspects set out above in this section in connection with EGFRvIII (e.g., an antibody that binds EGFRvIII, comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 85, HCDR 2 of SEQ ID NO: 86 and HCDR 3 of SEQ ID NO: 87, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 89, LCDR 2 of SEQ ID NO: 90 and LCDR 3 of SEQ ID NO: 91; or an antibody that binds TYRP-1 comprising a VH comprising the sequence of SEQ ID NO: 88 and a VL comprising the sequence of SEQ ID NO: 92).

In a further aspect, antibodies that bind to TYRP-1 or EGFRvIII according to any of the above aspects may have other characteristics, alone or in combination, described in connection with an antibody that binds to CD3 (if they do not clearly relate to an anti-CD3 antibody, such as binding sequences).

c) Charge modifications

The (multispecific) antibody of the invention may comprise amino acid substitutions in the Fab molecules contained therein that are particularly effective in reducing mispairing of light chains with mismatched heavy chains (Bence Jones-type side products) that may occur when producing Fab-based multispecific antibodies with VH/VL exchange in one (or more in the case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT Publication No. WO 2015/150447, incorporated herein by reference in its entirety, in particular the examples therein). The ratio of desired (multispecific) antibody versus undesirable side products, in particular Bence-Jones type side products that arise in multispecific antibodies with a VH/VL domain exchange in one of their 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 modifications").

Accordingly, in some aspects, wherein the first and second (and, if present, third) antigen-binding domains of the (multispecific) antibody are both Fab molecules, and in one of the antigen-binding domains (in particular, in the first antigen-binding domain) the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other,

i) in the CL constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is substituted with a positively charged amino acid (numbering according to Kabat), and in the CH1 constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbering according to the EU index according to Kabat); or

ii) in the CL constant domain of the first antigen-binding domain, the amino acid at position 124 is substituted with a positively charged amino acid (numbering according to Kabat), and in the CH1 constant domain of the first antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbering according to the EU index according to Kabat).

The (multispecific) antibody does not contain both modifications mentioned in i) and ii). The constant domains CL and CH1 of the antigen-binding domain containing the VH/VL exchange are not substituted for each other (i.e. remain unexchanged).

In a more specific aspect

i) in the CL constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the second (and, if present, third) 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 according to the EU index according to Kabat); or

ii) in the CL constant domain of the first antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the CH1 constant domain 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 according to the EU index according to Kabat).

In one such aspect, in the CL constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the CH1 constant domain of the second (and, if present, third) 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 according to the EU index according to Kabat).

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

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

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

In an even more preferred aspect, in the CL constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with arginine (R) (numbering according to Kabat), and in the CH1 constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat).

In preferred aspects, when the amino acid substitutions according to the above aspects are made in the CL constant domain and the CH1 constant domain of the second (and, if present, third) antigen-binding domain, the CL constant domain of the second (and, if present, third) antigen-binding domain is of the kappa isotype.

Alternatively, the amino acid substitutions according to the above aspects may be made in the CL constant domain and the CH1 constant domain of the first antigen-binding domain instead of the CL constant domain and the CH1 constant domain of the second (and, if present, third) antigen-binding domain. In preferred such aspects, the CL constant domain of the first antigen-binding domain is of the kappa isotype.

Accordingly, in one aspect, in the CL constant domain of the first antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the CH1 constant domain 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 according to the EU index according to Kabat).

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

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

In one aspect, in the CL constant domain of the first antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) (numbering according to Kabat), and in the CH1 constant domain of the first antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat).

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

In a preferred aspect, the (multispecific) antibody of the invention comprises

(a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10, and

(b) a second and, optionally, a third antigen-binding domain that binds a second antigen;

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

d) Formats of multispecific antibodies

The (multispecific) antibody according to the present invention can have various configurations. Typical configurations are illustrated in Fig. 1.

In preferred aspects, the antigen-binding domains contained in the (multispecific) antibody are Fab molecules. In such aspects, the first, second, third, etc. antigen-binding domain may be referred to herein as the first, second, third, etc. Fab molecule, respectively.

In one aspect, the first and second antigen-binding domains of the (multispecific) antibody are fused to each other, optionally via a peptide linker. In preferred aspects, each of the first and second antigen-binding domains is a Fab molecule. In one such aspect, 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 aspect, 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 aspects in which (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 further be fused to each other, optionally via a peptide linker.

A (multispecific) antibody with one antigen-binding domain (such as a Fab molecule) capable of specifically binding to a second antigen, such as a target cell antigen such as TYRP-1 or EGFRvIII (e.g. as shown in Fig. 1A, D, G, H, L, M) is particularly useful in cases where internalization of the second antigen can be expected following binding to the high-affinity antigen-binding domain. In such cases, the presence of more than one antigen-binding domain specific for the second antigen may enhance the internalization of the second antigen, thereby reducing its availability.

In other cases, however, it is advantageous to have a (multispecific) antibody comprising two or more antigen-binding domains (such as Fab molecules) specific for a second antigen, such as a target cell antigen (see the examples illustrated in Fig. 1B, 1C, 1E, 1F, 1I, 1K, 1H or 1O), such as to optimize targeting to the target site or to allow cross-linking of target cell antigens.

Accordingly, in preferred aspects, the (multispecific) antibody according to the present invention comprises a third antigen-binding domain.

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

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

In some aspects, each of the third and second antigen-binding domains is a Fab molecule, and the third antigen-binding domain is identical to the second antigen-binding domain. Thus, in these aspects, the second and third antigen-binding domains comprise the same heavy and light chain amino acid sequences and have the same domain arrangement (i.e., standard or crossover). Furthermore, in these aspects, the third antigen-binding domain comprises the same amino acid substitutions, if any, as the second antigen-binding domain. For example, amino acid substitutions described herein as "charge modifications" are made in the CL constant domain and the CH1 constant domain of each of the second antigen-binding domain and the third antigen-binding domain. Alternatively, said amino acid substitutions may be made in the CL constant domain and the CH1 constant domain of the first antigen-binding domain (which, in preferred aspects, is also a Fab molecule), but not in the CL constant domain and the CH1 constant domain of the second antigen-binding domain and the third antigen-binding domain.

Like the second antigen-binding domain, the third antigen-binding domain is preferably a standard Fab molecule. However, aspects are also provided in which the second and third antigen-binding domains are crossover Fab molecules (and the first antigen-binding domain is a standard Fab molecule). Thus, in preferred aspects, each of the second and third antigen-binding domains is a standard 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 heavy and light chains of the Fab are replaced/substituted with each other. In other aspects, each of the second and third antigen-binding domains is a crossover Fab molecule and the first antigen-binding domain is a standard Fab molecule.

In the case of a third antigen binding domain, in a preferred aspect the first antigen binding domain binds to CD3 and the second and third antigen binding domains bind to a second antigen, in particular a target cell antigen such as TYRP-1 or EGFRvIII.

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

The (multispecific) antibody according to the invention may have different configurations, i.e. the first, second (and optionally the third) antigen-binding domain 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. If the Fab molecule is fused to the N-terminus of the Fc domain subunit, the fusion typically occurs via the hinge region of the immunoglobulin.

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

In one aspect, each of the first and second antigen-binding domains is 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 Fab heavy chain of the first antigen-binding domain. In a particular aspect, the (multispecific) antibody consists essentially of first and second Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the 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 a configuration is schematically depicted in Fig. 1G and 1L (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 may be further fused to each other.

In another aspect, each of the first and second antigen-binding domains is a Fab molecule, and each of the first and second antigen-binding domains is fused at the C-terminus of a Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In a particular aspect, the (multispecific) antibody consists essentially of first and second Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein each of the first and second Fab molecules is fused at the C-terminus of a Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. Such a configuration is schematically depicted in Figs. 1A and 1D (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule and the second antigen-binding domain is a standard Fab molecule). The first and second Fab molecules can be fused to the Fc domain directly or via a peptide linker. In a preferred aspect, each of the first and second Fab molecules is fused to the Fc domain via a hinge region of an immunoglobulin. In a particular aspect, the hinge region of an immunoglobulin is a hinge region of human IgG ₁ , in particular when the Fc domain is the Fc domain of human IgG ₁ .

In some aspects, each of the first and second antigen-binding domains is 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 Fc domain subunit. In such aspects, the first antigen-binding domain may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain or (as described above) to the N-terminus of another Fc domain subunit. In such preferred aspects, said second antigen-binding domain is a standard 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 replaced/substituted with each other. In other such aspects, said second antigen-binding domain is a crossover Fab molecule and said first antigen-binding domain is a standard Fab molecule.

In one aspect, each of the first and second antigen-binding domains is 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 Fab heavy chain of the second antigen-binding domain. In a particular aspect, the (multispecific) antibody consists essentially of first and second Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second 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 a configuration is schematically depicted in Fig. 13 and 1M (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule and the second antigen-binding domain is a standard Fab molecule). 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.

In some aspects, the third antigen-binding domain, in particular the third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second Fc domain subunit. In such preferred aspects, each of said second and third antigen-binding domains is a standard 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 heavy and light chains of the Fab are replaced/substituted with each other. In other aspects, each of said second and third antigen-binding domains is a crossover Fab molecule, and the first antigen-binding domain is a standard Fab molecule.

In such a preferred aspect, each of the first and third antigen-binding domains is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, 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 aspect, the (multispecific) antibody consists essentially of the first, second and third Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein 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 Fc domain subunit, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit. Such a configuration is schematically depicted in Figs. 1B and 1E (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule and the second and third antigen-binding domains are a standard Fab molecule) and Figs. 1K and 1O (in these examples, the first antigen-binding domain is a standard Fab molecule and the second and third antigen-binding domains are a VH/VL crossover Fab molecule). The first and third Fab molecules can be fused to the Fc domain directly or via a peptide linker. In a preferred aspect, each of the first and third Fab molecules is fused to the Fc domain via an immunoglobulin hinge region. In a particular aspect, the immunoglobulin hinge region is a human IgG ₁ hinge region, in particular when the Fc domain is a human IgG ₁ Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

In another such aspect, each of the second and third antigen-binding domains is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. In a particular aspect, the (multispecific) antibody consists essentially of first, second and third Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein 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 Fc domain subunit, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit. Such a configuration is schematically depicted in Figs. 1B and 1E (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule and the second and third antigen-binding domains are a standard Fab molecule) and Figs. 1I and 1H (in these examples, the first antigen-binding domain is a standard Fab molecule and the second and third antigen-binding domains are a VH/VL crossover Fab molecule). The second and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a preferred aspect, each of the second and third Fab molecules is fused to the Fc domain via an immunoglobulin hinge region. In a particular aspect, the immunoglobulin hinge region is a human IgG ₁ hinge region, in particular when the Fc domain is a human IgG ₁ Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

In (multispecific) antibody configurations in which a Fab molecule is fused at the C-terminus of a Fab heavy chain to the N-terminus of each of the Fc domain subunits via an immunoglobulin hinge region, the two Fab molecules, the hinge regions and the Fc domain advantageously form an immunoglobulin molecule. In a preferred aspect, the immunoglobulin molecule is an immunoglobulin of the IgG class. In an even more preferred aspect, the immunoglobulin is an immunoglobulin of the IgG ₁ subclass. In another aspect, the immunoglobulin is an immunoglobulin of the IgG ₄ subclass. In a further preferred aspect, the immunoglobulin is a human immunoglobulin. In other aspects, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one aspect, the immunoglobulin comprises a human constant region, in particular a human Fc region.

In some of the (multispecific) antibodies of the invention, the Fab light chain of a first Fab molecule and the Fab light chain of a second Fab molecule are fused to each other, optionally via a peptide linker. Depending on the configuration of the first and second Fab molecules, the Fab light chain of the first molecule may be fused at the C-terminus to the N-terminus of the Fab light chain of the second Fab molecule or the Fab light chain of the second molecule may be fused at the C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. 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 expression of some of the (multispecific) antibodies of the invention.

The antigen binding domains can be fused to the Fc domain or to each other directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, ( _G4S ) _n , ( _SG4 ) _n , ( _G4S ) _n or _G4 ( _SG4 ) _n peptide linkers. "n" is generally an integer from 1 to 10, typically from 2 to 4. In one aspect, said peptide linker is at least 5 amino acids long, in one aspect from 5 to 100, in _a further aspect from 10 to 50 amino acids long. In one aspect, said peptide linker is (GxS) _n or (GxS) _nGm , where G=glycine, S=series, 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 aspect x=4 and n=2 or 3, in a further aspect x=4 and n=2. In one aspect, said peptide linker is (G ₄ S) ₂ . A particularly suitable peptide linker 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 joining the Fab heavy chains of the first and second Fab fragments comprises the sequence (D)-( _G4S ) ₂ (SEQ ID NOs 118 and 119). Another such suitable linker comprises the sequence ( _G4S ) ₄ . In addition, the linkers may comprise an immunoglobulin hinge region (or a portion thereof). In particular, if the Fab molecule is fused to the N-terminus of the Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the variable region of the Fab light chain of a first Fab molecule has a common carboxy-terminal peptide bond with a constant region of the Fab heavy chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the variable region of the heavy chain is replaced by the variable region of the light chain), which in turn has a common carboxy-terminal peptide bond with a subunit of the Fc domain (VL ₍₁₎ -CH1 ₍₁₎ -CH2-CH3 (-CH4)), and a polypeptide in which the Fab heavy chain of a second Fab molecule has a common carboxy-terminal peptide bond with a subunit of the Fc domain (VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3 (-CH4)). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule has a common carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH ₍₁₎ -CL ₍₁₎ ), and a Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In certain aspects, the polypeptides are covalently linked, such as by a disulfide bond.

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the variable region of the Fab heavy chain of a first Fab molecule has a common carboxy-terminal peptide bond with the constant region of the Fab light chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the constant region of the heavy chain is replaced by the constant region of the light chain), which in turn has a common carboxy-terminal peptide bond with a subunit of the Fc domain (VH ₍₁₎ -CL ₍₁₎ -CH2-CH3 (-CH4)), and a polypeptide in which the Fab heavy chain of a second Fab molecule has a common carboxy-terminal peptide bond with a subunit of the Fc domain (VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3 (-CH4)). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule has a common carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ ), and a Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In certain aspects, the polypeptides are covalently linked, such as by a disulfide bond.

In some aspects, the (multispecific) antibody comprises a polypeptide in which the Fab light chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -CH2 -CH3 (-CH4)). In other aspects, the (multispecific) antibody comprises a polypeptide in which the Fab heavy chain of a second Fab molecule shares a carboxy-terminal peptide bond with the variable region of the Fab light chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the constant region of the Fab heavy chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the variable region of the heavy chain is replaced by the variable region of the light chain), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ -CH2 -CH3 (-CH4)). In some of these aspects, the (multispecific) antibody further comprises a Fab crossover light chain polypeptide of a first Fab molecule, wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH ₍₁₎ -CL ₍₁₎ ), and a Fab light chain polypeptide of a second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In other aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain constant region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain polypeptide of a second Fab molecule (VH ₍₁₎ -CL ₍₁₎ -VL ₍₂₎ -CL ₍₂₎ ), or a polypeptide in which the Fab light chain polypeptide of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of the first Fab molecule (VL ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ), as the case may be. The (multispecific) antibody according to these aspects may further comprise (i) a polypeptide of an Fc domain subunit (CH2-CH3(-CH4)) or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule has a common carboxy-terminal peptide bond with an Fc domain subunit (VH ₍₃₎ -CH1 ₍₃₎ -CH2-CH3(-CH4)), and a Fab light chain polypeptide of a third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ). In certain aspects, the polypeptides are covalently linked, such as by a disulfide bond.

In some aspects, the (multispecific) antibody comprises a polypeptide in which the Fab heavy chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -CH2 -CH3 (-CH4)). In other aspects, the (multispecific) antibody comprises a polypeptide in which the Fab heavy chain of a second Fab molecule shares a carboxy-terminal peptide bond with the variable region of the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the constant region of the Fab light chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the constant region of the heavy chain is replaced by the constant region of the light chain), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ -CH2-CH3 (-CH4)). In some of these aspects, the (multispecific) antibody further comprises a Fab crossover light chain polypeptide of a first Fab molecule, wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ ), and a Fab light chain polypeptide of a second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In other aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the second Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CL ₍₂₎ ), or a polypeptide in which the Fab light chain polypeptide of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VL ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ), as appropriate. The (multispecific) antibody according to these aspects may further comprise (i) a polypeptide of an Fc domain subunit (CH2-CH3(-CH4)) or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH ₍₃₎ -CH1(3)-CH2-CH3(-CF£4)), and a Fab light chain polypeptide of a third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ). In certain aspects, the polypeptides are covalently linked, such as by a disulfide bond.

In certain aspects, the (multispecific) antibody does not comprise an Fc domain. In such preferred aspects, each of said second and, if present, third antigen-binding domains is a standard 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 heavy and light chains of the Fab are replaced/substituted with each other. In other such aspects, each of said second and, if present, third antigen-binding domains is a crossover Fab molecule, and the first antigen-binding domain is a standard Fab molecule.

In one such aspect, the (multispecific) antibody is preferably comprised of first and second antigen-binding domains and, optionally, one or more peptide linkers, wherein the first and second antigen-binding domains are both Fab molecules and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the heavy chain of the first antigen-binding domain. Such a configuration is schematically depicted in Figs. 1P and 1V (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule and the second antigen-binding domain is a standard Fab molecule).

In another such aspect, the (multispecific) antibody is preferably comprised of first and second antigen-binding domains and, optionally, one or more peptide linkers, wherein the first and second antigen-binding domains are both Fab molecules and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the heavy chain of the second antigen-binding domain. Such a configuration is schematically depicted in Fig. 1P and 1F (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule and the second antigen-binding domain is a standard Fab molecule).

In some aspects, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the (multispecific) antibody further comprises a third antigen-binding domain, in particular a third Fab molecule, wherein said third 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. In certain such aspects, the (multispecific) antibody consists essentially of the first, second and third Fab molecules and, optionally, one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the third 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. Such a configuration is schematically depicted in Fig. 1C and 1X (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule, and each of the second and third antigen-binding domains is a standard Fab molecule) and Figs. 1III and 1Y (in these examples, the first antigen-binding domain is a standard Fab molecule, and each of the second and third antigen-binding domains is a VH/VL crossover Fab molecule).

In some aspects, 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 a second Fab molecule, and the (multispecific) antibody further comprises a third antigen-binding domain, in particular a third Fab molecule, wherein said third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. In certain such aspects, the (multispecific) antibody consists essentially of the first, second and third Fab molecules and, optionally, one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. Such a configuration is schematically depicted in Fig. 1T and 1C (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule, and each of the second and third antigen-binding domains is a standard Fab molecule) and Figs. 1H and 1G (in these examples, the first antigen-binding domain is a standard Fab molecule, and each of the second and third antigen-binding domains is a VH/VL crossover Fab molecule).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain of a second Fab molecule shares a carboxy-terminal peptide bond with the variable region of the Fab light chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the constant region of the Fab heavy chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the variable region of the heavy chain is replaced by the variable region of the light chain) (VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of a first Fab molecule shares a common carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH ₍₁₎ -CL ₍₁₎ ), and a Fab light chain polypeptide of a second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the variable region of the Fab light chain of a first Fab molecule shares a carboxy-terminal peptide bond with a constant region of the Fab heavy chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the variable region of the heavy chain is replaced by a variable region of the light chain), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of a first Fab molecule shares a common carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH ₍₁₎ -CL ₍₁₎ ), and a Fab light chain polypeptide of a second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain of a second Fab molecule shares a carboxy-terminal peptide bond with the variable region of the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the constant region of the Fab light chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the constant region of the heavy chain is replaced by the constant region of the light chain) (VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a common carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ ), and a Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the variable region of the Fab heavy chain of a first Fab molecule has a common carboxy-terminal peptide bond with the constant region of the Fab light chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the constant region of the heavy chain is replaced by the constant region of the light chain), which in turn has a common carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule (VH ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a common carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ ), and a Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which a Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain variable region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the variable region of the heavy chain is replaced by the variable region of the light chain) (VH ₍₃₎ -CH1 ₍₃₎ -VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule has a common carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH ₍₁₎ -CL ₍₁₎ ), and a Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide of a third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which a Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region) (VH ₍₃₎ -CH1 ₍₃₎ -VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ). In some aspects,

The (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule has a common carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ ), and a Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide of a third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the variable region of the Fab light chain of a first Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab heavy chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the variable region of the heavy chain is replaced by the variable region of the light chain), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₃₎ -CH1 ₍₃₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule has a common carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH ₍₁₎ -CL ₍₁₎ ), and a Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide of a third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the variable region of the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab light chain of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the constant region of the heavy chain is replaced by the constant region of the light chain), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule (VH ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₃₎ -CH1 ₍₃₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule has a common carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ ), and a Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide of a third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the variable region of the Fab light chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the constant region of the Fab heavy chain of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the variable region of the heavy chain is replaced by the variable region of the light chain), which in turn shares a carboxy-terminal peptide bond with the variable region of the Fab light chain of a third Fab molecule, which in turn shares a carboxy-terminal peptide bond with the constant region of the Fab heavy chain of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the variable region of the heavy chain is replaced by the variable region of the light chain), ... substituted by a light chain variable region) (VH ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CH1 ₍₂₎ -VL ₍₃₎ -CH1 ₍₃₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain constant region of the second Fab molecule (VH ₍₂₎ -CL ₍₂₎ ), and a Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain constant region of the third Fab molecule (VH ₍₃₎ -CL ₍₃₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which a Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a third Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region) (VH ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CL ₍₂₎ -VH ₍₃₎ -CL ₍₃₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule has a common carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL ₍₂₎ -CH1 ₍₂₎ ), and the Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule has a common carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL ₍₃₎ -CH1 ₍₃₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the Fab light chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with a Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with a heavy chain of the first Fab molecule (VL ₍₃₎ -CH1 ₍₃₎ -VL ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH ₍₂₎ -CL ₍₂₎ ), and a Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (VH ₍₃₎ -CL ₍₃₎ ).

In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide in which the variable region of a Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with a constant region of a Fab light chain of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the constant region of the heavy chain is replaced by a constant region of the light chain), which in turn shares a carboxy-terminal peptide bond with the variable region of a Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the constant region of the heavy chain is replaced by a constant region of the light chain), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VH ₍₃₎ -CL ₍₃₎ -VH ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CH1 ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule has a common carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL ₍₂₎ -CH1 ₍₂₎ ), and the Fab light chain polypeptide of the first Fab molecule (VL ₍₁₎ -CL ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule has a common carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL ₍₃₎ -CH1 ₍₃₎ ).

In one aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain are substituted for each other, and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 2, HCDR 2 with SEQ ID NO: 3 and HCDR 3 with SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 8, LCDR 2 with SEQ ID NO: 9 and LCDR 3 with SEQ ID NO: 10;

b) a second antigen-binding domain that binds to a second antigen, in particular a target cell antigen, more particularly TYRP-1 or EGFRvIII, wherein the second antigen-binding domain is a (standard) Fab molecule;

c) Fc domain, consisting of the first and second subunits; wherein

(i) the first antigen-binding domain according to 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 according to b), and the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding domain according to b) 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 according to a), and the first antigen-binding domain according to 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 according to c).

In a preferred aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain are substituted for each other, and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 2, HCDR 2 with SEQ ID NO: 3 and HCDR 3 with SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 8, LCDR 2 with SEQ ID NO: 9 and LCDR 3 with SEQ ID NO: 10;

b) a second and third antigen-binding domains that bind to a second antigen, in particular a target cell antigen, in particular TYRP-1 or EGFRvIII, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule; and

c) Fc domain, consisting of the first and second subunits; wherein

(i) the first antigen-binding domain of 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 of b), and the second antigen-binding domain of b) and the third antigen-binding domain of b) 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 of c), or

(ii) the second antigen-binding domain of b) 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 of a), and the first antigen-binding domain of a) and the third antigen-binding domain of b) 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 of c).

In another aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain are substituted for each other, and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 2, HCDR 2 with SEQ ID NO: 3 and HCDR 3 with SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 8, LCDR 2 with SEQ ID NO: 9 and LCDR 3 with SEQ ID NO: 10;

b) a second antigen-binding domain that binds to a second antigen, in particular a target cell antigen, more particularly TYRP-1 or EGFRvIII, wherein the second antigen-binding domain is a (standard) Fab molecule;

c) Fc domain, consisting of the first and second subunits; and

(i) the first antigen-binding domain of a) and the second antigen-binding domain of b) 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 of c).

In all of the different configurations of the (multispecific) antibody according to the invention, the amino acid substitutions ("charge modifications") described herein, if present, may be in the CH1 and CL domains of the second and (if present) third antigen-binding domain/Fab molecule, or in the CH1 and CL domains of the first antigen-binding domain/Fab molecule. Preferably, they are in the CH1 and CL domains of the second and (if present) third antigen-binding domain/Fab molecule. According to the concept of the invention, if the amino acid substitutions described herein are made in the second (and, if present, third) antigen-binding domain/Fab molecule, such amino acid substitutions are not made in the first antigen-binding domain/Fab molecule. Conversely, if the amino acid substitutions described herein are made in the first antigen-binding domain/Fab molecule, no such amino acid substitutions are made in the second (and, if present, third) antigen-binding domain/Fab molecule. The amino acid substitutions are preferably made in (multispecific) antibodies comprising a Fab molecule in which the variable domains VL and VH1 of the Fab light chain and the Fab heavy chain are substituted for each other.

In preferred aspects of the (multispecific) antibody according to the invention, in particular wherein the amino acid substitutions described herein are made in the second (and, if present, third) antigen-binding domain/Fab molecule, the CL constant domain of the second (and, if present, third) Fab molecule is of the kappa isotype. In other aspects of the (multispecific) antibody according to the invention, in particular wherein the amino acid substitutions described herein are made in the first antigen-binding domain/Fab molecule, the CL constant domain of the first antigen-binding domain/Fab molecule is of the kappa isotype. In some aspects, the CL constant domain of the second (and, if present, third) antigen-binding domain/Fab molecule and the CL constant domain of the first antigen-binding domain/Fab molecule are of the kappa isotype.

In one aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 2, HCDR 2 with SEQ ID NO: 3 and HCDR 3 with SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 8, LCDR 2 with SEQ ID NO: 9 and LCDR 3 with SEQ ID NO: 10;

b) a second antigen-binding domain that binds to a second antigen, in particular a target cell antigen, more particularly TYRP-1 or EGFRvIII, wherein the second antigen-binding domain is a (standard) Fab molecule;

c) Fc domain, consisting of the first and second subunits;

wherein in the constant domain CL of the second antigen-binding domain according to b) the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most preferably arginine (R)), and wherein in the constant domain CH1 of the second antigen-binding domain according to b) the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat); and wherein

(i) the first antigen-binding domain according to 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 according to b), and the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding domain according to b) 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 according to a), and the first antigen-binding domain according to 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 according to c).

In a preferred aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 2, HCDR 2 with SEQ ID NO: 3 and HCDR 3 with SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 8, LCDR 2 with SEQ ID NO: 9 and LCDR 3 with SEQ ID NO: 10;

b) a second and third antigen-binding domains that bind to a second antigen, in particular a target cell antigen, in particular TYRP-1 or EGFRvIII, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule; and

c) Fc domain, consisting of the first and second subunits;

wherein in the constant domain CL of the second antigen-binding domain according to b) and the third antigen-binding domain according to b), the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most preferably arginine (R)), and wherein in the constant domain CH1 of the second antigen-binding domain according to b) and the third antigen-binding domain according to b), the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat); and wherein

(i) the first antigen-binding domain of 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 of b), and the second antigen-binding domain of b) and the third antigen-binding domain of b) 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 of c), or

(ii) the second antigen-binding domain according to b) 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 according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b) 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 according to c).

In another aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 2, HCDR 2 with SEQ ID NO: 3 and HCDR 3 with SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 8, LCDR 2 with SEQ ID NO: 9 and LCDR 3 with SEQ ID NO: 10;

b) a second antigen-binding domain that binds to a second antigen, in particular a target cell antigen, more particularly TYRP-1 or EGFRvIII, wherein the second antigen-binding domain is a (standard) Fab molecule;

c) Fc domain, consisting of the first and second subunits;

wherein in the constant domain CL of the second antigen-binding domain according to b) the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most preferably arginine (R)), and wherein in the constant domain CH1 of the second antigen-binding domain according to b) the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat); and

the first antigen-binding domain according to a) and the second antigen-binding domain according to b), each, are fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

According to any of the above aspects, the components of the (multispecific) antibody (e.g. Fab molecules, Fc domain) can be fused directly or via various linkers, in particular peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, which are described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, peptide linkers (G ₄ S) _n , (SG ₄ ) _n , (G ₄ S) _n or G ₄ (SG ₄ ) _n , where n is generally an integer from 1 to 10, typically from 2 to 4.

In a preferred aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10;

b) a second and a third antigen-binding domain that binds to TYRP-1, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 15, HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21;

c) Fc domain, consisting of the first and second subunits;

moreover

in the constant domain CL of the second and third antigen-binding domain according to b) the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most preferably arginine (R)), and wherein in the constant domain CH1 of the second and third antigen-binding domain according to b) the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat);

and additionally

the second antigen-binding domain according to b) 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 according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b), 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 according to c).

In a further preferred aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11;

b) a second and third antigen-binding domains that bind to TYRP-1, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22;

c) Fc domain, consisting of the first and second subunits; wherein

in the constant domain CL of the second and third antigen-binding domain according to b) the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most preferably arginine (R)), and wherein in the constant domain CH1 of the second and third antigen-binding domain according to b) the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat);

and additionally

the second antigen-binding domain according to b) 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 according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b), 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 according to c).

In a preferred aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10;

b) a second and a third antigen-binding domain that binds to EGFRvIII, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 85, HCDR 2 of SEQ ID NO: 86 and HCDR 3 of SEQ ID NO: 87, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 89, LCDR 2 of SEQ ID NO: 90 and LCDR 3 of SEQ ID NO: 91;

c) Fc domain, consisting of the first and second subunits;

moreover

in the constant domain CL of the second and third antigen-binding domain according to b) the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most preferably arginine (R)), and wherein in the constant domain CH1 of the second and third antigen-binding domain according to b) the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat);

and additionally

the second antigen-binding domain according to b) 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 according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b), 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 according to c).

In a further preferred aspect, the invention provides a (multispecific) antibody comprising

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11;

b) a second and third antigen-binding domains that bind to EGFRvIII, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 88 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 92;

c) Fc domain, consisting of the first and second subunits;

moreover

in the constant domain CL of the second and third antigen-binding domain according to b) the amino acid at position 124 is substituted with lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (most preferably arginine (R)), and wherein in the constant domain CH1 of the second and third antigen-binding domain according to b) the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat);

and additionally

the second antigen-binding domain according to b) 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 according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b), 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 according to c).

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

In a further aspect according to these aspects of the invention, in the first subunit of the Fc domain, additionally the serine residue at position 354 is substituted with a cysteine residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine residue (E356C) (in particular the serine residue at position 354 is substituted with a cysteine residue), and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is substituted with a cysteine residue (Y349C) (numbering according to the EU index according to Kabat).

In a further aspect according to these aspects of the invention, in each of the first and second Fc domain subunits, 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) (numbering according to the EU index according to Kabat).

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

In a preferred particular aspect, the (multispecific) 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: 23, 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: 24, 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: 25, and 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: 27. In a further preferred In a particular aspect, the (multispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 23, a polypeptide comprising the amino acid sequence of SEQ ID NO: 24, a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 25, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and TYRP-1, comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 23, 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: 24, 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: 25, and 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: 27. In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and TYRP-1, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 23, a polypeptide comprising the amino acid sequence of SEQ ID NO: 24, a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 25, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

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

In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and EGFRvIII, comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 109, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 110, 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: 111, and 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: 27. In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and EGFRvIII, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 109, a polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 111, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

8. Variants of Fc domains

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

The Fc domain of a (multispecific) 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, each subunit of which contains the heavy chain constant domains CH2 and CH3 of IgG. Two subunits of the Fc domain are capable of stable association with each other. In one aspect, a (multispecific) antibody of the invention contains no more than one Fc domain.

In one aspect, the Fc domain of the (multispecific) antibody is an IgG Fc domain. In a preferred aspect, the Fc domain is an IgG ₁ Fc domain. In another aspect, the Fc domain is an IgG ₄ Fc domain. In a more specific aspect, the Fc domain is an IgG ₄ Fc domain comprising an amino acid substitution at position S228 (numbering according to the EU index of Kabat), in particular an amino acid substitution S228P. This amino acid substitution reduces the in vivo turnover of the Fab arms of IgG ₄ antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further preferred aspect, the Fc domain is a human Fc domain. In an even more preferred aspect, the Fc domain is a human IgG ₁ Fc domain. A typical sequence of the Fc region of human IgG ₁ is given in SEQ ID NO: 117.

a) Modifications of the Fc domain that promote heterodimerization

The (multispecific) antibodies according to the invention comprise different antigen-binding domains which can be fused to one or the other of the two Fc domain subunits, so that the two Fc domain subunits are typically contained in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization lead to several possible combinations of the two polypeptides. Therefore, in order to improve the yield and purity of (multispecific) antibodies in recombinant production, it will be expedient to introduce a modification into the Fc domain of the (multispecific) antibody that facilitates the association of the desired polypeptides.

Accordingly, in preferred aspects, the Fc domain of the (multispecific) antibody according to the invention comprises a modification that facilitates the association of the first and second subunits of the Fc domain. The site of the most intense protein-protein interaction between the two subunits of the Fc domain of human IgG is the CH3 domain of the Fc domain. Thus, in one aspect, said modification is in the CH3 domain of the Fc domain.

There are several approaches for modification of the CH3 domain of the Fc domain to promote heterodimerization, which are well 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 2012058768, WO 2013157954, WO 2013096291. As a rule, in all such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit The Fc domains are designed in a complementary manner such that each CH3 domain (or the heavy chain containing it) can no longer form a homodimer with itself, but is forced to form a heterodimer with another complementarily engineered CH3 domain (so that heterodimerization of the first and second CH3 domains occurs and no homodimerization occurs between the first two or the second two CH3 domains). These different approaches to improve heavy chain heterodimerization are envisaged as different alternatives in combination with heavy-light chain modifications (e.g. swapping/replacing VH and VL in one binding arm and introducing substitutions of oppositely charged amino acids in the CH1/CL interface) in the (multispecific) antibody, which reduces heavy/light chain mispairing and Bence-Jones side products.

In a particular aspect, said modifications that facilitate the association of the first and second Fc domain subunits are a so-called knob-into-hole modification, comprising a modification of the knob in one of the two Fc domain subunits and a modification of the hole in the other of the two Fc domain subunits.

Knob-into-hole technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a bump ("knob") at the interface of a first polypeptide and a corresponding cavity ("cavity") at the interface of a second polypeptide, such that the bump can be positioned in the cavity to promote heterodimer formation and hinder homodimer formation. The bumps are constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensating cavities of identical or similar size to the bulges are created on the contact surface of the second polypeptide by replacing large amino acid side chains with smaller ones (for example, alanine or threonine).

Accordingly, in a preferred aspect, in the CH3 domain of the first subunit of the Fc domain of the (multispecific) antibody, the amino acid residue is replaced by an amino acid residue having a larger side chain volume, thereby creating a bulge in the CH3 domain of the first subunit that can be accommodated in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, the amino acid residue is replaced by an amino acid residue having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit that can accommodate the bulge in the CH3 domain of the first subunit.

Preferably, said 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).

Preferably, said 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).

The bulge and cavity can be created by altering the nucleic acid encoding the polypeptides, for example by site-directed mutagenesis, or by peptide synthesis.

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

In a further aspect, in the first subunit of the Fc domain, additionally the serine residue at position 354 is substituted with a cysteine residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine residue (E356C) (in particular, the serine residue at position 354 is substituted with a cysteine residue), and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is substituted with a cysteine residue (Y349C) (numbering according to the EU index according to Kabat). The introduction of two cysteine residues leads to the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In preferred aspects, the first Fc domain subunit comprises the amino acid substitutions S354C and T366W and the second Fc domain subunit comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the EU index according to Kabat).

In a preferred aspect, the antigen binding domain that binds to CD3 is fused (optionally via a second antigen binding domain that binds to a second antigen and/or a peptide linker) to a first subunit of the Fc domain (containing a "knob" modification). Without being limited by theory, fusing the antigen binding domain that binds to CD3 to a knob-containing subunit of the Fc domain will (further) minimize the creation of antibodies containing two antigen binding domains that bind to CD3 (due to steric hindrance of the two knob-containing polypeptides).

Other CH3 modification techniques for stimulating heterodimerization are provided as alternative embodiments according to the 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, WO 2013/096291.

In one aspect, the heterodimerization approach described in EP 1 870 459 is used as an alternative. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain. A particular aspect for the (multispecific) antibody of the invention are the amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and the amino acid mutations D399K; E357K in the other of the two CH3 domains of the Fc domain (numbering according to the EU index according to Kabat).

In another aspect, the (multispecific) antibody of the invention comprises 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, and additionally the amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numbering in accordance with the EU index according to Kabat).

In another aspect, the (multispecific) antibody of the invention comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the said (multispecific) antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally the amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numbering in accordance with the EU index according to Kabat).

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

In one aspect, the heterodimerization approach described in WO 2012/058768 is used as an alternative. In one aspect, the first CH3 domain comprises the amino acid mutations L351Y, Y407A, and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further aspect, the second CH3 domain comprises an additional amino acid mutation at position T411, D399, S400, F405, N390 or K392, such as selected from 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, e) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to the EU index according to Kabat). In a further aspect, the first CH3 domain comprises the amino acid mutations L351Y, Y407A, and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further aspect, the first CH3 domain comprises the amino acid mutation Y407A, and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further aspect, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the EU index according to Kabat).

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

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

In one aspect, the (multispecific) antibody or its Fc domain is of the IgG2 subclass, and alternatively the heterodimerization approach described in WO 2010/129304 is used.

In an alternative aspect, a modification that promotes association of the first and second Fc domain subunits includes a modification that mediates electrostatic interaction effects, such as those described in PCT Publication WO 2009/089004. In general, the method comprises replacing one of the amino acid residues at the interface of the Fc domain subunits with a charged amino acid residue such that homodimer formation becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable. In one such aspect, the first CH3 domain comprises an amino acid substitution K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), in particular K392D or N392D), and the second CH3 domain comprises an amino acid substitution D399, E356, D356 or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), in particular D399K, E356K, D356K or E357K, and more particularly D399K and E356K). In a further aspect, the first CH3 domain further comprises an amino acid substitution K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), in particular K409D or R409D). In a further aspect, the first CH3 domain additionally or alternatively comprises an amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g. glutamic acid (E) or aspartic acid (D)) (all numbering according to the EU index according to Kabat).

In a further aspect, the heterodimerization approach described in WO 2007/147901 is used as an alternative. In one aspect, the first CH3 domain comprises the amino acid mutations K253E, D282K and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K and K292D (numbering according to the EU index according to Kabat).

In another aspect, the heterodimerization approach described in WO 2007/110205 can be used as an alternative.

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

b) Fc domain modifications that reduce Fc receptor binding and/or effector function

The Fc domain confers favorable pharmacokinetic properties to the (multispecific) antibody, including a long serum half-life that facilitates good accumulation in the target tissue and a favorable tissue-to-blood distribution ratio. However, at the same time, it may result in undesirable targeting of the (multispecific) antibody to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. In addition, co-activation of Fc receptor signaling pathways may result in cytokine release, which, in combination with the T-cell activating properties and the long half-life of the (multispecific) antibody, leads to excessive cytokine receptor activation and severe side effects after systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce the effectiveness of the (multispecific) antibody due to potential destruction of T cells, for example by NK cells.

Accordingly, in preferred aspects, the Fc domain of the (multispecific) antibody according to the invention exhibits reduced Fc receptor binding affinity and/or reduced effector function compared to the native Fc domain of IgG ₁ . In one such aspect, the Fc domain (or a (multispecific) antibody comprising said Fc domain) exhibits less than 50%, in particular less than 20%, more particularly less than 10% and most particularly less than 5% of the binding affinity for the Fc receptor compared to the native Fc domain of IgG ₁ (or a (multispecific) antibody comprising a native Fc domain of IgG ₁ ), and/or less than 50%, in particular less than 20%, more particularly less than 10% and most particularly less than 5% of the effector function compared to the native Fc domain of IgG ₁ (or a (multispecific) antibody comprising a native Fc domain of IgG ₁ ). In one aspect, the Fc domain (or a (multispecific) antibody comprising said Fc domain) substantially does not bind to an Fc receptor and/or does not induce an effector function. In a preferred aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the effector function is one or more functions selected from the group consisting of CDC, ADCC, ADCP and cytokine secretion. In a preferred aspect, the effector function is ADCC. In one aspect, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native Fc domain of IgG ₁ . Substantially similar binding to FcRn is achieved when the Fc domain (or (multispecific) antibody comprising said Fc domain) exhibits greater than about 70%, in particular greater than about 80%, more particularly greater than about 90% of the binding affinity of the native Fc domain of IgG ₁ (or (multispecific) antibody comprising the native Fc domain of IgG ₁ ) to FcRn.

In certain aspects, the Fc domain is engineered to have reduced binding affinity for an Fc receptor and/or reduced effector function compared to a non-engineered Fc domain. In preferred aspects, the Fc domain of a (multispecific) antibody comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain for an 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 aspect, the amino acid mutation reduces the binding affinity of the Fc domain for an Fc receptor. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain for an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In aspects where more than one amino acid mutation is present that reduces 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 10-fold, at least 20-fold, or even at least 50-fold. In one aspect, the (multispecific) antibody comprising an engineered Fc domain exhibits less than 20%, in particular less than 10%, more particularly less than 5%, of the binding affinity to the Fc receptor compared to the (multispecific) antibody comprising a non-engineered Fc domain. In a preferred aspect, the Fc receptor is an Fcγ receptor. In some aspects, the Fc receptor is a human Fc receptor. In some aspects, the Fc receptor is an activating Fc receptor. In a particular aspect, the Fc receptor is a human activating Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some aspects, the binding affinity for a complement component is also reduced, in particular the binding affinity for C1q. In one aspect, the binding affinity for the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. maintaining the binding affinity of the Fc domain for said receptor is achieved when the Fc domain (or (multispecific) antibody comprising said Fc domain) exhibits more than about 70% of the binding affinity of the unengineered form of the Fc domain (or (multispecific) antibody comprising said unengineered form of the Fc domain) for FcRn. The Fc domain or (multispecific) antibodies of the invention comprising said Fc domain may exhibit more than about 80% and even more than about 90% of such affinity. In certain aspects, the Fc domain of the (multispecific) antibody is engineered to have reduced effector function compared to the unengineered Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular 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 cross-linking of target-bound antibodies, reduced maturation of dendritic cells, or reduced priming of T cells. In one aspect, the reduced effector function is one or more functions selected from the group of reduced CDC, reduced ADCC, reduced ADCF, and reduced cytokine secretion. In a preferred aspect, the reduced effector function is reduced ADCC. In one aspect, the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a (multispecific) antibody comprising a non-engineered Fc domain).

In one aspect, an amino acid mutation that reduces the binding affinity of an Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one aspect, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to the EU index of Kabat). In a more particular aspect, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numbering according to the EU index of Kabat). In some aspects, the Fc domain comprises the amino acid mutations L234A and L235A (numbering according to the EU index of Kabat). In one such aspect, the Fc domain is an IgG ₁ Fc domain, in particular a human IgG ₁ Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more particular aspect, the amino acid substitution is P329A or P329G, in particular P329G (numbering according to the EU index of Kabat). In one aspect, the Fc domain comprises an amino acid substitution at position P329 and an additional amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to the EU index of Kabat). In a more particular aspect, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In preferred aspects, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to the EU index of Kabat). In more preferred aspects, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). In particular, in preferred aspects, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (numbering according to the EU index according to Kabat), i.e., in each of 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) (numbering according to the EU index according to Kabat).

In one such aspect, the Fc domain is an IgG ₁ Fc domain, in particular a human IgG ₁ Fc domain. The amino acid substitution combination "P329G LALA" substantially completely abolishes Fcγ receptor (as well as complement) binding of the human IgG ₁ Fc domain, as described in PCT Publication No. WO 2012/130831, which is incorporated herein by reference in its entirety. WO 2012/130831 also describes methods for producing such mutant Fc domains and methods for determining properties thereof, such as Fc receptor binding or effector functions.

IgG ₄ antibodies exhibit reduced binding affinity for Fc receptors and reduced effector functions compared to IgG ₁ antibodies. Therefore, in some aspects, the Fc domain of the (multispecific) antibodies of the invention is the Fc domain of IgG ₄ , in particular the Fc domain of human IgG _4. In one aspect, the Fc domain of IgG ₄ comprises an amino acid substitution at position S228, in particular the amino acid substitution S228P (numbering according to the EU index according to Kabat). To further reduce the binding affinity for the Fc receptor and/or the effector function, in one aspect, the Fc domain of IgG ₄ comprises an amino acid substitution at position L235, in particular the amino acid substitution L235E (numbering according to the EU index according to Kabat). In another aspect, the Fc domain of IgG ₄ comprises an amino acid substitution at position P329, in particular the amino acid substitution P329G (numbering according to the EU index of Kabat). In a preferred aspect, the Fc domain of IgG ₄ comprises amino acid substitutions at positions S228, L235 and P329, in particular the amino acid substitutions S228P, L235E and P329G (numbering according to the EU index of Kabat). Such mutant Fc domains of IgG ₄ and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012/130831, the entirety of which is incorporated herein by reference.

In a preferred aspect, the Fc domain exhibiting reduced binding affinity for the Fc receptor and/or reduced effector function compared to a native IgGb Fc domain is the human IgG ₁ Fc domain comprising the amino acid substitutions L234A, L235A and, optionally, P329G, or the human IgG ₄ Fc domain comprising the amino acid substitutions S228P, L235E and, optionally, P329G (numbering according to the EU index according to Kabat).

In certain aspects, N-glycosylation of the Fc domain has been eliminated. In one such aspect, the Fc domain comprises an amino acid mutation at position N297, in particular an amino acid substitution with asparagine being replaced by alanine (N297A) or aspartic acid (N297D) (numbering according to the EU index according to Kabat).

In addition to the Fc domains described above herein and in PCT Publication No. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector function also include domains with a substitution of one or more Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbering according to the EU index according to Kabat). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with a substitution of residues 265 and 297 with alanine (U.S. Patent No. 7,332,581).

Mutant Fc domains can be produced by deleting, substituting, inserting or modifying amino acids using genetic or chemical methods well known in the art. Genetic methods can include site-specific mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

Binding to Fc receptors can be readily determined, for example, by ELISA or surface plasmon resonance (SPR), using standard equipment such as the BIAcore instrument (GE Healthcare) and Fc receptors that can be produced by recombinant expression. Alternatively, the binding affinity of Fc domains or (multispecific) Fc domain-containing antibodies for Fc receptors can be assessed using cell lines known to express specific Fc receptors, such as NK cells expressing the FcγIIIa receptor.

The effector function of an Fc domain or a (multispecific) antibody comprising an Fc domain can be determined by methods known in the art. Examples of in vitro assays for assessing 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 can be used (see, for example, the ACTI™ Non-Radioactive Flow Cytotoxicity Assay (CellTechnology, Inc. Mountain View, CA); and the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WD). Effector cells suitable for such an assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that described in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some aspects, the binding of the Fc domain to a complement component, in particular C1q, is reduced. Accordingly, in some aspects in which the Fc domain is engineered to have reduced effector function, said reduced effector function comprises reduced CDC. C1q binding assays can be performed to determine whether an Fc domain or a (multispecific) antibody comprising an Fc domain is capable of binding C1q and therefore has CDC activity. See, for example, the C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. Complement activation can be assessed using the CDC assay (see, for example, 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)).

Determination of FcRn binding and in vivo clearance/half-life can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759-1769 (2006); WO 2013/120929).

B. Polynucleotides

The invention further provides an isolated polynucleotide encoding the antibody of the invention. Said isolated polynucleotide may be a single polynucleotide or a plurality of polynucleotides.

Polynucleotides encoding the (multispecific) antibodies of the invention can be expressed as a single polynucleotide that encodes the entire antibody, or as multiple (e.g., two or more) co-expressed polynucleotides. The polypeptides encoded by the co-expressed polynucleotides can be combined by, for example, disulfide bonds or other means to form a functional antibody. For example, a portion of the light chain of an antibody can be encoded by a separate polynucleotide from the portion of the antibody comprising the heavy chain of the antibody. When co-expressed, the heavy chain polypeptides will combine with the light chain polypeptides to form the antibody. In another example, a portion of an antibody comprising one of the two Fc domain subunits and, optionally, one or more Fab molecules (or portions thereof) can be encoded by a separate polynucleotide from the portion of the antibody comprising the other of the two Fc domain subunits and, optionally, a Fab molecule (or portion thereof). When coexpressed, the Fc domain subunits will associate to form the Fc domain.

In some aspects, the isolated polynucleotide encodes an entire antibody molecule of the invention as described herein. In other aspects, the isolated polynucleotide encodes a polypeptide contained in an antibody of the invention as described herein.

In certain aspects, the polynucleotide or nucleic acid is DNA. In other aspects, the polynucleotide of the present invention is RNA, such as in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

B. Recombinant methods

The antibodies of the invention can be produced, for example, by solid-phase peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding an antibody, for example, as described above, are isolated and inserted into one or more vectors for subsequent cloning and/or expression in a host cell. Such a polynucleotide can be readily isolated and sequenced using conventional procedures. In one aspect, a vector is provided, in particular an expression vector, comprising a polynucleotide (including but not limited to one polynucleotide or a plurality of polynucleotides) of the invention. Methods well known to those skilled in the art can be used to construct expression vectors comprising an antibody coding sequence along with appropriate transcriptional/translational regulatory signals. These methods include in vitro recombinant DNA technologies, synthetic technologies, 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, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector may be part of a plasmid, a virus, or may be a nucleic acid fragment. Expression vectors include an expression cassette into which a polynucleotide encoding an antibody (i.e., the coding region) is cloned in operative linkage to a promoter and/or other transcriptional or translational regulatory elements. As used herein, a "coding region" is the portion of a nucleic acid that consists of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it may be considered part of the coding region, if present, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct, such as a single vector, or in separate polynucleotide constructs, such as separate (different) vectors. Furthermore, any vector may contain a single coding region or may contain two or more coding regions, such as a vector of the present invention may encode one or more polypeptides that are separated after or during translation into final proteins by proteolytic cleavage. In addition, the vector, polynucleotide or nucleic acid of the invention may encode heterologous coding regions, fused or not fused to a polynucleotide encoding an antibody of the invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. A functional linkage occurs when a coding region of a gene product, such as a polypeptide, is linked to one or more regulatory sequences such that expression of the gene product is influenced or controlled by these regulatory sequences. Two DNA fragments (such as a polypeptide coding region and an associated promoter) are "operably linked" if induction of the promoter results in transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or does not interfere with the ability to transcribe a DNA template. Thus, a promoter region is operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of influencing the transcription of the nucleic acid. The promoter may be a cell-specific promoter that causes sufficient transcription of DNA only in certain cells. In addition to the promoter, other transcriptional regulatory elements, such as enhancers, operators, repressors, and transcription termination signals, may be operably linked to the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional regulatory regions are described herein. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that are functional in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g., the immediate early promoter in combination with intron-A), simian virus 40 (e.g., the early promoter), and retroviruses (such as, for example, Rous sarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes, such as the actin, heat shock protein, and rabbit p-globin genes, as well as other sequences capable of regulating gene expression in eukaryotic cells. Additional suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., tetracycline-inducible promoters). Likewise, various translational regulatory elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly the internal ribosome entry site or IRES, also referred to as the CITE sequence). The expression cassette may also include other elements such as an origin of replication and/or chromosomal integration elements such as the retroviral long terminal repeats (LTRs) or the inverted terminal repeats (ITRs) of adeno-associated virus (AAV).

The coding regions of the polynucleotide and nucleic acid of the present invention may be linked to additional coding regions that encode secretory or signal peptides that direct the secretion of the polypeptide encoded by the polynucleotide of the present invention. For example, if secretion of an antibody is desired, DNA encoding a signal sequence may be placed upstream of a nucleic acid encoding an antibody of the invention or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein after initiation of export of the nascent protein chain through the rough endoplasmic reticulum. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to form the secreted or "mature" form of the polypeptide. In certain aspects, a native signal peptide, such as an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of such a sequence that retains the ability to direct secretion of a polypeptide that is operably linked to it, is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence can be replaced by a human tissue plasminogen activator (TPA) leader sequence or a mouse β-glucuronidase.

DNA encoding a short protein sequence that can be used to facilitate subsequent purification (e.g., a histidine tag) or labeling of the antibody can be included within or at the ends of the antibody-encoding (fragment) polypeptide.

In a further aspect, a host cell is provided comprising a polynucleotide (i.e., one polynucleotide or a plurality of polynucleotides) of the invention. In certain aspects, a host cell is provided comprising a vector of the invention. Polynucleotides and vectors may include any of the elements, individually or in combination, described herein in connection with polynucleotides and vectors, respectively. In one such aspect, the host cell comprises (e.g., has been transformed or transfected with) one or more vectors comprising one or more polynucleotides that encode an antibody (or portion thereof) of the invention. As used herein, the term "host cell" refers to any type of cellular system that can be engineered to generate an antibody of the invention or fragments thereof. Host cells suitable for replicating and supporting expression of antibodies are well known in the art. Such cells can be transfected or transduced with a specific expression vector if desired, and large quantities of vector-containing cells can be grown to seed large-scale fermenters to produce sufficient quantities of antibodies for clinical applications. Suitable host cells include prokaryotic microorganisms such as E. coli or various eukaryotic cells such as Chinese hamster ovary (CHO) cells, insect cells, etc. For example, the polypeptides can be produced in bacteria, particularly if glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cell paste into a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for cloning or expression of polypeptide-encoding vectors, including strains of fungi and yeast whose glycosylation pathways have been "humanized", resulting in a polypeptide with a partially or fully human glycosylation profile. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). Host cells for expression of (glycosylated) polypeptides are also derived 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 in combination 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 producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines include the SV40-transformed CV1 monkey kidney cell line (COS-7), the human embryonic kidney cell line (293 or 293T cells, described, for example, in Graham et al., J Gen Virol 36, 59 (1977)), newborn hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, described, for example, in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), brown rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT) 060562), TRI cells, described, for example, in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells and FS4 cells. Other suitable 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, NSO, P3X63, and Sp2/0. For a review of some mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, to name a few, but also cells contained within a transgenic animal, a transgenic plant, or a cultured plant or animal tissue. In one aspect, the host cell is a eukaryotic cell, particularly a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., a Y0, NSO, Sp20 cell). In one aspect, the host cell is not a cell in the human body.

Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide containing either the heavy or light chain of an antigen-binding domain, such as an antibody, can be engineered to also express the other chain of the antibody so that the expressed product is an antibody that has both a heavy and a light chain.

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

The components of the (multispecific) antibody of the invention may be genetically fused to each other. The (multispecific) antibody may be designed so that its components are directly fused to each other or indirectly via a linker sequence. The composition and length of the linker may be determined according to methods well known in the art and may be tested for effectiveness. Examples of linker sequences between different components of the (multispecific) antibodies are provided herein. Additional sequences may also be included if necessary to include a cleavage site to separate the individual fusion components, such as an endopeptidase recognition sequence.

Antibodies prepared as described herein can be purified by methods known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. In the case of purification by affinity chromatography, the antibody, ligand, receptor, or antigen to which the antibody binds can be used. For example, a protein A or protein G matrix can be used to affinity chromatograph the antibodies of the invention. The sequential steps of protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the antibody substantially as described in the examples. Antibody purity can be determined by any of a number of well-known analytical methods, including gel electrophoresis, high-pressure liquid chromatography, etc.

G. Methods of analysis

Identification, screening or characterization of the antibodies provided herein with respect to their physical/chemical properties and/or biological activity can be carried out using various assays known in the art.

1. Binding assays

Binding (affinity) to an Fc receptor or target antigen can be determined, for example, by surface plasmon resonance (SPR) using standard equipment such as the BIAcore instrument (GE Healthcare) and receptors or target proteins that can be produced by recombinant expression. Alternatively, binding of antibodies to different receptors or target antigens can be assessed using cell lines expressing a particular receptor or target antigen, for example by flow cytometry (FACS). A specific illustrative and exemplary aspect for determining CD3 binding activity is described below.

In one aspect, CD3 binding activity is determined using SPR as follows:

SPR was performed on a Biacore T200 instrument (GE Healthcare). The capture anti-Fab antibody (GE Healthcare, #28958325) was immobilized on a CM5 S-series sensor chip (GE Healthcare) using standard amine coupling chemistry at 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 running and dilution buffers. Anti-CD3 antibodies at a concentration of 2 μg/ml (in 20 mM His, 140 mM NaCl, pH 6.0) were injected over approximately 60 s at a flow rate of 5 μl/min. The CD3 antigen used is a heterodimer of the ectodomains of CD3 delta and CD3 epsilon fused to a human Fc domain with knob-into-hole modifications and a C-terminal Avi tag (see SEQ ID NOs 28 and 29). The CD3 antigen is injected at a concentration of 10 μg/mL for 120 s and dissociation is monitored at a flow rate of 5 μL/min for about 120 s. The chip surface is restored by two successive injections of 10 mM glycine, pH 2.1, for about 60 s each. Bulk refractive index differences are corrected by subtracting blank injections and by subtracting the response obtained from the flow cell blank control. The binding response is assessed 5 s after the end of the injection. To normalize the binding signal, CD3 binding is divided by the anti-Fab response (the signal (RE) obtained after capture of the anti-CD3 antibody on the immobilized anti-Fab antibody). The binding activity of an antibody to CD3 after a particular treatment compared to the binding activity of an antibody to CD3 after another treatment (also called the relative active concentration (RAC)) is calculated by comparing the binding activity of an antibody sample after a particular treatment to the binding activity of the corresponding antibody sample after another treatment.

2. Activity analysis

The biological activity of the (multispecific) antibodies of the invention can be measured using various assays, as described in the examples. The biological activity can, for example, include induction of T cell proliferation, induction of signaling 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 improved survival.

D. Compositions, formulations and routes of administration

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

Additionally, a method is provided for producing an antibody of the invention in a form suitable for in vivo administration, comprising (a) producing an antibody in accordance with the invention and (b) formulating the antibody with at least one pharmaceutically acceptable carrier, wherein the antibody preparation is formulated for in vivo administration.

The pharmaceutical compositions of the present invention comprise an effective amount of an antibody dissolved or dispersed in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" refers to molecular entities and compositions that are generally nontoxic to recipients at the dosages and concentrations employed, i.e., do not produce an adverse, allergic or other undesirable reaction when administered to an animal, such as, for example, a human, as the case may be. The method of preparing a pharmaceutical composition that contains an antibody and, optionally, a further active ingredient, should be apparent to one of skill in the art in light of the present invention and as illustrated in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Furthermore, when administered to an animal (e.g., a human), it should be understood that the preparations must meet the standards of sterility, pyrogen-freeness, general safety and purity required by the FDA's Office of Biological Standards or the appropriate authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavors, colors, the like materials, and combinations thereof, as would be known to one skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except where any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is envisaged.

The antibody of the invention (and any additional therapeutic agent) can be administered by any suitable routes, including parenteral, intrapulmonary and intranasal, and, if local treatment is desired, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing can be by any convenient route, for example by injections such as intravenous or subcutaneous injections, depending in part on whether the administration is short-term or chronic.

Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the antibodies of the invention can be formulated in aqueous solutions, in particular physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer. The solution can contain auxiliary agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibodies can be in powder form for constitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antibodies of the invention in the required amount into a suitable solvent with various other ingredients listed below, as needed. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. In general, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional required ingredient from a previously sterile-filtered liquid medium. If necessary, the liquid medium should be suitably buffered and the liquid diluent first rendered isotonic with a sufficient amount of saline or glucose before injection. The composition should be stable under the conditions of manufacture and storage and protected against the contaminating action of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to the minimum safe level, for example, less than 0.5 ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal-containing complexes (e.g. Zn-protein complexes) and/or nonionic surfactants such as polyethyleneglycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol, dextran and the like. Optionally, the suspension may also contain suitable stabilizers or substances which increase the solubility of the compounds to enable the preparation of highly concentrated solutions. In addition, suspensions of the active compounds can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.

The active ingredients can also be incorporated into microcapsules obtained, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, the matrices being in the form of shaped articles, for example films or microcapsules. In certain aspects, prolonged absorption of the injectable composition can be achieved by using absorption delaying agents in the compositions, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

In addition to the compositions described above, the antibodies can also be formulated as depot preparations. Such prolonged-release formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, the antibodies can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives, e.g., as a poorly soluble salt.

Pharmaceutical compositions containing the antibodies of the invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, embedding or lyophilizing processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients that facilitate processing of proteins into preparations that can be used for pharmaceutical purposes. The appropriate formulation depends on the chosen route of administration.

Antibodies can be formulated as a composition in free acid or base form, neutral form, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include acid addition salts, for example, formed with free amino groups of the protein composition or formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with carboxyl groups can also be prepared from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts are generally more soluble in aqueous and other protic solvents than the corresponding free base forms.

E. Therapeutic methods and compositions

Any of the antibodies provided herein can be used in therapeutic methods. The antibodies of the invention can be used as immunotherapeutic agents, for example, in the treatment of cancers.

For use in therapeutic methods, the antibodies of the invention should be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration, and other factors known to practitioners.

In one aspect, antibodies of the invention are provided for use as a medicine. In further aspects, antibodies of the invention are provided for use in treating a disease. In certain aspects, antibodies of the invention are provided for use in a method of treatment. In one aspect, the invention provides an antibody of the invention for use in treating a disease in an individual in need thereof. In certain aspects, the invention provides an antibody for use in a method of treating an individual having a disease, comprising administering to the individual an effective amount of the antibody. In certain aspects, the disease to be treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In certain aspects, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as an anti-cancer agent if the disease to be treated is cancer. In further aspects, the invention provides an antibody of the invention for use in inducing lysis of a target cell, in particular a tumor cell. In certain aspects, the invention provides an antibody of the invention for use in a method for inducing lysis of a target cell, in particular a tumor cell, in an individual, comprising administering to the individual an effective amount of the antibody to induce lysis of the target cell. The "individual" according to any of the above aspects is a mammal, preferably a human.

In a further aspect, the invention provides the use of an antibody of the invention in the manufacture or preparation of a medicament. In one aspect, the medicament is for treating a disease in an individual in need thereof. In a further aspect, the medicament is for use in a method of treating a disease, comprising administering to an individual having the disease an effective amount of the medicament. In certain aspects, the disease to be treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In one aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example an anti-cancer agent if the disease to be treated is cancer. In a further aspect, the medicament is for inducing lysis of a target cell, in particular a tumor cell. In a further aspect, the medicament is for use in a method of inducing lysis of a target cell, in particular a tumor cell, in an individual, comprising administering to the individual an effective amount of a medicament for inducing lysis of the target cell. An "individual" under any of the above aspects may be a mammal, preferably a human.

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

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

In certain aspects, the disease being treated is a proliferative disorder, in particular cancer. Non-limiting examples of cancers 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, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferative disorders that can be treated with an antibody of the present invention include, but are not limited to, neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic organs, skin, soft tissue, spleen, chest area and genitourinary system. Also included are precancerous conditions or lesions and cancer metastases. In certain aspects, the cancer is selected from the group consisting of kidney cancer, bladder cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer and prostate cancer. In one aspect, in particular, wherein the antibody is a multispecific antibody binding to TYRP-1 as a second antigen, the cancer is a cancer expressing TYRP-1. In one aspect, in particular, wherein the antibody is a multispecific antibody binding to TYRP-1 as a second antigen, the cancer is a skin cancer, in particular melanoma. In one aspect, in particular, wherein the antibody is a multispecific antibody binding to EGFRvIII as a second antigen, the cancer is a cancer expressing EGFRvIII. In one aspect, in particular, wherein the antibody is a multispecific antibody binding to EGFRvIII as a second antigen, the cancer is a brain cancer, in particular glioblastoma. One skilled in the art will appreciate that in many cases the antibody may not provide a cure, but may only provide a partial beneficial effect. In some aspects, a physiological change that has some beneficial effect is also considered therapeutically beneficial. Thus, in some aspects, the amount of an antibody that provides a physiological change is considered an "effective amount". The subject, patient, individual in need of treatment is typically a mammal, in particular a human.

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

For the prevention or treatment of a disease, the appropriate dose of an antibody of the invention (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 body weight of the patient, the type of antibody, the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically, previous or concurrent therapeutic interventions, the patient's medical history and response to the antibody, and the judgment of the attending physician. The practitioner responsible for administration will in any event determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for each individual subject. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at different times, bolus administration, and pulse infusion.

The antibody may conveniently be administered to a patient in a single dose or over a series of treatment courses. Depending on the type and severity of the disease, a candidate initial dosage for administration to a patient may be from about 1 mcg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg) of the antibody, such as in one or more separate administrations or as a continuous infusion. A single typical daily dosage may range from about 1 mcg/kg to 100 mg/kg or more, depending on the factors mentioned above. In the case of repeated administrations over several days or more, depending on the condition, treatment is typically continued until the desired degree of suppression of disease symptoms is achieved. A single typical dosage of the antibody ranges from about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, the dose can also be from about 1 microgram/kg body weight, about 5 micrograms/kg body weight, about 10 micrograms/kg body weight, about 50 micrograms/kg body weight, about 100 micrograms/kg body weight, about 200 micrograms/kg body weight, about 350 micrograms/kg body weight, about 500 micrograms/kg body weight, about 1 milligram/kg body weight, about 5 milligrams/kg body weight, about 10 milligrams/kg body weight, about 50 milligrams/kg body weight, about 100 milligrams/kg body weight, about 200 milligrams/kg body weight, about 350 milligrams/kg body weight, about 500 milligrams/kg body weight to about 1000 mg/kg body weight or more per administration, and any range resulting therefrom. In non-limiting examples of the range obtained from the values listed herein, a dose in the range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 micrograms/kg body weight to about 500 milligrams/kg body weight, etc. can be administered, taking into account the above numerical values. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to a patient. Such doses can be administered at intervals, such as once a week or once every three weeks (e.g., so that the patient receives from about two to about twenty, or, for example, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more smaller doses. At the same time, other dosing schedules can be used. The effectiveness of such therapy can be easily monitored using traditional techniques and assays.

The antibodies of the invention are generally used in an amount effective to achieve the intended purpose. When used to treat or prevent a disease state, the antibodies of the invention or pharmaceutical compositions thereof are administered or used in an effective amount.

For systemic administration, the effective dose can first be estimated from an in vitro assay, such as a cell culture assay. The dose for use in animal models can then be formulated to achieve a circulating concentration range that includes the IC ₅₀ determined in cell culture. Such information can be used to more accurately determine doses useful in humans.

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

The amount and interval of dosage can be individually adjusted to provide plasma levels of antibodies sufficient to maintain a therapeutic effect. Usual dosages for patients when administered by injection are in the range of about 0.1 to 50 mg/kg/day, typically about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses every day. Plasma levels can be measured, for example, by HPLC.

An effective dose of the antibodies of the invention will generally provide a therapeutic benefit without significant toxicity. The toxicity and therapeutic efficacy of an antibody can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD ₅₀ /ED ₅₀ . Antibodies that exhibit high therapeutic indices are preferred. In one aspect, an antibody of the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range suitable for human use. The dosage is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on a number of factors, such as the dosage form employed, the route of administration employed, the condition of the subject, etc. The exact composition, route of administration, and dosage may be selected by the individual physician taking into account the patient's condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).

The treating physician of patients treated with antibodies of the invention should know how and when to stop, interrupt or adjust the administration due to toxicity, organ dysfunction, etc. Conversely, the treating physician should also know how to adjust the treatment to higher levels if the clinical response has been insufficient (avoiding toxicity). The amount of the administered dose in the treatment of the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition can, for example, be assessed in part by standard assessment methods. In addition, the dose and possibly the frequency of dosing will also vary according to the age, body weight and response of each individual patient.

Antibodies of the invention can be administered in combination with one or more other agents in a therapy. For example, an antibody of the invention can be administered together with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat a symptom of a disease in an individual in need of such treatment. Such an additional therapeutic agent can include any active ingredients suitable for the particular disease being treated, preferably those that have complementary activity and do not negatively affect each other. In certain aspects, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to inducers of apoptosis. In a preferred aspect, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an antiangiogenic agent.

Such other agents are suitably present in the combination in amounts that are 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 dosages and by the routes described herein, or in dosages that are from about 1% to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

Such combination therapies noted above include combined administration (when two or more therapeutic agents are included in one or separate compositions) as well as separate administration, in which case the administration of the antibody of the invention may occur before, simultaneously and/or after the administration of an additional therapeutic agent and/or adjuvant. The antibodies of the invention may also be used in combination with radiation therapy.

F. Finished products

In another aspect of the invention, there is provided a finished article containing materials intended for the treatment, prevention and/or diagnosis of the above-described disorders. The finished article comprises a container and a label or package insert located thereon or associated therewith. Suitable containers include, for example, bottles, vials, syringes, bags for intravenous solution, etc. The containers can be made of a number of materials, such as glass or plastic. The container contains a composition that is effective alone or in combination with another composition for the treatment, prevention and/or diagnosis of a pathological condition, and can have a sterile entry port (for example, the container can be a bag for intravenous solution or a vial with a stopper pierced by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the selected pathological condition. In addition, the finished article may comprise (a) a first container containing a composition, wherein said composition comprises an antibody of the invention; and (b) a second container containing a composition, wherein said composition comprises an additional cytotoxic or other therapeutic agent. The finished article in this aspect may further comprise a package insert indicating that the compositions can be used to treat a particular pathological condition. Alternatively or additionally, the finished article may further comprise 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 also comprise other materials required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.

Z. Methods and compositions for diagnostics and detection

In certain aspects, any of the antibodies provided herein are useful for detecting the presence of their target (e.g., CD3, TYRP-1, EGFRvIII) in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In certain aspects, the biological sample includes a cell or tissue, such as prostate tissue.

In one embodiment, an antibody of the invention is provided for use in a diagnostic or detection method. In a further aspect, a method is provided for detecting the presence of CD3, TYRP-1 or EGFRvIII in a biological sample. In certain aspects, the method comprises contacting the biological sample with an antibody of the present invention under conditions that permit binding of the antibody to CD3, TYRP-1 or EGFRvIII and detecting whether a complex is formed between the antibody and CD3, TYRP-1 or EGFRvIII. Such a method may be an in vitro or in vivo method. In one embodiment, an antibody of the invention is used to select subjects for therapy with an antibody that binds CD3, TYRP-1 and/or EGFRvIII, for example, where CD3, TYRP-1 and/or EGFRvIII is a biomarker for patient selection.

Typical disorders that can be diagnosed using the antibody of the invention include cancer, in particular skin cancer or brain cancer.

In certain aspects, an antibody according to the present invention is provided, wherein the antibody is labeled. Labels include, but are not limited to, labels or moieties that are directly detectable (e.g., fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive labels), as well as moieties such as enzymes or ligands that are indirectly detectable, such as by an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioactive isotopes ^32P , ^14C , ^125I , ^3H , 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-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, etc.

III. SEQUENCES

IV. EXAMPLES

Examples of methods and compositions according to the invention are given below. It should be understood that in practice a number of other aspects can be realized taking into account the general description given above.

Example 1 - Creation of an optimized CD3 binding fragment

Starting from a previously described (see, e.g., WO 2014/131712, incorporated herein by reference) CD3 binding fragment, herein referred to as “CD3 _orig ”, and comprising the VH and VL sequences of SEQ ID NOs 6 and 11, respectively, we aimed to optimize the properties of this binding fragment by removing two asparagine deamidation sequence motifs at positions 97 and 100 of the Kabat CDR3 of the heavy chain.

To this end, we generated a phage display-ready heavy chain antibody library with both asparagines at Kabat positions 97 and 100 deleted and additionally randomized HI, H2, and H3 CDRs to compensate for the loss of affinity caused by the substitution of Asn97 and Asn100 through an affinity maturation process.

This library was applied to filamentous phage via fusion with the minimal coat protein p3 (Marks et al. (1991) J Mol Biol 222, 581-597) and selected for binding to recombinant CD3ε.

During the initial screening, 10 candidate clones were identified that demonstrated acceptable binding to the recombinant antigen as measured by SPR, in the form of Fab fragments (produced by E. coli).

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

The selected clone, herein referred to as “CD3 _opt ” and containing the VH and VL sequences of SEQ ID NO 7 and 11, respectively, was further evaluated and converted to a bispecific format as described below.

Example 2 - Binding of an optimized CD3 binding fragment to CD3

Binding to recombinant CD3

Binding to recombinant CD3 was determined by surface plasmon resonance (SPR) for the optimized CD3 binding fragment "CD3 _opt " and the original CD3 binding fragment "CD3 _orig ", both in the human IgG ₁ format with the P329G L234A L235A mutations ("PGLALA", EU numbering) in the Fc region (SEQ ID NOs 12 and 14 (CD3 _orig ) and SEQ ID NOs 13 and 14 (CD3 _opt )).

To evaluate the effect of deamidation site removal and its impact on antibody stability, binding of the original and optimized CD3 binding fragment to recombinant CD3 was studied after temperature stress for 14 days at 37°C or 40°C. Samples stored at -80°C were used as reference. Reference and 40°C-stressed samples were in 20 mM His, 140 mM NaCl, pH 6.0, and 37°C-stressed samples were in PBS, pH 7.4, all at a concentration of 1.2-1.3 mg/mL. After the stress period (14 days), samples were back-dialyzed into PBS in 20 mM His, 140 mM NaCl, pH 6.0 for additional analysis.

The relative active concentration (RAC) of the samples was determined by the SPR method as follows.

SPR was performed on a Biacore T200 instrument (GE Healthcare). The capture anti-Fab antibody (GE Healthcare, #28958325) was immobilized on a CM5 S-series sensor chip (GE Healthcare) using standard amine coupling chemistry, resulting in a surface density of 4000-6000 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% P20 surfactant) was used as running and dilution buffer. Anti-CD3 antibodies at a concentration of 2 μg/ml were injected over 60 s at a flow rate of 5 μl/min. CD3 antigen (see below) was injected at 10 μg/ml for 120 s and dissociation was monitored at a flow rate of 5 μl/min for 120 s. The chip surface was restored by two consecutive injections of 10 mM glycine, pH 2.1, for 60 s each. Bulk refractive index differences were corrected by subtracting blank injections and by subtracting the response obtained from the flow cell blank control. For binding assessment, the binding response was determined 5 s after the end of injection. To normalize the binding signal, CD3 binding was divided by the anti-Fab response (the signal (PE) obtained after capture of the anti-CD3 antibody on the immobilized anti-Fab antibody). The relative active concentration was calculated by comparing each temperature-stressed sample with the corresponding unstressed sample.

The antigen used was a heterodimer of the ectodomains of CD3 delta and CD3 epsilon fused to a human Fc domain with knob-into-hole modifications and a C-terminal Avi tag (see SEQ ID NOs 28 and 29).

The results of this experiment are illustrated in Fig. 2. As can be seen, the optimized CD3 binding fragment CD3 _opt exhibited markedly improved binding to CD3 after temperature stress (2 weeks at 37°C, pH 7.4) compared to the original CD3 binding fragment CD3 _orig. This result demonstrates that the removal of the deamidation site was successful and resulted in an antibody with superior stability properties related to its in vivo half-life as well as the antibody composition at neutral pH.

CD3 binding on Jurkat cells

CD3 binding on the human Jurkat NFAT reporter T cell line was determined by FACS for the optimized CD3 binding fragment "CD3 _opt " and the original CD3 binding fragment "CD3 _orig ", both in the human IgG ₁ format with P329G L234A L235A mutations ("PGLALA", EU numbering) in the Fc region (SEQ ID NOs 12 and 14 (CD3 _orig ) and SEQ ID NOs 13 and 14 (CD3 _opt )).

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphocytic leukemia reporter cell line with the NFAT promoter expressing human CD3. Cells were cultured in RPMI1640, 2 g/L glucose, 2 g/L _NaHCO3 , 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1 x NEAA, 1 x sodium pyruvate at 0.1-0.5 million cells/ml. A final concentration of 200 μg/ml hygromycin B was added each time the cells were passaged.

For binding analysis, Jurkat NFAT cells were harvested, washed in PBS and resuspended in FACS buffer. Antibody staining was performed in a 96-well round-bottom plate. Therefore, 100,000 to 200,000 cells were seeded per well. The plates were centrifuged for 4 min at 400 x g and the supernatant was removed. The antibodies to be studied were diluted in FACS buffer and 20 μl of the antibody solution were added to the wells for 30 min at 4°C. To remove unbound antibody, cells were washed twice with FACS buffer before addition of diluted secondary antibody (PE-conjugated AffiniPure E(ab')2 fragment goat anti-human IgG, Fcg fragment-specific; Jackson ImmunoResearch #109-116-170). After 30 min of incubation at 4°C, unbound secondary antibody was washed away. Before measurement, cells were resuspended in 200 μl FACS buffer and analyzed by flow cytometry using a BD Canto II.

As shown in Fig. 3, the optimized CD3 binding fragment “CD3 _opt ” and the original CD3 binding fragment “CD3 _orig ” bound comparably well to CD3 on Jurkat cells.

Example 3 - Functional activity of the CD3 binding fragment

The functional activity of the optimized CD3 binding fragment "CD3 _opt " was examined in a Jurkat reporter cell assay and compared with that of the original CD3 binding fragment "CD3 _orig ". To examine the functional IgG activity, anti-PGLALA expressing CHO cells were co-incubated with Jurkat NFAT reporter cells in the presence of increasing concentrations of human IgG ₁ PGLALA to CD3 _opt or human IgG ₁ PGLALA to CD3 _orig . CD3 activation on Jurkat NFAT reporter cells following T cell cross-linking induces luciferase production and luminescence can be measured as a marker of activation. Human IgG ₁ wt to CD3 _orig was included as a negative control, which cannot bind to anti-PGLALA expressing CHO cells and therefore cannot cross-link on Jurkat NFAT cells. A schematic illustration of this analysis is shown in Fig. 4.

Anti-PGLALA expressing CHO cells are CHO-K1 cells engineered to express on their surface an antibody that specifically binds to human IgG ₁ Fc(PGLALA) (see WO 2017/072210, incorporated herein by reference). These cells were cultured in DMEM/F12 containing 5% FBS + 1% GluMax. Jurkat NFAT reporter cells are as described in Example 2.

Upon simultaneous binding of CD3 hulgGI PGLALA to anti-PGLALA expressed on CHO and CD3 expressed on Jurkat-NFAT reporter cells, the NFAT promoter is activated and results in the expression of active firefly luciferase. The intensity of the luminescence signal (obtained after addition of luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Jurkat-NFAT reporter cells were grown in suspension and cultured in RPMI1640, 2 g/L glucose, 2 g/L NaHCO ₃ , 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1 x NEAA, 1 x sodium pyruvate at 0.1-0.5 million cells/ml, 200 μg/ml hygromycin. For the assay, CHO cells were harvested and viability was determined using ViCell. 30,000 target cells/well were seeded in a flat-bottomed 96-well white-walled plate (Greiner bio-one #655098) in 100 µl of medium and 50 µl/well of diluted antibodies or medium (for controls) were added to the CHO cells. Jurkat-NFAT reporter cells were then harvested and viability was determined using ViCell. Cells were resuspended at 1.2 million cells/ml in cell culture medium without hygromycin B and added to CHO cells at 60,000 cells/well (50 µl/well) to give a final effector to target (E:T) ratio of 2:1 and a final volume of 200 µl per well. Then 4 µl of GloSensor (Promega #E1291) (2% of the final volume) were added to each well. The cells were incubated for 24 h at 37°C in a humidified incubator. At the end of the incubation time, luminescence was recorded using a TECAN Spark 10M.

As shown in Fig. 5, the optimized CD3 binding fragment CD3 _opt had activity on Jurkat NFAT cells after cross-linking similar to CD3 _orig .

Example 4 - Generation of a T-cell bispecific antibody containing an optimized CD3 binding fragment

TYRP1 TSV

The optimized CD3 binding fragment identified in Example 1 (“CD3 _opt ”, SEQ ID NO 7 (VH) and 11 (VL)) was used to generate a T-cell bispecific antibody (TCB) targeting CD3 and TYRP1 (“TYRP1 TCB”).

The TYRP1 binding fragment contained in this TCB was created by humanization of the TYRP1 binding fragment "TA99" (see GenBank entries AXQ57811 and AXQ57813 for the heavy and light chain, respectively), and contained the heavy and light chain variable region sequences set forth in SEQ ID NOs 18 and 22, respectively.

A schematic illustration of the TCV molecule is shown in Fig. 6, and its complete sequences are given in SEQ ID NOs 23, 24, 25, and 27.

A similar molecule with the original CD3 binding sequences (SEQ ID NO 23, 24, 25 and 26) was also obtained.

Bispecific molecules were generated by transient transfection of HEK293 cells with EBNA. Cells were transfected with the respective expression vectors 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)). Cells were centrifuged and the medium was replaced with pre-warmed CD CHO medium (Thermo Fisher, #10743029). Expression vectors were mixed in CD CHO medium, PEI (polyethyleneimine, Polysciences, #23966-1) was added, the solution was vortexed and incubated for 10 min at room temperature. After that, the cells (2 million/ml) were mixed with the vector/PEI solution, transferred to a flask and incubated for 3 hours at 37°C in a shaking incubator with an atmosphere of 5% CO2. After incubation, Excell medium with supplements (80% of the total volume) was added. One day after transfection, supplements (Feed, 12% of the total volume) were added. Cell supernatants were collected after 7 days by centrifugation and subsequent filtration (0.2 μm filter).

Proteins were purified from filtered cell culture supernatants using standard methods. Briefly, Fc-containing proteins were purified from cell culture supernatants using protein A affinity chromatography (MabSelect Sure, GE Healthcare: equilibration 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 performed at pH 3.0 followed by immediate neutralization of sample pH. The protein was concentrated by centrifugation (Millipore Amicon® ULTRA-15 (#UFC903096)) and aggregated protein was separated from monomeric protein by size exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

The concentration of purified proteins was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated from the amino acid sequence according to Pace et al. (1995), Protein Science 4, 2411–23. The purity and molecular weight of proteins were analyzed by CE-SDS in the presence or absence of reducing agent using a LabChipGXII (Perkin Elmer) (Table 1). Determination of the aggregate content was performed by HPLC chromatography at 25°C using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (25 mM K ₂ HPO ₄ , 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7, or 200 mM KH ₂ PO ₄ , 250 mM KCl, pH 6.2, respectively) (Table 2).

Example 5 - Binding of a T-cell bispecific antibody containing an optimized CD3 binding fragment to CD3 and TYRP1

Binding to recombinant CD3

Binding of TYRP1 TCB to recombinant CD3 was assessed by SPR using TCBs with optimized (TYRP1 TCB CD3 _opt ) or original (TYRP1 TCB CD3 _orig ) CD3 binding sequences.

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

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

Human and cynomolgus monkey antigens were run at a concentration of 12.35 3000 nM at a flow rate of 30 μl/min through flow cells for 240 s. The dissociation phase was monitored for 240 s and initiated by switching from sample solution to HBS-EP. The chip surface was recovered after each cycle using a single injection of 10 mM glycine, pH 2.0, for 30 s.

The antigen used was heterodimers of human or cynomolgus monkey CD3 delta and CD3 epsilon ectodomains fused to a human Fc domain with knob-into-hole modifications 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)).

Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell (without trapped TCB). Affinity constants were obtained from kinetic rate constants by fitting a 1:1 Langmuir binding model using BIAeval software (GE Healthcare).

The K^ values for binding to human and cynomolgus monkey CD3 were determined to be 50 nM and 20 nM, respectively, for TYRP1 TCB CD3 _opt and were similar for TYRP1 TCB CD3orjg (50 nM and 40 nM, respectively).

This shows that under stress-free conditions, both CD3 _opt or CD3 _orig containing TCBs bound recombinant CD3 comparably well.

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

The results of this experiment are illustrated in Fig. 7.

As can be seen in Fig. 7, TCBs containing the optimized CD3 binding fragment CD3 _opt showed markedly improved binding to CD3 after stress (2 weeks at 37°C, pH 7.4) compared to TCBs containing the original CD3 binding fragment CD3 _orig . This result confirms that the improved properties of the optimized CD3 binding fragment (see Example 2) are maintained at the TCB level.

Binding to recombinant TYRP1

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

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

An antibody that specifically binds to human IgG ₁ Fc(PGLALA) (see WO 2017/072210, incorporated herein by reference) was directly coupled to the CM5 sensor chip at pH 5.0 using a standard amine coupling kit (GE Healthcare). Antigen capture (see below) was performed at a flow rate of 10 μL/min for 30 s. 3-fold serial dilutions of TYRP1 Fab fragments were run through the flow cells at 30 μL/min for 180 s to record the association phase. The dissociation phase was monitored for 180 s or 1200 s and initiated by switching from the sample solution to HBS-EP. The chip surface was restored after each cycle using a single injection of 10 mM glycine, pH 2, for 30 s at 30 µl/min.

The antigens used were monomeric fusions of the extracellular domain (ECD) of human, cynomolgus, or mouse TYRP1 with the human Fc domain with knob-into-hole modifications (and PG LALA) and a C-terminal Avi tag (see SEQ ID NOs 32 and 35) (human TYRP1), SEQ ID NOs 33 and 35 (cynomolgus TYRP1), or SEQ ID NOs 34 and 35 (mouse TYRP1)).

Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell (without captured antigen). Affinity constants (K _D ) were obtained from kinetic rate constants by fitting a 1:1 Langmuir binding model using BIAeval software (GE Healthcare).

The Kd values for binding to human, cynomolgus monkey and mouse TYRP1 were determined to be 130 pM, 180 pM and 530 pM, respectively, and were similar to the parent antibody TA99 (90 pM, 120 pM and 310 pM, respectively).

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

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

The results of this experiment are illustrated in Fig. 8 and confirm that binding to human TYRP1 for both TSVs (as well as for the corresponding TYRP1 binding fragment in IgG format) is not affected by stress conditions.

CD3 binding on Jurkat cells

CD3 binding on the human Jurkat NFAT reporter T cell line was determined by FACS for TYRP1 TCBs containing the optimized CD3 binding fragment “CD3 _opt ” or the original CD3 binding fragment “CD3 _orig ” as described above in Example 2.

As shown in Fig. 9, TCB containing the optimized CD3 binding fragment "CD3 _opt " binds to CD3 on Jurkat cells at least as well as TCB containing the original CD3 binding fragment "CD3 _orig ".

Example 6 - Functional activity of a T-cell bispecific antibody containing an optimized CD3 binding fragment

CD3 activation

TYRP1 TCBs containing the optimized CD3 binding fragment CD3 _opt or the original CD3 binding fragment CD3 _orig (Example 4) were tested in a Jurkat NFAT reporter cell assay (see Example 3) in the presence of TYRP1-positive melanoma cells M150543 (a primary melanoma cell line obtained from the Dermatology Cell Bank of the University of Zurich).

Upon simultaneous binding of TYRP1 TCB to TYRP1-positive target cells and CD3 antigen (expressed on Jurkat-NFAT reporter cells), the NFAT promoter is activated and results in the expression of active firefly luciferase. The intensity of the luminescence signal (obtained after addition of luciferase substrate) is proportional to the intensity of CD3 activation and signaling. This assay was performed as described in Example 3, using Ml50543 instead of anti-PGLALA-expressing CHO cells.

As seen for IgG (Example 3), both CD3 _opt or CD3 _orig containing TCBs had similar functional activity on NFAT reporter cells and induced CD3 activation in a concentration-dependent manner (Fig. 10).

Destruction of target cells

In the next step, both TCB molecules were tested in a tumor cell killing assay with freshly isolated human PBMCs from three different donors, which were co-incubated with the human melanoma cell line M150543. Tumor cell lysis was determined by LDH release at 24 h and 48 h. CD4 and CD8 T cell activation was analyzed by CD69 and CD25 upregulation in both cell subsets at 48 h.

Briefly, target cells were harvested with trypsin/EDTA, washed, and plated at a density of 30,000 cells/well using flat-bottomed 96-well plates. Cells were allowed to attach overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density gradient centrifugation of fresh blood obtained from healthy human donors. Fresh blood was diluted in sterile PBS and stratified in a Histopaque gradient (Sigma #H8889). After centrifugation (450 x g, 30 min, room temperature), the plasma above the PBMC-containing intermediate phase was discarded, and the PBMCs were transferred to a new Falcon tube, which was then filled with 50 ml PBS. The mixture was centrifuged (400 x g, 10 min, room temperature), the supernatant was discarded, and the pelleted PBMCs were washed twice with sterile PBS (350 x g, 10 min centrifugation steps). The resulting PBMC population was automatically counted (ViCell) and stored in RPMI1640 medium containing 10% FBS and 1% G-alanyl-E-glutamine (Biochrom #K0302) at 37°C, 5% _CO2 in a cell incubator until further use (not exceeding 24 h). For killing assays, antibodies were added at the indicated concentrations in triplicate. PBMCs were added to target cells at a final effector to target (E:T) ratio of 10:1. Target cell killing was analyzed after 24 h of incubation at 37°C, 5% _CO2 by quantifying LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science #11 644 793 001). Maximum target cell lysis (= 100%) was achieved by incubating target cells with 1% Triton X-100. Minimum lysis (= 0%) refers to target cells that were co-incubated with effector cells without the bispecific construct.

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

For all three donors, both TCBs containing the optimized or original CD3 binding fragment induced T cell activation and tumor cell lysis in a comparable manner (Fig. 11). The EC50 values for tumor cell lysis for all three donors at 48 h are shown in Table 3.

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

The pharmacokinetics (PK) of TYRP1 TCB with different CD3 binding fragments (CD3 _orig and CD3 _opt ) were studied following intravenous bolus administration at 1 mg/kg to human FcRn transgenic (strain 32, homozygous) and FcRn knockout (Jackson Laboratory strain numbers 003982 and 014565) mice (n = 3/strain/test compound). Serial blood microsamples were collected from human FcRn transgenic (tg) mice for up to 672 h (9 samples/mouse from 5 min to 672 h post dose) and from FcRn knockout (ko) mice for up to 96 h (8 samples/mouse from 5 min to 96 h post dose). Serum was obtained and stored frozen until analysis. Mouse serum samples were analyzed by a general ECLIA method specific for human Ig/Fab CHI/kappa domain using the cobas® e411 instrument (Roche) under non-GLP conditions. Pharmacokinetic evaluation was performed using a standard non-compartmental assay.

The results of this study are illustrated in Table 4. They show that CDR engineering does not result in different sequence properties that would affect antibody clearance. CD3 _opt is equally effective as CD3 _orig in terms of serum half-life, but has the added benefit of increased CDR stability.

Example 8 - Creation of an additional T-cell bispecific antibody containing an optimized

CD3 binding fragment

The optimized CD3 binding fragment identified in Example 1 (“CD3 _opt ”, SEQ ID NO 7 (VH) and 11 (VL)) was used to generate a T-cell bispecific antibody (TCB) targeting CD3 and EGFRvIII (“EGFRvIII TCB”).

The EGFRvIII binding fragment contained in this TCB (P063.056) was obtained from phage display after affinity maturation (see below) and contained the heavy and light chain variable region sequences set forth in SEQ ID NOs 88 and 92, respectively.

A schematic illustration of the TCV molecule is shown in Fig. 6, and its complete sequences are given in SEQ ID NOs 109, 110, 111, and 27.

A similar molecule with the original CD3 binding sequences (SEQ ID NOs 109, 110, 111 and 26) was also obtained.

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

In addition, phage display-derived anti-EGFRvIII antibodies were produced in human _IgG1 format in a similar manner (transfection of HEK293 EBNA cells with IgG heavy and light chain expression vectors at a 1:1 ratio) for use as described below.

All IgG and TSV constructs were purified to comparable quality with monomer content greater than 95% as determined by size exclusion chromatography.

Selection of antibodies to EGFRvIII

Anti-EGFRvIII antibodies were generated from phage display and affinity matured. Antibodies exhibiting high binding affinity and specificity for EGFRvIII (P056.021 (SEQ ID NOs 40 and 44), P056.052 (SEQ ID NOs 48 and 52), P047.019 (SEQ ID NOs 56 and 60), P057.012 (SEQ ID NOs 64 and 68), P057.011 (SEQ ID NOs 72 and 76), P056.027 (SEQ ID NOs 80 and 84)) were tested for binding to cell surface-expressed EGFRvIII using CHO cells stably expressing EGFRvIII and the EGFRvIII-positive human glioblastoma cell line DK-MG. To confirm specificity and exclude cross-reactivity with wild-type EGFR (EGFRwt), selected antibodies were tested for binding to the EGFRwt-positive human tumor cell line MKN-45 (Fig. 12). Cetuximab was included as a positive control for binding to EGFRwt and non-targeted DP47 IgG as a negative control. All selected antibodies specifically bound EGFRvIII without cross-reactivity with EGFRwt and were considered for further study.

In the next step, the functional activity of these anti-EGFRvIII antibodies in the form of IgG ₁ PGLALA (a human IgG ₁ format with P329G L234A L235A mutations (“PGLALA”, EU numbering) in the Fc region) was analyzed on DK-MG cells that were co-incubated with Jurkat NFAT reporter cells expressing the anti-PGLALA chimeric antigen receptor (CAR) by measuring luminescence (CAR J assay, see PCT Application No. PCT/EP2018/086038, incorporated herein by reference in its entirety). DP47 IgG ₁ PGLALA was included as a negative control. All anti-EGFRvIII antibodies tested induced strong activation of CAR-expressing Jurkat NFAT reporter cells (Fig. 13). All EGFRvIII antibodies tested, except P047.019, which demonstrated the weakest binding and activation, were selected for conversion to the TCB format (with CDorig as the CD3 binding fragment).

Binding of selected anti-EGFRvIII antibodies converted to TCB format to CHO-EGFRvIII cells was compared with that of the corresponding IgG (Fig. 14) to confirm that conversion to TCB format does not affect the binding ability of anti-EGFRvIII antibodies. Most of the EGFRvIII clones tested retained their ability to bind to EGFRvIII after conversion to TCB format; only clone P057.011 showed slightly reduced binding to EGFRvIII in TCB format compared to the corresponding IgG (Table 5).

The functional activity of EGFRvIII TSVs was then examined in the Jurkat NFAT reporter cell assay on EGFRvIII-positive DK-MG cells (Fig. 15). All EGFRvIII TSVs tested were active in the Jurkat NFAT reporter cell assay, with P056.021 being the most active, followed by P056.027, P056.052, and P057.012, which had similar activity, and P057.011, which had the lowest activity. EGFRvIII TSVs were then examined in a tumor cell lysis assay with PBMCs cocultured with DK-MG or MKN-45 cells to exclude cross-reactivity of EGFRvIII TSVs with EGFRwt (Fig. 16). In this assay, in addition to tumor cell lysis, T cell activation (Fig. 17) and cytokine release (Fig. 18) were measured as additional outcomes. As seen in the previous reporter cell assay, EGFRvIII TCB P056.021 had the highest activity on EGFRvIII-positive cells, with no activity on EGFRwt-positive cells. EGFRvIII TCB P057.011 showed non-specific activity on EGFRwt cells and was therefore excluded. EGFRvIII TCBs P056.027, P056.052, and P057.012 had comparable activities. Based on these results, EGFRvIII binding fragments P056.021 and P057.012 were selected for an additional round of affinity maturation.

No good binding fragments could be obtained from P057.012 (results not shown). The affinity and specificity for EGFRvIII of the selected EGFRvIII binding fragments obtained from P056.021, determined by SPR, are shown in Table 6.

The affinity matured EGFRvIII binding fragments (P063.056 (SEQ ID NOs 88 and 92), P064.078 (SEQ ID NOs 96 and 100), P065.036 (SEQ ID NOs 104 and 108)) were also compared with the parental binding fragment for specific binding to EGFRvIII on U87MG-EGFRvIII and MKN-45 cells (Fig. 19). The best EGFRvIII binding fragment in terms of affinity and specificity for EGFRvIII, P063.056, was selected for conversion to TCB format with CD3orjg or CD3 _opt as the CD3 binding fragment.

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

Methods

Surface Plasmon Resonance

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

To determine specificity, EGFRvIII and EGFRwt ECD antigens were captured with anti-his (Penta His, Qiagen) immobilized on a CM5 chip for 40 s at 100 nM. A single injection of anti-EGFRvIII antibodies was performed at 500 nM for 60 s before reduction with 10 mM glycine, pH 2.0, for 60 s. Response units greater than 50 for EGFRvIII binding were observed. Responses greater than 5 response units (RU) were considered positive for EGFRwt binding, and IgGs were categorized as specific if they were less than 5 RU for EGFRwt.

Cell lines

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphocytic leukemia reporter cell line with the NFAT promoter expressing human CD3. Cells were cultured in RPMI1640, 2 g/L glucose, 2 g/L _NaHCO3 , 10% FBS, 25 mM HEPES, 1% GlutaMAX, 1 x NEAA, 1 x sodium pyruvate at 0.1-0.5 million cells/ml. A final concentration of 200 μg/ml hygromycin B was added each time the cells were passaged.

Jurkat NFAT cells with the PGLALA CAR were generated in the laboratory. The original cell line (Jurkat NFAT; Signosis) is a human acute lymphocytic leukemia reporter cell line with an NFAT promoter that results in luciferase expression upon activation by human CD3. They were engineered to express a chimeric antigen receptor capable of recognizing the P293G LALA mutation. For culture, cells were grown in suspension in RPMI1640 supplemented with 10% FBS and 1% glutamine and maintained at 0.4–1.5 million cells per ml.

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

DK-MG (DSMZ #ACC 277) is a human glioblastoma cell line. DK-MG cells were enriched for EGFRvIII expression by cell sorting. Cells were cultured in RPMI 1860, 10% FBS, and 1% GlutaMAX.

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

MKN-45 (DSMZ ACC 409) are human gastric adenocarcinoma cells expressing high levels of EGFRwt. Cells were cultured in extended RPMI1640 containing 2% FBS and 1% GlutaMAX.

Target binding as determined by flow cytometry Cells used for binding experiments were harvested, washed with PBS and resuspended in FACS buffer. Antibody staining was performed in a 96-well round-bottom plate. Cells were harvested, counted and seeded at 100,000 to 200,000 cells per well. Plates were centrifuged for 4 min at 400 x g and the supernatant was removed. Antibodies to be tested were diluted in FACS buffer and 20 μl of antibody solution were added to the wells for 30 min at 4°C. To remove unbound antibody, cells were washed twice with FACS buffer before addition of diluted secondary antibody, PE-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG specific for the Fcg fragment (Jackson ImmunoResearch #109-116-170 or #109-116-098). After 30 min of incubation at 4°C, unbound secondary antibody was washed away. Before measurement, cells were resuspended in 200 μl FACS buffer and analyzed by flow cytometry using a BD Canto II or BD FACS Fortessa. CAR J NFAT reporter cell assay with EGFRvIII PGLALA IgG The ability of EGFRvIII PGLALA IgG to induce T cell activation was assessed using the CAR J NFAT reporter cell assay. The principle of this assay is to co-culture engineered Jurkat-NFAT effector cells with tumor antigen-expressing cancer cells. Only upon simultaneous binding of IgG to the CAR via the PGLALA mutation and the EGFRvIII target antigen, the NFAT promoter is activated and leads to increased luciferase expression in Jurkat effector cells. Upon addition of sufficient substrate, active firefly luciferase results in the emission of luminescence, which can be measured as a signal of CAR-mediated activation. Briefly, target cells were harvested and viability was determined. 30,000 target cells/well were seeded in a white-walled, flat-bottomed 96-well plate (Greiner bio-one #655098) in 100 µl of medium the day before the assay. The following day, the medium was removed and 25 µl/well of diluted antibodies or medium (for controls) were added to the target cells. Jurkat-NFAT reporter cells were then harvested and viability was determined using ViCell. Cells were resuspended at 1.5 million cells/ml in cell culture medium and added to tumor cells at 75,000 cells/well (50 µl/well) to give a final effector to target (E:T) ratio of 2.5:1 and a final volume of 75 µl per well. Then, 4 μL GloSensor (Promega #E1291) (2% of the final volume) were added to each well. The cells were incubated for 24 h at 37°C in a humidified incubator. At the end of the incubation time, the plates were adapted to room temperature (approximately 15 min). Then, 25 μL/well One-Glo luciferase (Promega, #E6120) were added and the plate was incubated for 15 min in the dark before recording the luminescence with TECAN Spark. Jurkat NFAT reporter cell assay with EGFRvIII TCB The ability of EGFRvIII TCB with improved CD3 binding fragment or original CD3 binding fragment to induce T cell cross-linking and subsequent T cell activation was assessed using EGFRvIII-positive cells and Jurkat-NFAT reporter cells. Upon simultaneous binding of EGFRvIII TCV to EGFRvIII-positive target cells and CD3 antigen (expressed on Jurkat-NFAT reporter cells), the NFAT promoter is activated and results in expression of active firefly luciferase. The intensity of the luminescence signal (obtained after addition of luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Target cells were harvested for analysis and viability was determined. 30,000 target cells/well were seeded in a flat-bottomed 96-well white-walled plate (Greiner bio-one #655098) in 100 µl of medium and 50 µl/well of diluted antibodies or medium (for controls) were added to the target cells. Jurkat-NFAT reporter cells were then harvested and viability was determined using ViCell. Cells were resuspended at 1.2 million cells/mL in cell culture medium without hygromycin B and added to tumor cells at 60,000 cells/well (50 μL/well) to give a final effector to target (E:T) ratio of 2:1 and a final volume of 200 μL per well. Then, 4 μL of GloSensor (Promega #E1291) (2% of the final volume) were added to each well. Cells were incubated for 24 h at 37°C in a humidified incubator. At the end of the incubation time, luminescence was recorded using a TECAN Spark.

T cell-mediated killing of tumor cells Target cells were harvested with trypsin/EDTA, washed, and seeded at a density of 30,000 cells/well using flat-bottomed 96-well plates. Cells were allowed to attach overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density gradient centrifugation of fresh blood obtained from healthy human donors. Fresh blood was diluted in sterile PBS and stratified in a Histopaque gradient (Sigma, #H8889). After centrifugation (450 x g, 30 min, room temperature), the plasma above the PBMC-containing intermediate phase was discarded, and the PBMCs were transferred to a new Falcon tube, which was then filled with 50 ml PBS. The mixture was centrifuged (400 x g, 10 min, room temperature), the supernatant was discarded, and the pelleted PBMCs were washed twice with sterile PBS (350 x g, 10 min centrifugation steps). The resulting PBMC population was automatically counted (ViCell) and stored in RPMI1640 medium containing 10% FBS and 1% GlutaMAX at 37°C, 5% _CO2 in a cell incubator until further use (not exceeding 24 h). For killing assays, antibody was added at the indicated concentrations in triplicate. PBMCs were added to target cells at a final effector to target (E:T) ratio of 10:1. Target cell killing was analyzed after 24 h of incubation at 37°C, 5% _CO2 by quantifying LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644793001). Maximum target cell lysis (=100%) was achieved by incubating target cells with 1% Triton X-100. Minimum lysis (=0%) refers to target cells that were co-incubated with effector cells without the bispecific construct.

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

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

Example 9 - Binding of a T-cell bispecific antibody containing an optimized CD3 binding fragment to CD3 and EGFRvIII

Binding to recombinant CD3

Binding of EGFRvIII TCB to recombinant CD3 was assessed by SPR using TCBs with optimized (EGFRvIII TCB CD3 _opt ) or original (EGFRvIII TCB CD3 _orig ) CD3 binding sequences as described for TYRP1 TCB in Example 5 above. The capture antibody was coupled to the sensor chip surface by direct immobilization of approximately 5200 resonance units (RU) at pH 5.0 using a standard amine coupling kit (GE Healthcare) and the TCB molecules were captured for 30 s at 20 nM with a flow rate of 10 μl/min.

The KD values for binding to human and cynomolgus monkey CD3 were determined to be 30 nM and 20 nM, respectively, for TYRP1 TCB CD3 _opt and were similar for TYRP1 TCB CD3orjg (40 nM and 30 nM, respectively).

This shows that under stress-free conditions, both CD3 _opt or CD3 _orig containing TCBs bound recombinant CD3 comparably well.

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

The results of this experiment are illustrated in Fig. 21.

As can be seen in Fig. 21, TCBs containing the optimized CD3 binding fragment CD3 _opt showed markedly improved binding to CD3 after stress (2 weeks at 37°C, pH 7.4) compared to TCBs containing the original CD3 binding fragment CD3 _orig . This result again confirms that the improved properties of the optimized CD3 binding fragment (see Example 2) are maintained at the TCB level.

Binding to recombinant EGFRvIII

Binding of EGFRvIII TSV to recombinant EGFRvIII was analyzed by SPR.

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

Anti-Fc antibody (GE Healthcare) was directly coupled to the CM5 sensor chip at pH 5.0 using a standard amine coupling kit (GE Healthcare). EGFRvIII TCB capture (5 nM) was performed at a flow rate of 10 μl/min for 30 s. 3-fold serial dilutions of EGFRvIII antigen were run through the flow cells at 30 μl/min for 200 s to record the association phase. The dissociation phase was monitored for 300 s and initiated by switching from sample solution to HBS-EP. The chip surface was recovered after each cycle using a single injection of 3 M MgCL2 for 30 s at 20 μl/min.

The antigen used contained the extracellular domain of human EGFRvIII fused to an Avi tag and a His tag at the C-terminus (EGFRvIII-ECD avi his; SEQ ID NO: 36).

Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell (without captured TCB). Affinity constants ( _KD ) were obtained from kinetic rate constants by fitting a 1:1 Langmuir binding model using BIAeval software (GE Healthcare). The apparent avidity constant KD was fitted by kinetic analysis through the rate constants using a 1:1 binding fit for this 2:1 interaction.

Kd (affinity) values for binding to human EGFRvIII were determined to be 6 nM for EGFRvIII TCBs containing both CD3 _opt and CD3 _orig .

Binding of EGFRvIII TCB to recombinant EGFRvIII was also assessed after 14 days of temperature stress at 37°C or 40°C using TCB with optimized or original CD3 binding sequences. This experiment was performed as described above in Example 5, using EGFRvIII-ECD avi his as the antigen (see above).

The results of this experiment are shown in Fig. 22 and confirm that binding to EGFRvIII for both TCBs is not affected by stress conditions.

CD3 binding on Jurkat cells

CD3 binding on the human Jurkat NFAT reporter T cell line was determined by FACS for EGFRvIII TCBs containing the optimized CD3 binding fragment “CD3 _opt ” or the original CD3 binding fragment “CD3 _orig ” as described above in Example 2.

As shown in Fig. 23, TCBs containing the optimized CD3 binding fragment “CD3 _opt ” and the original CD3 binding fragment “CD3 _orig ” bound comparably well to CD3 on Jurkat cells.

Binding to EGFRvIII on U87MG-EGFRvIII cells

Binding to EGFRvIII on the human glioblastoma cell line U87MG-EGFRvIII was determined by FACS for EGFRvIII TCBs containing the EGFRvIII binding fragment P063.056 with CD3 _opt or CD3orjg, or the EGFRvIII clone P056.021 with _CD3orig . The EGFRvIII binding fragment P063.056 was also included in IgG format.

As shown in Fig. 24, all three TCBs bind with high affinity to EGFRvIII expressed on U87MG-EGFRvIII cells, and conversion to TCB format does not affect binding.

Example 10 - Functional activity of a T-cell bispecific antibody containing an optimized CD3 binding fragment

CD3 activation

EGFRvIII TCBs containing the selected EGFRvIII binding fragment (P063.056) and the optimized CD3 binding fragment CD3 _opt or the original CD3 binding fragment CD3 _orig were tested in a Jurkat NFAT reporter cell assay in the presence of EGFRvIII-positive DK-MG, U87MG-huEGFRvIII glioblastoma cells and EGFRwt-positive MKN45 cells as described above in Example 8.

As seen for IgG (Example 3), both CD3 _opt or CD3 _orig containing TCBs had similar functional activity on NFAT reporter cells and induced CD3 activation in a concentration-dependent manner (Fig. 20).

Destruction of target cells

EGFRvIII TCBs containing the selected EGFRvIII binding fragment (P063.056) and the optimized CD3 binding fragment CD3 _opt or the original CD3 binding fragment CD3 _orig were compared with EGFRvIII TCBs containing the parental EGFRvIII binding fragment P056.021 and the CD3 binding fragment CD3 _orig in a tumor cell killing experiment in the presence of the U87MG-EGFRvIII glioblastoma cell line and PBMCs as described above in Example 8. As seen in Jurkat NFAT reporter cells, the functional activity of TCBs with CD3 _opt or CD3 _orig is similar in inducing tumor cell lysis and activating CD4 and CD8 T cells as measured by CD69 upregulation (Fig. 25).

In addition, the functional activity of EGFRvIII TCB with the EGFRvIII binding fragment P063.056 and the optimized CD3 binding fragment CD3 _opt in a “2+1 format” (as illustrated in Fig. 6) was compared with EGFRvIII TCB with the same EGFRvIII and CD3 binding fragments in a “1+1 head-to-tail format” (schematically depicted in Fig. 1G). These two EGFRvIII TSVs were tested in a Jurkat NFAT reporter cell assay and a tumor cell killing assay with the U87MG-EGFRvIII glioblastoma cell line as described in Example 8. The EGFRvIII TSV in the 2+1 format had superior functional activity in both CD3 activation measured in the Jurkat NFAT reporter cell assay (Fig. 26) and induction of tumor cell killing and T cell activation in the PBMC killing assay (Fig. 27).

Example 11 - Functional characteristics of T-cell bispecific antibodies containing an optimized CD3 binding fragment

Proliferation and activation of T cells by EGFRvIII TCB

The functional activity of EGFRvIII TCBs containing the selected EGFR binding fragment (P063.056) and the optimized CD3 binding fragment CD3 _opt or the original CD3 binding fragment CD3 _orig was compared with EGFRvIII TCBs containing the parental EGFRvIII binding fragment P056.021 and the CD3 binding fragment CD3 _orig in a T cell proliferation assay on U87MG-EGFRvIII cells (Fig. 28). All three TCBs induced strong proliferation and activation of CD4 T cells and CD8 T cells. P063.056 EGFRvIII TCB with CD3 _opt had higher activity than the other two EGFRvIII TCBs.

Lysis of tumor cells by EGFRvIII TCB

Subsequently, EGFRvIII TCBs containing the selected EGFR binding fragment (P063.056) and the optimized CD3 binding fragment CD3 _opt , and EGFRvIII TCBs containing the parental EGFRvIII binding fragment P056.021 and the CD3 binding fragment CD3 _orig were tested in a tumor cell lysis assay with PBMCs co-cultured with DK-MG cells (Fig. 29). In this assay, in addition to tumor cell lysis, T cell activation and cytokine release were measured as additional outcomes. As seen previously, P063.056 EGFRvIII TCB with CD3 _opt had higher activity than P056.021 EGFRvIII TCB with CD3 _orig in tumor cell lysis, T cell activation and IFNγ and TNFα release.

T cell activation and tumor cell lysis by TYRP1 TSV The functional property of TYRP1 TSV to induce cytokine release was investigated by co-cultivation of the primary melanoma cell line M150543 with PBMCs isolated from a healthy donor. T cell-mediated tumor cell lysis via TYRP1 TSV was analyzed at 24 h and 48 h after treatment (Fig. 30). The release of IFNγ and TNFα into the supernatant as well as the activation of CD4 and CD8 T cells were analyzed at 48 h after treatment. TYRP1 TSV was able to induce active tumor cell lysis as early as 24 h. This was accompanied by a strong activation of CD4 and CD8 T cells, as determined by CD25 upregulation, as well as a significant release of IFNγ and TNFα.

Methods

Isolation of the MCPC

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density gradient centrifugation of fresh blood obtained from healthy human donors. Fresh blood was diluted in sterile PBS and stratified in a Histopaque gradient (Sigma, #H8889). After centrifugation (450 x g, 30 min, room temperature), the plasma above the PBMC-containing intermediate phase was decanted and the PBMCs were transferred to a new Falcon tube, which was then filled with 50 ml PBS. The mixture was centrifuged (400 x g, 10 min, room temperature), the supernatant was decanted, and the pelleted PBMCs were washed twice with sterile PBS (350 x g, 10 min centrifugation steps). The resulting PBMC population was automatically counted (ViCell) and stored in RPMI1640 medium containing 10% FBS and 1% GlutaMAX at 37°C, 5% _CO2 in a cell incubator until further use (not longer than 24 h) or frozen and stored in liquid nitrogen for further use. The day before use, frozen PBMC were thawed and cultured overnight at 37°C.

T cell proliferation

Briefly, target cells were harvested, counted, and washed twice with PBS. Cells were resuspended at 5 million cells/ml in PBS. Cells were stained with eFluor 670 Cell Proliferation Assay Dye (eBioscience, #65-0840-85) at a final concentration of 5 μM for 10 min at 37°C. To stop the staining reaction, cells were added with 4 volumes of complete cell culture medium and incubated for 5 min at 4°C, followed by three washes with medium. Labeled target cells were counted and adjusted to 0.1 million cells/ml in RPMI1640, 10% FBS, and 1% GlutaMax. 10,000 target cells per well were seeded in a 96-well plate. The treatments were then added at the indicated concentrations, and finally 100,000 PBMCs isolated from a healthy donor were added per well. The cells were incubated for 5 days at 37°C, then PBMCs were collected and stained with CD3 BUV395 (BioLegend, #563548), CD4 PE (BioLegend, #300508), CD8 APC (BioLegend, #344722), CD25 PE/Cy7 (BioLegend, #302612). Proliferation was determined by diluting eFluor 670 dye in CD4 T cells and CD8 T cells measured by flow cytometry (FACS Fortessa, BD Bioscience), and CD4 T cell and CD8 T cell activation was measured by measuring CD25 upregulation.

T cell-mediated killing of tumor cells Target cells were harvested with trypsin/EDTA, washed and seeded at a density of 30,000 cells/well using flat-bottomed 96-well plates. Cells were allowed to attach overnight. For killing assays, antibodies were added at the indicated concentrations in triplicate. PBMCs were added to target cells at a final effector to target (E:T) ratio of 10:1. Target cell killing was analyzed after 24 h of incubation at 37°C, 5% _CO2 by quantifying LDH released into cell supernatants by apoptotic/necrotic cells (LDH Detection Kit, Roche Applied Science, #11 644 793 001). Maximal target cell lysis (=100%) was achieved by incubating target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells that were co-incubated with effector cells without the bispecific construct.

T cell activation

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

Secretion of cytokines

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

Example 13 - In vivo efficacy of T-cell bispecific antibodies containing an optimized CD3 binding fragment

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

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

Fully humanized female NSG mice (Roche Glycart AG, Switzerland) were maintained under specific pathogen-free conditions with 12 h light/12 h dark cycles according to mandatory guidelines (GV-Solas; Felasa; TierschG). The experimental protocol was reviewed and approved by the local regulatory authority (ZH223/2017). Continuous health monitoring was performed on an ongoing basis.

On day 0 of the study, mice were injected subcutaneously with 2 x 10 ⁶ IGR-1 cells, then randomized and weighed. Twenty days after tumor cell injection (tumor volume > 200 mm ³ ), mice were injected intravenously twice weekly with 10 μg (0.5 mg/kg) TYRP1 TCB for five weeks. All mice were injected intravenously with 200 μl of the respective solution. Mice in the vehicle group were injected with histidine buffer, and mice in the treatment group were injected with the TYRP1 TCB construct. To obtain the appropriate amount of antibody per 200 μl, stock solutions were diluted with histidine buffer as needed. Tumor size was measured three times weekly with a caliper, and volume in mm ³ +/- SES was plotted using GrahPad Prism software. Statistical analysis was performed using JMP12 software.

Fig. 31 shows that TYRP1 TSV mediated significant efficacy in terms of tumor growth inhibition compared to the vehicle group (68% IRO, p=0.0058*).

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

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

Fully humanized female NSG mice (Roche Glycart AG, Switzerland) were maintained under specific pathogen-free conditions with 12 h light/12 h dark cycles according to mandatory guidelines (GV-Solas; Felasa; TierschG). The experimental protocol was reviewed and approved by the local regulatory authority (ZH223/2017). Continuous health monitoring was performed on an ongoing basis.

On day 0 of the study, mice were injected subcutaneously with 5 × 10 ⁵ U87-EGFRvIII cells, then randomized and weighed. Two weeks after tumor cell injection (tumor volume > 200 mm ³ ), mice were injected intravenously with 10 μg (0.5 mg/kg) EGFRvIII TCB twice weekly for five weeks. All mice were injected intravenously with 200 μl of the respective solution. Mice in the vehicle group received histidine buffer, and mice in the treatment group received the EGFRvIII TCB construct. To obtain the appropriate amount of antibody per 200 μl, stock solutions were diluted with histidine buffer as needed. Tumor size was measured three times weekly with calipers and plotted as a function of volume in mm ³ +/- SES using GrahPad Prism software.

Figure 32 shows that EGFRvIII TSV mediated significant efficacy in terms of tumor growth control, with all mice achieving complete remission.

Example 12 - PK study with EGFRvIII TSV in mice

The pharmacokinetics (PK) of EGFRvIII TSV containing the optimized CD3 binding fragment CD3 _opt were studied following intravenous bolus administration at 1 mg/kg in human FcRn transgenic (line 32, homozygous) and NOD-SCID mice. Serial blood microsamples were collected from human FcRn transgenic (tg) and NOD-SCID mice for up to 672 h (9 samples/mouse from 5 min to 672 h post-dose). Mouse serum samples treated with EGFRvIII TSV were analyzed using a specific enzyme-linked immunosorbent assay (ELISA) under GLP-free conditions. Capture of EGFRvIII TSV was performed with biotinylated EGFRvIII antigen (huEGFRvIII his biotin) on streptavidin-coated microtiter plates (SA-MTP). Detection of EGFRvIII-bound TCB was performed using digoxigenin-labeled anti-human IgG ₁ Fc(PGLALA) monoclonal antibody (see Example 3) followed by the addition of anti-digoxigenin-POD secondary antibody for detection. The signal was generated by adding peroxidase substrate (ABTS). The calibration range was 2.35 ng/mL to 150 ng/mL, and 2.5 ng/mL corresponded to the lower limit of quantification (LLOQ).

The results of this study are shown in Table 7. The PK profile of EGFRvIII TCB is within the expected range for both mouse strains studied, indicating that the design of the CDR or CD3 binding fragment does not result in different sequence properties that would affect antibody clearance.

Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be taken as limiting the scope of the invention. The disclosure of all patents and scientific literature cited herein is expressly incorporated by reference in its entirety.

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SEQUENCE LIST

<110> F. HOFFMANN-LA ROCHE AG

<120> CD3-BINDING ANTIBODIES

<130> P35214

<150> EP18214994.8

<151> 2018-12-21

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Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro

450

<210> 13

<211> 453

<212> Protein

<213> Artificial sequence

<220>

<223> CD3opt IgG HC

<400> 13

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr

100 105 110

Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro

450

<210> 14

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> CD3orig / CD3opt IgG LC

<400> 14

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 15

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 HCDR1

<400> 15

Asp Tyr Phe Leu His

1 5

<210> 16

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 HCDR2

<400> 16

Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 17

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 HCDR3

<400> 17

Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr

1 5 10

<210> 18

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 VH

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Phe Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 LCDR1

<400> 19

Arg Ala Ser Gly Asn Ile Tyr Asn Tyr Leu Ala

1 5 10

<210> 20

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 LCDR2

<400> 20

Asp Ala Lys Thr Leu Ala Asp

1 5

<210> 21

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 LCDR3

<400> 21

Gln His Phe Trp Ser Leu Pro Phe Thr

1 5

<210> 22

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 VL

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile Tyr Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln His Phe Trp Ser Leu Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 674

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 VH-CH1(EE) - CD3orig/CD3opt VL-CH1 - Fc (lug, PGLALA)

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Phe Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr

225 230 235 240

Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr

245 250 255

Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp

260 265 270

Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr

275 280 285

Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

290 295 300

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu

305 310 315 320

Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly

325 330 335

Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro

340 345 350

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

355 360 365

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

370 375 380

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

385 390 395 400

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

405 410 415

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

420 425 430

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

435 440 445

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

580 585 590

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Pro

<210> 24

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 VH-CH1(EE) -Fc (hollow, PGLALA)

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr

20 25 30

Phe Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 25

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 VL-CL(RK)

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile Tyr Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln His Phe Trp Ser Leu Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 26

<211> 232

<212> Protein

<213> Artificial sequence

<220>

<223> CD3orig VH-CL

<400> 26

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 27

<211> 232

<212> Protein

<213> Artificial sequence

<220>

<223> CD3opt VH-CL

<400> 27

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr

100 105 110

Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 28

<211> 360

<212> Protein

<213> Artificial sequence

<220>

<223> Human CD3 epsilon stem - Fc (protrusion) - Avi

<400> 28

Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys

1 5 10 15

Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro

20 25 30

Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp

35 40 45

Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys

50 55 60

Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg

65 70 75 80

Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg

85 90 95

Val Ser Glu Asn Cys Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser

100 105 110

Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

115 120 125

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

130 135 140

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

145 150 155 160

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

165 170 175

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

180 185 190

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

195 200 205

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

210 215 220

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

225 230 235 240

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

245 250 255

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

260 265 270

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

275 280 285

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

290 295 300

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

305 310 315 320

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

325 330 335

Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu

340 345 350

Ala Gln Lys Ile Glu Trp His Glu

355 360

<210> 29

<211> 325

<212> Protein

<213> Artificial sequence

<220>

<223> Human CD3 delta stem - Fc (valley) - Avi

<400> 29

Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys

1 5 10 15

Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser

20 25 30

Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly

35 40 45

Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr

50 55 60

Val Gln Val His Tyr Arg Met Cys Arg Ser Glu Gln Leu Tyr Phe Gln

65 70 75 80

Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

85 90 95

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

100 105 110

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

115 120 125

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

130 135 140

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

145 150 155 160

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

165 170 175

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

180 185 190

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

195 200 205

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

210 215 220

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

225 230 235 240

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

245 250 255

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

260 265 270

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

275 280 285

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

290 295 300

Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys

305 310 315 320

Ile Glu Trp His Glu

325

<210> 30

<211> 351

<212> Protein

<213> Artificial sequence

<220>

<223> Cynomolgus Macaque CD3 Epsilon Stem - Fc (Protrusion) - Avi

<400> 30

Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr Gln

1 5 10 15

Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu

20 25 30

Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser

35 40 45

Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly

50 55 60

Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His

65 70 75 80

His Leu Tyr Leu Lys Ala Arg Val Ser Glu Asn Cys Val Asp Glu Gln

85 90 95

Leu Tyr Phe Gln Gly Gly Ser Pro Lys Ser Ala Asp Lys Thr His Thr

100 105 110

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

115 120 125

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

130 135 140

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

145 150 155 160

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

165 170 175

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

180 185 190

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

195 200 205

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

210 215 220

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

225 230 235 240

Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

245 250 255

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

260 265 270

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

275 280 285

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

290 295 300

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

305 310 315 320

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly

325 330 335

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

340 345 350

<210> 31

<211> 334

<212> Protein

<213> Artificial sequence

<220>

<223> Cynomolgus monkey CD3 delta stem - Fc (hollow) - Avi

<400> 31

Phe Lys Ile Pro Val Glu Glu Leu Glu Asp Arg Val Phe Val Lys Cys

1 5 10 15

Asn Thr Ser Val Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Thr

20 25 30

Asn Asn Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly

35 40 45

Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Ala

50 55 60

Val Gln Val His Tyr Arg Met Ser Gln Asn Cys Val Asp Glu Gln Leu

65 70 75 80

Tyr Phe Gln Gly Gly Ser Pro Lys Ser Ala Asp Lys Thr His Thr Cys

85 90 95

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

100 105 110

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

115 120 125

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

130 135 140

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

145 150 155 160

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

165 170 175

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

180 185 190

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

195 200 205

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser

210 215 220

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys

225 230 235 240

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

245 250 255

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

260 265 270

Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

275 280 285

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

290 295 300

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly

305 310 315 320

Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

325 330

<210> 32

<211> 699

<212> Protein

<213> Artificial sequence

<220>

<223> Human TYRP1 ECD - Fc (knob) - Avi

<400> 32

Gln Phe Pro Arg Gln Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met

1 5 10 15

Cys Cys Pro Asp Leu Ser Pro Val Ser Gly Pro Gly Thr Asp Arg Cys

20 25 30

Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser

35 40 45

Arg Pro His Ser Pro Gln Tyr Pro His Asp Gly Arg Asp Asp Arg Glu

50 55 60

Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn

65 70 75 80

Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala

85 90 95

Ala Cys Asp Gln Arg Val Leu Ile Val Arg Arg Asn Leu Leu Asp Leu

100 105 110

Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys

115 120 125

Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu

130 135 140

Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile

145 150 155 160

Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe

165 170 175

Leu Gly Val Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu

180 185 190

Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu

195 200 205

Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr

210 215 220

Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp

225 230 235 240

Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn

245 250 255

Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr

260 265 270

Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Asp Gly Pro Ile Arg

275 280 285

Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro

290 295 300

Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr

305 310 315 320

Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu

325 330 335

Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu

340 345 350

His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His

355 360 365

Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp

370 375 380

Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr

385 390 395 400

Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met

405 410 415

Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala

420 425 430

Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Ile Gln Trp Pro Ser Arg Glu

435 440 445

Phe Ser Val Pro Glu Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys

450 455 460

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

465 470 475 480

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

485 490 495

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

500 505 510

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

515 520 525

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

530 535 540

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

545 550 555 560

Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

565 570 575

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu

580 585 590

Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

595 600 605

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

610 615 620

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

625 630 635 640

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

645 650 655

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

660 665 670

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp

675 680 685

Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

690 695

<210> 33

<211> 698

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 cynomolgus monkey VCD - Fc (protrusion) - Avi

<400> 33

Gln Phe Pro Arg Glu Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met

1 5 10 15

Cys Cys Pro Asp Leu Ser Pro Met Ser Gly Pro Gly Thr Asp Arg Cys

20 25 30

Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser

35 40 45

Arg Pro His Ser Pro Arg Tyr Pro His Asp Gly Arg Asp Asp Arg Glu

50 55 60

Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn

65 70 75 80

Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala

85 90 95

Ala Cys Asp Gln Arg Val Leu Val Val Arg Arg Asn Leu Leu Asp Leu

100 105 110

Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys

115 120 125

Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu

130 135 140

Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile

145 150 155 160

Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe

165 170 175

Leu Gly Ala Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu

180 185 190

Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu

195 200 205

Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr

210 215 220

Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp

225 230 235 240

Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn

245 250 255

Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr

260 265 270

Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Ser Gly Pro Ile Arg

275 280 285

Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro

290 295 300

Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr

305 310 315 320

Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu

325 330 335

Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu

340 345 350

His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His

355 360 365

Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp

370 375 380

Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr

385 390 395 400

Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met

405 410 415

Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala

420 425 430

Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Val Gln Trp Pro Ser Arg Glu

435 440 445

Phe Ser Val Pro Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

450 455 460

Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

465 470 475 480

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

485 490 495

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

500 505 510

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

515 520 525

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

530 535 540

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

545 550 555 560

Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

565 570 575

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu

580 585 590

Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro

595 600 605

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

610 615 620

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

625 630 635 640

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

645 650 655

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

660 665 670

Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile

675 680 685

Phe Glu Ala Gln Lys Ile Glu Trp His Glu

690 695

<210> 34

<211> 699

<212> Protein

<213> Artificial sequence

<220>

<223> Mouse TYRP1 ECD - Fc (protrusion) - Avi

<400> 34

Gln Phe Pro Arg Glu Cys Ala Asn Ile Glu Ala Leu Arg Arg Gly Val

1 5 10 15

Cys Cys Pro Asp Leu Leu Pro Ser Ser Gly Pro Gly Thr Asp Pro Cys

20 25 30

Gly Ser Ser Ser Gly Arg Gly Arg Cys Val Ala Val Ile Ala Asp Ser

35 40 45

Arg Pro His Ser Arg His Tyr Pro His Asp Gly Lys Asp Asp Arg Glu

50 55 60

Ala Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys Gln Cys Asn Asp Asn

65 70 75 80

Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala

85 90 95

Ala Cys Asn Gln Lys Ile Leu Thr Val Arg Arg Asn Leu Leu Asp Leu

100 105 110

Ser Pro Glu Glu Lys Ser His Phe Val Arg Ala Leu Asp Met Ala Lys

115 120 125

Arg Thr Thr His Pro Gln Phe Val Ile Ala Thr Arg Arg Leu Glu Asp

130 135 140

Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Val

145 150 155 160

Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe

165 170 175

Leu Gly Thr Gly Gln Glu Ser Phe Gly Asp Val Asp Phe Ser His Glu

180 185 190

Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Gln Leu Glu

195 200 205

Arg Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr

210 215 220

Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Val Cys Thr Asp Asp

225 230 235 240

Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn

245 250 255

Ser Val Phe Ser Gln Trp Arg Val Val Cys Glu Ser Leu Glu Glu Tyr

260 265 270

Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Gly Gly Pro Ile Arg

275 280 285

Arg Asn Pro Ala Gly Asn Val Gly Arg Pro Ala Val Gln Arg Leu Pro

290 295 300

Glu Pro Gln Asp Val Thr Gln Cys Leu Glu Val Arg Val Phe Asp Thr

305 310 315 320

Pro Pro Phe Tyr Ser Asn Ser Thr Asp Ser Phe Arg Asn Thr Val Glu

325 330 335

Gly Tyr Ser Ala Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu

340 345 350

His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His

355 360 365

Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp

370 375 380

Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr

385 390 395 400

Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met

405 410 415

Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala

420 425 430

Pro Asp Asn Leu Gly Tyr Ala Tyr Glu Val Gln Trp Pro Gly Gln Glu

435 440 445

Phe Thr Val Ser Glu Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys

450 455 460

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

465 470 475 480

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

485 490 495

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

500 505 510

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

515 520 525

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

530 535 540

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

545 550 555 560

Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

565 570 575

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu

580 585 590

Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

595 600 605

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

610 615 620

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

625 630 635 640

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

645 650 655

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

660 665 670

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp

675 680 685

Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

690 695

<210> 35

<211> 225

<212> Protein

<213> Artificial sequence

<220>

<223> Fc (valley)

<400> 35

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 36

<211> 393

<212> Protein

<213> Artificial sequence

<220>

<223> ECD EGFRvIII - Avi - His

<400> 36

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys

1 5 10 15

Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val

20 25 30

Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly

35 40 45

Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn

50 55 60

Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile

65 70 75 80

Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu

85 90 95

Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly

100 105 110

Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala

115 120 125

Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln

130 135 140

Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg

145 150 155 160

Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys

165 170 175

Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr

180 185 190

Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys

195 200 205

Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys

210 215 220

Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg

225 230 235 240

Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg

245 250 255

Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu

260 265 270

Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys

275 280 285

Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys

290 295 300

Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala

305 310 315 320

Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly

325 330 335

Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile

340 345 350

Pro Ser Val Asp Gly Gly Ser Pro Thr Pro Pro Thr Pro Gly Gly Gly

355 360 365

Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

370 375 380

Ala Arg Ala His His His His His

385 390

<210> 37

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.021 HCDR1

<400> 37

Ser Tyr Trp Ile Ala

1 5

<210> 38

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.021 HCDR2

<400> 38

Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 39

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.021 HCDR3

<400> 39

Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr

1 5 10

<210> 40

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P056.021 VH

<400> 40

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 41

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.021 LCDR1

<400> 41

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 42

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.021 LCDR2

<400> 42

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 43

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.021 LCDR3

<400> 43

Gln Gln Val His Ser Gly Pro Pro Val Thr

1 5 10

<210> 44

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P056.021 VL

<400> 44

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Val His Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu

100 105 110

Ile Lys

<210> 45

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.052 HCDR1

<400> 45

Asn Tyr Trp Ile Gly

1 5

<210> 46

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.052 HCDR2

<400> 46

Thr Ile Tyr Pro Gly Asp Ser Asp Arg Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 47

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.052 HCDR3

<400> 47

Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr

1 5 10

<210> 48

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P056.052 VH

<400> 48

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Met Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Ser Asp Arg Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Leu Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 49

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.052 LCDR1

<400> 49

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 50

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.052 LCDR2

<400> 50

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 51

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.052 LCDR3

<400> 51

Gln Gln Val His Ser Gly Pro Pro Val Thr

1 5 10

<210> 52

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P056.052 VL

<400> 52

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Val His Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu

100 105 110

Ile Lys

<210> 53

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P047.019 HCDR1

<400> 53

Ser Ile Trp Ile His

1 5

<210> 54

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P047.019 HCDR2

<400> 54

Thr Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 55

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P047.019 HCDR3

<400> 55

Thr Gly Pro Gly Leu Ala Phe Asp Tyr

1 5

<210> 56

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P047.019 VH

<400> 56

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Pro Ser Ile

20 25 30

Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Pro Gly Leu Ala Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 57

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P047.019 LCDR1

<400> 57

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 58

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P047.019 LCDR2

<400> 58

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 59

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P047.019 LCDR3

<400> 59

Gln Gln Ser Tyr Ser Thr Pro Ile Thr

1 5

<210> 60

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P047.019 VL

<400> 60

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Ser Thr Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 61

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.012 HCDR1

<400> 61

Asn Tyr Trp Ile Ala

1 5

<210> 62

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.012 HCDR2

<400> 62

Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 63

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.012 HCDR3

<400> 63

Ala Thr Asn Ile Ala Ser Gly Gly Tyr Phe Asp Tyr

1 5 10

<210> 64

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P057.012 VH

<400> 64

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ala Asn Tyr

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Ala Thr Asn Ile Ala Ser Gly Gly Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 65

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.012 LCDR1

<400> 65

Lys Ser Ser Gln Ser Val Leu Trp Asn Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 66

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.012 LCDR2

<400> 66

Trp Ala Ser Lys Arg Glu Ser

1 5

<210> 67

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.012 LCDR3

<400> 67

Gln Gln Ser Tyr Ser Ala Pro Ile Thr

1 5

<210> 68

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P057.012 VL

<400> 68

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Trp Asn

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Lys Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Ser Ala Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 69

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.011 HCDR1

<400> 69

Arg Arg Trp Ile Ala

1 5

<210> 70

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.011 HCDR2

<400> 70

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 71

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.011 HCDR3

<400> 71

Ala Thr Asn Ile Ala Ser Gly Gly Tyr Phe Asp Tyr

1 5 10

<210> 72

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P057.011 VH

<400> 72

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Asn Phe Gly Arg Arg

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Ala Thr Asn Ile Ala Ser Gly Gly Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 73

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.011 LCDR1

<400> 73

Lys Ser Ser Gln Ser Val Leu Trp Asn Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 74

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.011 LCDR2

<400> 74

Trp Ala Ser Lys Arg Glu Ser

1 5

<210> 75

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P057.011 LCDR3

<400> 75

Gln Gln Ser Tyr Ser Ala Pro Ile Thr

1 5

<210> 76

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P057.011 VL

<400> 76

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Trp Asn

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Lys Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Ser Tyr Ser Ala Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 77

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.027 HCDR1

<400> 77

Asn Asn Trp Ile Ala

1 5

<210> 78

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.027 HCDR2

<400> 78

Val Ile Tyr Pro Gly Asp Ser Asp Lys Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 79

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.027 HCDR3

<400> 79

Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr

1 5 10

<210> 80

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P056.027 VH

<400> 80

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Gly Asn Asn

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Tyr Pro Gly Asp Ser Asp Lys Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 81

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.027 LCDR1

<400> 81

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 82

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.027 LCDR2

<400> 82

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 83

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P056.027 LCDR3

<400> 83

Gln Gln Val His Ser Gly Pro Pro Val Thr

1 5 10

<210> 84

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P056.027 VL

<400> 84

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Val His Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu

100 105 110

Ile Lys

<210> 85

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P063.056 HCDR1

<400> 85

Ser Tyr Trp Ile Ala

1 5

<210> 86

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P063.056 HCDR2

<400> 86

Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 87

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P063.056 HCDR3

<400> 87

Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr

1 5 10

<210> 88

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P063.056 VH

<400> 88

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 89

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P063.056 LCDR1

<400> 89

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 90

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P063.056 LCDR2

<400> 90

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 91

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P063.056 LCDR3

<400> 91

Gln Gln Gln Arg Asp Gly Pro Pro Val Thr

1 5 10

<210> 92

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P063.056 VL

<400> 92

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Gln Arg Asp Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu

100 105 110

Ile Lys

<210> 93

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P064.078 HCDR1

<400> 93

Ser Tyr Trp Ile Ala

1 5

<210> 94

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P064.078 HCDR2

<400> 94

Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 95

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P064.078 HCDR3

<400> 95

Val Ser Arg Leu Ser Tyr Ala Leu Asp Tyr

1 5 10

<210> 96

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P064.078 VH

<400> 96

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Arg Leu Ser Tyr Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 97

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P064.078 LCDR1

<400> 97

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 98

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P064.078 LCDR2

<400> 98

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 99

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P064.078 LCDR3

<400> 99

Gln Gln Val His Ser Gly Pro Pro Val Thr

1 5 10

<210> 100

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P064.078 VL

<400> 100

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Val His Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu

100 105 110

Ile Lys

<210> 101

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P065.036 HCDR1

<400> 101

Ser Tyr Trp Ile Ala

1 5

<210> 102

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P065.036 HCDR2

<400> 102

Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 103

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P065.036 HCDR3

<400> 103

Val Ser Arg Ser Ser Tyr Ala Leu Asp Tyr

1 5 10

<210> 104

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P065.036 VH

<400> 104

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Arg Ser Ser Tyr Ala Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 105

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P065.036 LCDR1

<400> 105

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 106

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P065.036 LCDR2

<400> 106

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 107

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII P065.036 LCDR3

<400> 107

Gln Gln Val Tyr Ser Gly Pro Pro Val Thr

1 5 10

<210> 108

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII P065.036 VL

<400> 108

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Val Tyr Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu

100 105 110

Ile Lys

<210> 109

<211> 672

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII VH-CH1(EE) - CD3orig/CD3opt VL-CH1 - Fc (lug, PGLALA)

<400> 109

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly

210 215 220

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu

225 230 235 240

Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly

245 250 255

Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln

260 265 270

Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys

275 280 285

Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly

290 295 300

Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu

305 310 315 320

Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly

325 330 335

Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

340 345 350

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

355 360 365

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

370 375 380

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

385 390 395 400

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

405 410 415

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

420 425 430

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

435 440 445

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

450 455 460

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

465 470 475 480

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

485 490 495

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

500 505 510

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

515 520 525

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

530 535 540

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

545 550 555 560

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

565 570 575

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

580 585 590

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

595 600 605

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

610 615 620

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

625 630 635 640

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

645 650 655

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 110

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> EGFRvIII VH-CH1(EE) -Fc (hollow, PGLALA)

<400> 110

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr

20 25 30

Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 111

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223>EGFRvIII VL-CL(RK)

<400> 111

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Gln Arg Asp Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu

100 105 110

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

115 120 125

Asp Arg Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

130 135 140

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

145 150 155 160

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

165 170 175

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

180 185 190

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

195 200 205

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 112

<211> 186

<212> Protein

<213> Homo sapiens

<400> 112

Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys

1 5 10 15

Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro

20 25 30

Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp

35 40 45

Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys

50 55 60

Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg

65 70 75 80

Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg

85 90 95

Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile

100 105 110

Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr

115 120 125

Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly

130 135 140

Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro

145 150 155 160

Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp

165 170 175

Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

180 185

<210> 113

<211> 177

<212> Protein

<213> Macaca fascicularis

<400> 113

Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr Gln

1 5 10 15

Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu

20 25 30

Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser

35 40 45

Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly

50 55 60

Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His

65 70 75 80

His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp

85 90 95

Val Met Ala Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Leu

100 105 110

Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys

115 120 125

Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly

130 135 140

Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro

145 150 155 160

Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg

165 170 175

Ile

<210> 114

<211> 513

<212> Protein

<213> Homo sapiens

<400> 114

Gln Phe Pro Arg Gln Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met

1 5 10 15

Cys Cys Pro Asp Leu Ser Pro Val Ser Gly Pro Gly Thr Asp Arg Cys

20 25 30

Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser

35 40 45

Arg Pro His Ser Pro Gln Tyr Pro His Asp Gly Arg Asp Asp Arg Glu

50 55 60

Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn

65 70 75 80

Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala

85 90 95

Ala Cys Asp Gln Arg Val Leu Ile Val Arg Arg Asn Leu Leu Asp Leu

100 105 110

Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys

115 120 125

Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu

130 135 140

Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile

145 150 155 160

Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe

165 170 175

Leu Gly Val Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu

180 185 190

Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu

195 200 205

Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr

210 215 220

Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp

225 230 235 240

Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn

245 250 255

Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr

260 265 270

Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Asp Gly Pro Ile Arg

275 280 285

Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro

290 295 300

Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr

305 310 315 320

Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu

325 330 335

Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu

340 345 350

His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His

355 360 365

Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp

370 375 380

Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr

385 390 395 400

Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met

405 410 415

Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala

420 425 430

Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Ile Gln Trp Pro Ser Arg Glu

435 440 445

Phe Ser Val Pro Glu Ile Ile Ala Ile Ala Val Val Gly Ala Leu Leu

450 455 460

Leu Val Ala Leu Ile Phe Gly Thr Ala Ser Tyr Leu Ile Arg Ala Arg

465 470 475 480

Arg Ser Met Asp Glu Ala Asn Gln Pro Leu Leu Thr Asp Gln Tyr Gln

485 490 495

Cys Tyr Ala Glu Glu Tyr Glu Lys Leu Gln Asn Pro Asn Gln Ser Val

500 505 510

Val

<210> 115

<211> 919

<212> Protein

<213> Homo sapiens

<400> 115

Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys

1 5 10 15

Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val

20 25 30

Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly

35 40 45

Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn

50 55 60

Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile

65 70 75 80

Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu

85 90 95

Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly

100 105 110

Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala

115 120 125

Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln

130 135 140

Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg

145 150 155 160

Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys

165 170 175

Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr

180 185 190

Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys

195 200 205

Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys

210 215 220

Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg

225 230 235 240

Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg

245 250 255

Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu

260 265 270

Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys

275 280 285

Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys

290 295 300

Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala

305 310 315 320

Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly

325 330 335

Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile

340 345 350

Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val

355 360 365

Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His Ile Val Arg

370 375 380

Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val Glu Pro

385 390 395 400

Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg Ile Leu

405 410 415

Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly Ala Phe

420 425 430

Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu Lys Val Lys

435 440 445

Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser Pro Lys Ala

450 455 460

Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser Val Asp Asn

465 470 475 480

Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln

485 490 495

Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg

500 505 510

Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn Trp Cys Val

515 520 525

Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg Leu Val His

530 535 540

Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln His Val

545 550 555 560

Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys

565 570 575

Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu

580 585 590

Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp Val Trp Ser

595 600 605

Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser Lys Pro Tyr

610 615 620

Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu Lys Gly Glu

625 630 635 640

Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met

645 650 655

Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys Phe Arg Glu

660 665 670

Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln Arg Tyr Leu

675 680 685

Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro Thr Asp Ser

690 695 700

Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp Asp Val Val

705 710 715 720

Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe Ser Ser Pro

725 730 735

Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala Thr Ser Asn

740 745 750

Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln Ser Cys Pro

755 760 765

Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp Pro Thr Gly

770 775 780

Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro Val Pro Glu

785 790 795 800

Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser Val Gln Asn

805 810 815

Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro

820 825 830

His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu

835 840 845

Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala

850 855 860

His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp

865 870 875 880

Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe

885 890 895

Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln

900 905 910

Ser Ser Glu Phe Ile Gly Ala

915

<210> 116

<211> 1186

<212> Protein

<213> Homo sapiens

<400> 116

Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln

1 5 10 15

Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn

20 25 30

Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg

35 40 45

Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr

50 55 60

Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu

65 70 75 80

Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala

85 90 95

Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro

100 105 110

Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser Asn

115 120 125

Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val

130 135 140

Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His Leu

145 150 155 160

Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp

165 170 175

Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala

180 185 190

Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys

195 200 205

His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys

210 215 220

Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr Cys

225 230 235 240

Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn

245 250 255

Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro

260 265 270

Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly

275 280 285

Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys

290 295 300

Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu

305 310 315 320

Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys

325 330 335

Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe

340 345 350

Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu

355 360 365

Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln

370 375 380

Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu

385 390 395 400

Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val

405 410 415

Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile

420 425 430

Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala

435 440 445

Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr

450 455 460

Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln

465 470 475 480

Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro

485 490 495

Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val

500 505 510

Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn

515 520 525

Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn

530 535 540

Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His

545 550 555 560

Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met

565 570 575

Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val

580 585 590

Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly

595 600 605

Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr

610 615 620

Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile

625 630 635 640

Gly Leu Phe Met Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg

645 650 655

Arg Leu Leu Gln Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly

660 665 670

Glu Ala Pro Asn Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe

675 680 685

Lys Lys Ile Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

690 695 700

Gly Leu Trp Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile

705 710 715 720

Lys Glu Leu Arg Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

725 730 735

Asp Glu Ala Tyr Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg

740 745 750

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu

755 760 765

Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn

770 775 780

Ile Gly Ser Gln Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly

785 790 795 800

Met Asn Tyr Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala

805 810 815

Arg Asn Val Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe

820 825 830

Gly Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu

835 840 845

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His

850 855 860

Arg Ile Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

865 870 875 880

Trp Glu Leu Met Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala

885 890 895

Ser Glu Ile Ser Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

900 905 910

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

915 920 925

Ile Asp Ala Asp Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe

930 935 940

Ser Lys Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp

945 950 955 960

Glu Arg Met His Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala

965 970 975

Leu Met Asp Glu Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr

980 985 990

Leu Ile Pro Gln Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr

995 1000 1005

Pro Leu Leu Ser Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val

1010 1015 1020

Ala Cys Ile Asp Arg Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu

1025 1030 1035

Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp Pro Thr Gly Ala Leu

1040 1045 1050

Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro Val Pro Glu Tyr

1055 1060 1065

Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser Val Gln Asn

1070 1075 1080

Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser Arg Asp

1085 1090 1095

Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro Glu

1100 1105 1110

Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp

1115 1120 1125

Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu

1130 1135 1140

Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys

1145 1150 1155

Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr

1160 1165 1170

Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala

1175 1180 1185

<210> 117

<211> 225

<212> Protein

<213> Homo sapiens

<400> 117

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 118

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 118

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 119

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 119

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 120

<211> 107

<212> Protein

<213> Homo sapiens

<400> 120

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 121

<211> 105

<212> Protein

<213> Homo sapiens

<400> 121

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 122

<211> 328

<212> Protein

<213> Homo sapiens

<400> 122

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<---

Claims (42)

1. An antibody that binds to CD3 and to TYRP-1, wherein the antibody comprises a) a first antigen-binding domain that binds CD3 comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10 and b) a second and optionally third antigen-binding domain that binds TYRP-1 comprising a VH comprising HCDR 1 of SEQ ID NO: 15, HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17, and a VL comprising LCDR 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21. 2. The antibody of claim 1, wherein 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 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: 11. 3. The antibody of claim 1 or 2, wherein the VH of the second and wherein the third antigen-binding domain is present 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: 18, and/or the VL of the second and wherein the third antigen-binding domain is present 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: 22. 4. An antibody that binds to CD3 and to TYRP-1, wherein the antibody comprises a) a first antigen-binding domain that binds CD3, comprising a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 11; b) a second and optionally third antigen-binding domain that binds TYRP-1, comprising the VH sequence of SEQ ID NO: 18 and the VL sequence of SEQ ID NO: 22. 5. The antibody of any one of claims 1 to 4, wherein the first antigen-binding domain is a Fab molecule. 6. An antibody according to any one of claims 1-5, comprising an Fc domain consisting of a first and a second subunit. 7. The antibody of any one of claims 1 to 6, wherein the second and/or, if present, third antigen-binding domain is a Fab molecule. 8. The antibody of any one of claims 1 to 7, wherein the first antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH of the Fab light chain and the Fab heavy chain, are substituted for each other. 9. The antibody of any one of claims 1 to 8, wherein the second and, if present, the third antigen-binding domain is a standard Fab molecule. 10. The antibody of any one of claims 1 to 9, wherein the second and, if present, the third antigen-binding domain is a Fab molecule, wherein in the CL constant domain the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H), numbering according to Kabat, and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H), numbering according to Kabat, and in the CH1 constant domain the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D), numbering according to the EU index according to Kabat, and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D), numbering according to the EU index according to Kabat. 11. The antibody of any one of claims 1 to 10, wherein the first and second antigen-binding domains are fused to each other, optionally via a peptide linker. 12. The antibody of any one of claims 1-11, wherein each of the first and second antigen-binding domains is a Fab molecule and either (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, 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 antibody of any one of claims 1 to 12, wherein each of the first, second and, if present, third antigen-binding domains is a Fab molecule, and the antibody comprises an Fc domain consisting of a first and a second subunit; and wherein either (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and the first antigen-binding domain is 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 Fab heavy chain 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. The antibody of claim 13, wherein the antibody consists of a first, second and third antigen-binding domain, an Fc domain and optionally one or more peptide linkers, wherein the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the 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, and 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. 15. The antibody of any one of claims 6 to 14, wherein the Fc domain is the Fc domain of IgG, in particular IgG1. 16. The antibody of any one of claims 6 to 15, wherein the Fc domain is a human Fc domain. 17. The antibody of any one of claims 6-16, wherein the Fc domain is an IgG1 Fc domain. 18. The antibody of any one of claims 6-17, wherein the Fc domain comprises a modification that facilitates association of the first and second subunits of the Fc domain. 19. The antibody of any one of claims 6 to 18, wherein in the CH3 domain of the first subunit of the Fc domain the amino acid residue is replaced by an amino acid residue having a larger side chain volume, thereby creating a bulge in the CH3 domain of the first subunit that can be placed in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain the amino acid residue is replaced by an amino acid residue having a smaller side chain volume, thus creating a cavity in the CH3 domain of the second subunit that can accommodate the bulge in the CH3 domain of the first subunit. 20. The antibody according to any one of claims 6-19, wherein in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and where optionally (a) in the second subunit of the Fc domain, the threonine residue at position 366 is replaced by a serine residue (T366S) and the leucine residue at position 368 is replaced by an alanine residue (L368A), numbering according to the EU index according to Kabat, and/or (b) in the first subunit of the Fc domain, additionally the serine residue at position 354 is replaced by a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine residue (E356C), and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C), numbering in accordance with the EU index according to Kabat. 21. The antibody of any one of claims 6 to 20, wherein 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 EU index according to Kabat. 22. The antibody of any one of claims 6-21, wherein the Fc domain comprises one or more amino acid substitutions that reduce Fc receptor binding and/or effector function. 23. The antibody according to any one of claims 6 to 22, wherein the Fc domain comprises substitutions at positions selected from the group E233, L234, L235, N297, P331 and P329, in particular at positions selected from the group L234, L235 and P329, numbered according to the EU Kabat index. 24. The antibody of any one of claims 6 to 23, wherein each subunit of the Fc domain comprises L234A, L235A and P329G, numbered according to the EU Kabat index. 25. The antibody of any one of claims 1 to 24, wherein the 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: 23, 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: 24, 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: 25, and 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: 27. 26. An isolated polynucleotide encoding an antibody according to any one of claims 1-25. 27. A host cell for producing an antibody, containing the isolated polynucleotide according to claim 26. 28. A method for producing an antibody that binds to CD3 and to TYRP-1, comprising the steps of (a) culturing the host cell of claim 27 under conditions suitable for expression of the antibody, and, optionally, (b) isolating the antibody. 29. A pharmaceutical composition for treating cancer, comprising an antibody according to any one of claims 1-25 and a pharmaceutically acceptable carrier. 30. Use of an antibody according to any one of claims 1-25 or a pharmaceutical composition according to claim 29 as a medicinal product for the treatment of cancer. 31. The use according to claim 30, wherein the cancer is a TYRP-1 expressing cancer and/or wherein the cancer is a skin cancer, in particular melanoma. 32. Use of an antibody according to any one of claims 1-25 or a pharmaceutical composition according to claim 29 for the production of a medicinal product for the treatment of cancer. 33. The use according to claim 32, wherein the cancer is a TYRP-1 expressing cancer and/or wherein the cancer is a skin cancer, in particular melanoma. 34. A method of treating cancer in an individual, comprising administering to said individual an effective amount of an antibody according to any one of claims 1-25 or a pharmaceutical composition according to claim 29. 35. The method of claim 34, wherein the cancer is a TYRP-1 expressing cancer and/or wherein the cancer is a skin cancer, in particular melanoma.