RU2810924C2 - Antibodies binding to cd3 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an antibody that binds to CD3 and EGFRvIII, a method of its production, as well as a composition containing it. An isolated polynucleotide encoding the above antibody, as well as a cell containing it, are also disclosed.

EFFECT: invention is effective for the treatment of cancer.

43 cl, 32 dwg, 7 tbl, 13 ex

Description

The present invention generally relates to antibodies that bind to CD3, including multispecific antibodies, for example, to activate T cells. In addition, the present invention 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 methods of using them in the treatment of a disease.

BACKGROUND OF THE ART

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 ζ chain, generating an activation signal in T cells.

CD3 has been extensively studied as a drug target. Monoclonal antibodies targeting CD3 have been used as immunosuppressive therapy for autoimmune diseases such as type I diabetes or in the treatment of transplant rejection. The anti-CD3 antibody, muromonab-CD3 (OCT3), was the first monoclonal antibody approved for clinical use in humans in 1985.

The more recent use of anti-CD3 antibodies refers to a form of bispecific antibodies that binds CD3 on one side and a tumor cell antigen on the other. 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 either 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 storage of 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 function.

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

SUMMARY OF THE INVENTION

The present invention provides antibodies, including multispecific (eg, bispecific) antibodies, that bind to CD3 and are resistant to degradation by asparagine deamidation and are thus exceptionally stable as 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 multispecific antibodies that bind to CD3, provided in the present invention retain more than about 90% CD3 binding activity after 2 weeks at pH 7.4, 37°C, compared to the activity binding 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, the antibody comprising a first antigen binding domain comprising 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) containing 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. In one aspect, VH contains 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 VL contains 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.

In a further aspect, the invention provides an antibody that binds to CD3, the antibody comprising 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 first and second subunits.

In one aspect, the antibody contains 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 VL and VH variable domains or the CL and CH1 constant domains, in particular the VL and VH variable domains of the Fab light chain and the Fab heavy chain are substituted with each other.

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

In one aspect, the second and, if present, third antigen binding domain is a Fab molecule, wherein in the CL constant domain, the amino acid at position 124 is independently replaced by 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 ) (Kabat EU numbering) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU numbering).

In one aspect, the first and second antigen binding domains are fused to each other, optionally through 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 with 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 first and second subunits; 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 a 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, in particular an IgG ₁ . In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc contains a modification that promotes association of the first and second subunits of the Fc domain. In one aspect, the Fc domain contains 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, 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 with SEQ ID NO: 19, LCDR 2 with SEQ ID NO: 20, and LCDR 3 with SEQ ID NO: 21. In one aspect, the second and, if present, a 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 VL containing an amino acid sequence that is at least about 95%, 96%, 97% identical , 98%, 99% or 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, 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 with SEQ ID NO: 89, LCDR 2 with SEQ ID NO: 90, and LCDR 3 with SEQ ID NO: 91. In one aspect, the second and, if present, 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 VL containing an amino acid sequence that is at least about 95%, 96%, 97% identical , 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 92.

In accordance with a further aspect of the invention, an isolated polynucleotide encoding an antibody of the invention and a host cell containing the isolated polynucleotide of the invention are provided.

In another aspect, a method of 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 produced by the method of the invention.

The invention further provides a pharmaceutical composition containing 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 in accordance with the invention for use as a medicament. In one aspect, an antibody or pharmaceutical composition of the invention is provided for use in treating a disease. In a specific aspect, the disease is cancer.

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

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1. Typical configurations of (multispecific) antibodies according to the invention. (A, D) Illustration of the “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”). (B, E) Illustration of the “2+1 IgG Crossfab” molecule. (G, L) Illustration of a “1+1 IgG Crossfab” molecule with an alternative order of the Crossfab and Fab components (“inverted molecule”). (3, M) Illustration of the “1+1 IgG Crossfab” molecule. (I, H) Illustration of a “2+1 IgG Crossfab” molecule with two Cross Fabs. (K,O) Illustration of a “2+1 IgG Crossfab” molecule with two CrossFabs and an alternative order of the Crossfab and Fab components (“inverted molecule”). (P, U) Illustration of the "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 molecule "Crossfab-(Fab) ₂ ". (H, Sh) Illustration of the “Fab-(Crossfab) ₂ ” molecule. (Ш, Ш) Illustration of the molecule “(Crossfab) ₂ -Fab”. Black dot: optional modification in the Fc domain promoting heterodimerization. ++, --: amino acids with opposite charges, optionally included in the CH1 and CL domains. The Crossfab molecules depicted contain interchangeable VH and VL regions, but may, in aspects where charge modifications are made to the CH1 and CL domains, alternatively comprise interchangeable CH1 and CL domains.

Fig. 2. Relative binding activity of original and optimized CD3 binding fragments CD3 _orig and 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 (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 associated with 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 (TCA) molecules produced in the Examples. All tested TCB antibody molecules were obtained in the form of “2+1 IgG CrossFab, inverted” with charge modifications (mutual replacement of VH/VL in the CD3 binding molecule, charge modifications in target antigen binding molecules, EE=147E, 213E; RK=123R, 124K).

Fig. 7. Relative binding activity of TYRP1 TCB 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 TCB containing original or optimized CD3 binding fragments CD3 _orig or CD3 _opt , or the corresponding TYRP1 IgG with recombinant TYRP1 as measured 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 original or optimized CD3 binding fragments CD3 _orig or CD3 _opt to Jurkat NFAT cells as measured by flow cytometry. Detection of TSV associated with Jurkat NFAT cells was performed using a fluorescently labeled anti-human Fc-specific secondary antibody.

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

Fig. 11. Destruction of tumor cells and activation of T cells TYRP1 TSV containing original or optimized CD3 binding fragments. Killing of the melanoma cell line M150543 after treatment with TYRP1 TSV CD3 _orig and TYRP1 TSV 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, G, N) and 48 h (B, 3, O). In parallel, as a marker of T cell activation, upregulation of CD25 (B, D, I, L, P, S) and CD69 (G, E, K, M, R, T) on CD8 (D, E, L, M, S, T) and CD4 (B, D, I, L, P, R) T cells in PBMCs by flow cytometry after 48 hours.

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. Activation of CAR J 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. The 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. Activation of Jurkat NFAT by TSV to EGFRvIII. 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 EGFRvIII TSV. The 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. Activation of T cells EGFRvIII 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, 3), using activation markers CD25 (A, C, E, G) or CD69 (B, D, F, 3) on CD4 T cells (A D) or CD8 T cells (E-H).

Fig. 18. Release of cytokines from EGFRvIII TSV. Induction of the release of IFNγ (A, D), TNFα (B, E) and granzyme B (C, F) EGFRvIII TSV was determined after co-cultivation with freshly isolated PBMCs and EGFRvIII-positive DK-MG cells (A B) or EGFRwt-positive MKN cells -45 (G-E).

Fig. 19. Specific binding of EGFRvIII IgG PGLALA with affinity matured. Specific binding of affinity-matured anti-EGFRvIII antibodies to EGFRvIII was compared to the parental EGFRvIII binding molecule on U87MG-EGFRvIII cells (A) and on the EGFRwt-positive cell line MKN-45 (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 TCB 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 as measured 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. 23. Binding of EGFRvIII TCBs containing original or optimized CD3 binding fragments CD3 _orig or CD3 _opt to Jurkat NFAT cells as measured by flow cytometry. Detection of TSV associated with 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 TSV associated with U87MG-EGFRvIII cells was performed using a fluorescently labeled anti-human Fc-specific secondary antibody.

Fig. 25. Lysis of tumor cells and activation of T cells EGFRvIII TSV. Induction of specific lysis of tumor cells (A, B) and activation of T cells (C, D) EGFRvIII TSV was determined after co-cultivation 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. Activation of Jurkat NFAT with comparison of 2+1 format and 1+1 format EGFRvIII TSV. Jurkat NFAT activation was determined as a marker of CD3 interaction with EGFRvIII TSV in the inverted 2+1 format and the head-to-tail 1+1 format in the presence of EGFRvIII-positive U87MG-EGFRvIII cells.

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

Fig. 28. Activation and proliferation of T cells by EGFRvIII TSV. The 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 PBMCs isolated from healthy donors.

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

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 (E, F) by TYRP-1 TSV was determined after co-culturing the patient-derived melanoma cell line M150543 with PBMCs. Tumor cell lysis was determined at 24 hours and 48 hours after treatment, and T cell activation and cytokine release were determined at 48 hours.

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

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

DETAILED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

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

As used herein, the terms "first", "second" or "third" with respect to antigen binding domains, etc. are used to facilitate discrimination when more than one of each type of fragment is present. The use of these terms does not imply any particular order or orientation of the fragment unless expressly 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 can be used 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 non-CD3 protein is less than about 10% of the binding of the antibody to CD3 as measured, for example, by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to CD3 has a dissociation constant ( _KD ) 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 by, for example, SPR. In certain aspects, an anti-CD3 antibody binds to a CD3 epitope that is conserved among CD3s from different species.

The term “antibody” is used herein in its broadest sense to include a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the required antigen binding activity.

"Antibody fragment" refers to a molecule, other than the intact antibody, that contains a portion of the 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 (eg, 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).

As used herein, the terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably to refer to an antibody having a structure substantially similar to that of a native antibody.

As used herein, the term “monoclonal antibody” refers to an antibody derived from a population of essentially homogeneous antibodies, i.e. the individual antibodies composing the population are identical and/or bind the same epitope, with the exception of possible variant antibodies, for example those containing natural mutations or arising during the production of the monoclonal antibody preparation, such variants generally being present in small quantities. 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 designation “monoclonal” refers to the specificity of the antibody as being derived from an essentially homogeneous population of antibodies, and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be produced by various methods, including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci, such methods and other typical methods for producing monoclonal antibodies. 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 definition, e.g., electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC, affinity chromatography, size exclusion chromatography) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. In 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 another source or species.

A “humanized” antibody refers to a chimeric antibody containing 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 the non-human antibody, and all or substantially all of the FRs correspond to those of the human antibody . Such variable domains are referred to herein as a “humanized variable region.” The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. In some aspects, certain FR residues in a humanized antibody are replaced with corresponding residues of a non-human antibody (eg, 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, for example 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 the sequence of an antibody produced by a human or a human cell, or obtained from a non-human source that uses human antibody repertoires or other sequences encoding human antibodies. This definition of a human antibody expressly excludes a humanized antibody containing non-human antigen-binding moieties. In certain aspects, the human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain aspects, the human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered herein to be human antibodies or human antibody fragments.

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

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in the binding of the antibody to an antigen. The heavy chain and light chain variable domains (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, for example, 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. In addition, antibodies that bind a particular antigen can be isolated 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, for example, Portolano et al., J. Immunol. 750:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As used herein 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).

For the purposes of this document, the amino acid positions of all constant regions and heavy and light chain domains are numbered in accordance with 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 constant domain light chain CL isotype kappa or lambda, 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 herein by the name “Kabat EU numbering” or “Kabat EU numbering”.

As used herein, the term “hypervariable region” or “HVR” refers to each of the regions of an antibody variable domain that are hypervariable in sequence and that determine antigen-binding specificity, such as “complementarity determining regions” (“CDRs”). In general, antibodies contain six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). Typical CDRs for this document include:

(a) hypervariable loops located in 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 in 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 in amino acid residues 27 c-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 stated, CDRs are defined in accordance with Kabat et al., supra. One skilled in the art will recognize that CDR assignment may also be determined in accordance with Chothia, supra, McCallum, supra, or any other scientifically accepted system of nomenclature.

"Framework region" or "FR" refers to residues of the variable domain other than the complementarity determining regions (CDRs). The FR variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Accordingly, the HVR and FR sequences are generally located 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 (eg, FR residues) are numbered herein in accordance with Kabat et al., supra.

For the purposes of this document, a “human acceptor framework” is a framework containing the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, defined below. A human acceptor framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain 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, the 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 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, a subset of sequences is a subset of Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

As used herein, the term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, IgG immunoglobulins are heterotetrameric glycoproteins weighing about 150,000 Daltons, consisting of two light chains and two heavy chains that are linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain contains a variable domain (VH), also called a heavy chain variable domain or heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3), also called a constant region. heavy chain. Similarly, in the N-terminal to C-terminal direction, each light chain contains a variable domain (VL), also called a light chain variable domain or light chain variable region, followed by a light chain constant domain (CL), also called a light chain constant region. chains. 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 can be further divided into subtypes, e.g. γ ₁ (IgG ₁ ), γ ₂ (IgG ₂ ), γ ₃ (IgG3), γ ₄ (IgG4), α ₁ (IgA ₁ ) and α ₂ (IgA ₂ ). An 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 molecules, a Fab and an Fc domain, linked through 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 main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins 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 Fab heavy and light chain are exchanged (i.e., replaced with each other), i.e. The Fab crossover molecule contains a peptide chain consisting of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, in the N- to C-terminal direction), and a peptide chain consisting of a heavy chain variable domain VH and a constant domain light chain domain CL (VH-CL, in the direction from N- to C-terminus). For clarity, in a Fab crossover molecule in which the Fab light chain and Fab heavy chain variable domains 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. Meanwhile, in a Fab crossover molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the VH heavy chain variable domain is herein referred to as the “heavy chain” of the (crossover) Fab molecule.

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

As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the Fc region of the human IgG heavy chain extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. In this case, 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 through expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain a full-length heavy chain or may contain a cleaved version of a full-length heavy chain. This can occur when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, Kabat EU numbering). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) Fc regions may or may not be present. Heavy chain amino acid sequences including the Fc region (or Fc domain subunit as defined herein ) are indicated herein without the C-terminal glycine-lysine dipeptide unless otherwise noted. In one aspect, the heavy chain, including the Fc region (subunit) as defined herein, contained in an antibody according to the invention contains an additional C-terminal glycine-lysine dipeptide (G446 and K447, Kabat EU index numbering) . In one aspect, the heavy chain, including the Fc region (subunit) as defined herein, contained in an antibody according to the invention contains an additional C-terminal glycine residue (G446, Kabat EU numbering). Unless otherwise indicated herein, 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, a “subunit” of an Fc domain refers to one of the two polypeptides that form the dimeric Fc domain, i.e. a polypeptide containing the C-terminal constant regions of the immunoglobulin heavy chain, capable of stable self-association. For example, the Fc domain subunit of an IgG contains the IgG CH2 and IgG CH3 constant domains.

By "fused" is meant that the components (eg, a Fab molecule and an 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. The multispecific antibody may, for example, be a bispecific antibody. Typically, a bispecific antibody contains two antigen binding sites, each of which is specific for a different antigenic determinant. In certain aspects, a multispecific (eg, bispecific) antibody is capable of simultaneously binding two antigenic determinants, in particular, two antigenic determinants expressed on two different cells.

As used herein, the term “valence” refers to the presence of a certain 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-specific antigen-binding site 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 complementarity determining regions (CDRs). A native immunoglobulin molecule, as a rule, has two antigen-binding sites; a Fab molecule, as a rule, has one antigen-binding site.

As used herein, the term “antigenic determinant” or “antigen” refers to a site (e.g., a contiguous 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 disease-affected cells, on the surface of immune cells, free in blood 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 (eg, humans), non-human primates (eg, cynomolgus monkeys), and rodents (eg, mice and rats), unless otherwise indicated. This term covers "full-length" full-length CD3 as well as any form of CD3 resulting from 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, particularly the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in SEQ ID NO: 112 (without signal peptide). Also see UniProt (www.uniprot.org) accession number P07766 (version 189) or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. In another aspect, the CD3 is cynomolgus CD3 (Macaca fascicularis), particularly cynomolgus CD3ε. The amino acid sequence of cynomolgus CD3ε is shown in SEQ ID NO: 113 (without signal peptide). Also see NCBI GenBank No. BAB71849.1. In certain aspects, an antibody of the invention binds to a CD3 epitope that is conserved among CD3 antigens from different species, particularly human and cynomolgus CD3. In preferred aspects, the antibody binds to human CD3.

As used herein, “target cell antigen” refers to an antigenic determinant present on the surface of a target cell, for example, a cell in a tumor, such as a cancer cell or tumor stroma 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" stands for 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 shown in SEQ ID NO: 114 (without signal peptide). Also see UniProt entry #P17643 (version 185). As used herein, "TYRP1" refers to any native TYRP1 from any vertebrate source, including mammals such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys), and rodents (eg, mice and rats) ), unless otherwise specified. This term covers "full-length" full-length TYRP1 as well as any form of TYRP1 resulting from cellular processing. The term also includes naturally occurring variants of TYRP1, 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 shown in SEQ ID NO: 115 (without signal peptide). The wild-type human EGFR sequence is shown in SEQ ID NO: 116 (without signal peptide). Also see UniProt entry #P00533 (version 258). As used herein, "EGFRvIII" refers to any native EGFRvIII from any vertebrate source, including mammals such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys), and rodents (eg, mice and rats) ), unless otherwise specified. This term covers "full-length" full-length EGFRvIII (but not wild-type EGFR), as well as any form of EGFRvIII resulting from cellular processing (eg, EGFRvIII without a signal peptide). In one aspect, EGFRvIII is human EGFRvIII.

"Affinity" refers to the strength of the net noncovalent interactions between one binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise specified, as used herein, “binding affinity” refers to the characteristic binding affinity that reflects the 1:1 interaction between members of a binding pair (eg, antibody and 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. The preferred method for measuring affinity is surface plasmon resonance (SPR).

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

“Reduced binding,” eg, reduced binding to the Fc receptor, refers to a decrease in affinity for the corresponding interaction as defined, eg, SPR. For clarity, this term also includes a reduction in affinity to zero (or below the detection limit of the analytical method), i.e. complete elimination of interaction. Conversely, “increased binding” refers to increasing the binding affinity for the corresponding interaction.

As used herein, “T cell activation” refers to one or more cellular responses of a lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, 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.

“Modification to promote association of the first and second Fc domain subunits” is manipulation of the peptide backbone or post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide containing an Fc domain subunit with an identical polypeptide to form a homodimer. As used herein, the 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 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 subunits of the Fc domain so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing the first subunit of the Fc domain and a polypeptide containing the second subunit of the Fc domain, which may not be identical in the sense that additional components fused to each subunit (e.g., antigen binding domains ), are not identical. In some aspects, the modification that promotes association of the first and second subunits of the Fc domain includes an amino acid mutation in the Fc domain, particularly an amino acid substitution. In a preferred aspect, the modification promoting 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 provided by the Fc region of an antibody, which vary depending on the isotype of the antibody. Examples of antibody effector functions include: Clq 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 antigen uptake by antigen presenting cells, down regulation of cell surface receptors (eg, 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, induces signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (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 derivatives thereof containing an Fc region specifically bind, generally through a protein moiety 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 environment surrounding the target cells, through the ADCC mechanism defined above, and/or as an increase in the concentration of antibody in the environment surrounding the target cells. target cells, necessary to ensure lysis of a given number of target cells in a given time through the ADCC mechanism. ADCC reduction is defined relative to ADCC mediated by the same antibody produced by the same host cell type, using the same standard preparation, purification, formulation and storage methods (which are well known to those skilled in the art), but which has not been engineered. For example, a reduction in ADCC mediated by an antibody containing an amino acid substitution in the Fc domain that reduces ADCC is determined relative to ADCC mediated by the same antibody without an 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).

As used herein, the terms “design, engineer, engineer” are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Design involves modifications to the amino acid sequence, glycosylation profile or side chain groups of individual amino acids, as well as combinations of these approaches.

As used herein, the term “amino acid mutation” is intended to include amino acid substitutions, deletions, insertions and modifications. Any combination of substitution, deletion, insertion and modification can be made to produce the final construct, as long as the final construct has the desired characteristics, for example, reduced binding to the 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 change, for example, the binding characteristics of the Fc region, non-conservative amino acid substitutions are particularly preferred, i.e. substitution of one amino acid with another amino acid having different structural and/or chemical properties. Amino acid substitutions include substitution with non-naturally occurring amino acids or naturally occurring amino acid derivatives of twenty standard amino acids (eg, 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 may 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, may also be applicable. Various designations may be used herein to refer to the same amino acid mutation. For example, a replacement of proline at position 329 of the Fc domain with glycine may be designated 329G, G329, _G329 , P329G, or Pro329Gly.

"Percentage (%) 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 the reference polypeptide sequence, after sequence alignment and gapping, if necessary, to achieve maximum percentage identity sequences, and without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FAS TA software package . Those skilled in the art can determine appropriate parameters for sequence alignment, 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 has been filed with user documentation at the United States Copyright Office, Washington, DC 20559, where it is registered under United States Copyright Number TXU510087, and described in WO 2001/007611.

Unless otherwise noted, for the purposes of this document, % amino acid sequence identity values are obtained using the ggsearch program from FASTA version 36.3.8 or later with the BLOSUM50 comparison matrix. The authorship of the FASTA software package belongs to 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 the package itself is open access 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; discovery: -10; extension : -2; Ktup=2) to ensure that the alignment is global rather than local. The percentage of amino acid identity is output in the alignment output header file.

The term "polynucleotide" or "nucleic acid molecule" includes any compound and/or substance that contains 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, with said bases representing the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually presented from 5' to 3'. As used herein, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers, containing two or more of these molecules. A nucleic acid molecule can be linear or circular. In addition, the term "nucleic acid molecule" includes both sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecule described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars, or phosphate backbones, 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, for example, in a host or patient. Such DNA (eg, cDNA) or RNA (eg, mRNA) vectors may be unmodified or modified. For example, the mRNA can be chemically modified to increase 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, for example, 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 contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal position that differs from its natural chromosomal position.

"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 one vector or separate vectors and such polynucleotide molecules present in one or more locations in the 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. The term includes a vector in the form of a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of the host cell into which it is introduced. Some vectors are capable of controlling the expression of the nucleic acids to which they are operably 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 "transformants" and "transformed cells", which include the primary transformed cells and their progeny, regardless of the number of passages. The offspring may not be completely identical to the parent cell in nucleic acid content and may contain mutations. This includes mutant progeny that have the same function or biological activity against 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, for example, cultured mammalian cells, such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 murine myeloma cells, PER cells, PER.C6 cells or cells hybridomas, yeast cells, insect cells and plant cells, to name a few, but also cells present in the body of a transgenic animal, transgenic plant or 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 a human body.

The term "pharmaceutical composition" or "pharmaceutical composition" refers to a preparation that is in a form that provides effective biological activity of the active ingredient contained therein, and which does not contain additional components that are unacceptably toxic to the subject to whom the composition will be administered.

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

In the context of this document, the term "treatment" (and its grammatical variations such as "treat" or "treating") refers to a clinical intervention with the purpose of changing the natural history of a disease in the individual being treated, and can be carried out either for prevention or treatment. time of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of onset or recurrence of disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or temporary relief of disease, and 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.

"Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (for example, cows, sheep, cats, dogs and horses), primates (for example, humans and non-human primates such as monkeys), rabbits and rodents (for example, mice and rats ). In certain aspects, the individual or subject is a human being.

An "effective amount" of an agent, for example, a pharmaceutical composition, refers to an amount effective in doses 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 packaging of therapeutic products that contain information regarding the indications, use, 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 efficacy and safety, pharmacokinetics, and manufacturing feasibility. 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, isolated antibodies are provided that bind to CD3. In one aspect, the invention provides antibodies that specifically bind to CD3. In certain aspects, anti-CD3 antibodies retain more than about 90% CD3 binding activity after 2 weeks at pH 7.4, 37°C, compared to 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, the antibody comprising a first antigen binding domain comprising 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) containing 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.

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

In one aspect, VH and/or VL comprises an acceptor human framework region, for example, a human immunoglobulin framework region or a human consensus framework region.

In one aspect, VH comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3 and/or FR4 sequence) of the heavy chain variable region sequence SEQ ID NO: 7. In one aspect, VH comprises a sequence that is at least at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In one aspect, VH contains a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7 In one aspect, 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 that is at least 95%, 96%, 97%, 98%, or 99% identical identity, contains substitutions (eg, conservative substitutions), insertions or deletions compared to the reference sequence, but the antibody containing this sequence retains the ability to bind to CD3. In certain aspects, a substitution, insertion, and/or deletion has been made of a total of 1-10 amino acids in the amino acid sequence of SEQ ID NO: 7. In certain aspects, the substitutions, insertions, or deletions are in regions outside the CDR (i.e., in the FR) . 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 to that sequence.

In one aspect, VL comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3 and/or FR4 sequence) of the light chain variable region sequence SEQ ID NO: 11. In one aspect, VL comprises a sequence that is at least at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In one aspect, VL contains a sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 11 In one aspect, 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 that is at least 95%, 96%, 97%, 98%, or 99% identical identity, contains substitutions (eg, conservative substitutions), insertions or deletions compared to the reference sequence, but the antibody containing this sequence retains the ability to bind to CD3. In certain aspects, a substitution, insertion, and/or deletion has been made of a total of 1-10 amino acids in the amino acid sequence of SEQ ID NO: 11. In certain aspects, the substitutions, insertions, or deletions are in regions outside the CDR (i.e., in the FR) . In one aspect, VL comprises the amino acid sequence of SEQ ID NO: 11. Optionally, VL comprises the amino acid sequence of SEQ ID NO: 11, including post-translational modifications to that sequence.

In one aspect, VH contains 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 contains 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 contains the amino acid sequence of SEQ ID NO: 11.

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

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

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

In one aspect, VH comprises 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, VL comprises 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, the antibody comprising 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 containing a human constant region, in particular an IgG immunoglobulin molecule containing a human CH1, CH2, CH3 and/or CL domain. Exemplary human constant domain sequences are shown in SEQ ID NO: 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, particularly 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 contain amino acid mutations in the Fc domain, as described herein.

In one aspect, the first antigen binding domain comprises a human constant region. In one aspect, the first antigen binding fragment is a Fab molecule containing 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 contain amino acid mutations described herein as "charge modifications" and/or may contain deletion or substitution of one or more (in in particular two) N-terminal amino acids, if it belongs to the 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 CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 122. In particular, the heavy chain constant region (in particular the CH1 domain) may contain 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, particularly an IgG ₁ antibody. In one aspect, the antibody is a full-length antibody.

In another aspect, the antibody is an antibody fragment selected from the group of an Fv molecule, a scFv molecule, a Fab molecule, and an F(ab') ₂ molecule; in particular Fab molecules. In another aspect, the antibody fragment is a diabody, tribody, 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 VL and VH variable domains or the CL and CH1 constant domains, in particular the VL and VH variable domains of the Fab light chain and the Fab heavy chain are substituted for each other (i.e., the first antigen binding domain represents is a Fab crossover molecule).

In a further aspect, an antibody according to any of the above aspects may have any of the features, individually or in combination, described in sections P.A. 1.-8.below.

In a preferred aspect, the antibody comprises an Fc domain, in particular an IgG Fc domain, more specifically 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 consists of first and second subunits and may have any of the features, individually or in combination, described below herein with respect to Fc domain variants (Section II. A. 8.).

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

1. Antibody fragments

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

In one aspect, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab') ₂ molecule, particularly a Fab molecule as described herein. A "Fab'molecule" is distinguished 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 antibody hinge region. Fab'-SH are Fab' molecules in which the cysteine residue(s) of the constant domains bear(s) a free thiol group. Treatment with pepsin results in the production of an F(ab') ₂ molecule, which contains two antigen-binding sites (two Fab molecules) and part of the Fc region.

In another aspect, the antibody fragment is a diabody, tribody, or tetrabody. "Dibodies" are antibody fragments with two antigen-binding sites, which can be bivalent or bispecific. See for example 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). Tribodies 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" 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. These single-chain Fab molecules are stabilized by a natural disulfide bond between the CL domain and the CH1 domain. In addition, single-chain Fab molecules can be further stabilized by creating interchain disulfide bonds through the insertion of cysteine residues (eg, at position 44 of the heavy chain variable domain and position 100 of the light chain variable domain 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 heavy (VH) and light (VL) chain variable domains of an antibody connected by a linker. Specifically, the linker is a short polypeptide of 10-25 amino acids and is typically enriched with 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. This protein retains the specificity of the parent antibody despite to remove constant regions and add a linker. For a review of scFv fragments, see, for example, 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 US patents Ms. 5571894 and 5587458.

In another aspect, the antibody fragment is a single domain antibody. “Single-domain antibodies” are antibody fragments containing all or part of the variable domain of the heavy chain of an antibody, or all or part of the variable domain of the light chain of an antibody. In certain aspects, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, US Pat. No. 6,248,516 B1).

Antibody fragments can be produced by various methods, including, but not limited to, proteolytic cleavage of the intact antibody, as well as recombinant production by recombinant host cells (eg, E. coli), as described herein.

2. Humanized antibodies

In certain aspects, an 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. In general, a humanized antibody contains one or more variable domains, in which the CDRs (or portions thereof) are derived from non-human antibody sequences and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody optionally also contains at least a portion of the human constant region. In some aspects, certain FR residues in a humanized antibody are replaced with corresponding residues of a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

Humanized antibodies and methods for their production are discussed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US patents Ms 5821337, 7527791, 6982321 and 7087409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface change"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the "directed selection" approach of FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, best-fit framework regions (see, for example, Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the human antibody consensus sequence of a particular subset of light or heavy chain variable regions (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol, 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

3. Options for no glycosylation

In certain aspects, an antibody provided herein is modified to increase or decrease the degree of glycosylation of the antibody. The addition or removal of glycosylation sites to an antibody is conveniently accomplished by changing the amino acid sequence so as to create or remove one or more glycosylation sites.

If the antibody contains an Fc region, the oligosaccharide attached to it can be modified. Native antibodies produced by mammalian cells typically contain a branched biantennary oligosaccharide, which is usually 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 may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the “trunk” of the biantennary oligosaccharide structure. In some aspects, modifications to the oligosaccharide in an antibody of the invention can be made to create variants of the antibody with certain improved properties.

In one aspect, antibody variants are provided comprising a non-fucosylated oligosaccharide, i.e. an oligosaccharide structure lacking fucose attached (directly or indirectly) to the Fc region. Specifically, 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 the 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 the 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% (ie, no fucosylated oligosaccharides). The percentage of non-fucosylated oligosaccharides is the (average) number 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 mass TOF-MALDI spectrometry, for example 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 Fc region residues); in this case, Asn297 can also be located about ±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 Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially in Example 11), and knockout cell lines such as CHO cells with a knockout of the alpha-1,6-fucosyltransferase gene, FUT8 (see, for example, 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 GDP-fucose or carrier protein synthesis activity ( see for example US2004259150, US2005031613, US2004132140, US2004110282).

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

Variants of antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region are also proposed. 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 antibodies constructed using cysteine, for example, TNIOMAB™ antibodies, in which one or more residues are replaced with cysteine residues. In preferred aspects, the substituted residues are at accessible sites on the antibody. By replacing 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. Antibodies constructed using cysteine can be created as described, for example, in US patent No. 7521541, 830930, 7855275, 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 are readily available. Fragments suitable for antibody derivatization 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic copolymer anhydride, polyamino acids (homopolymers or random copolymers), dextran or poly(p-vinylpyrrolidone)polyethylene glycol, propylene glycol homopolymers, oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer can have any molecular weight and can 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 amount and/or type of polymers used for derivatization can be determined based on considerations that include, but are not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be useful in therapy under certain conditions etc.

6. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CD3 antibody as herein 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 given 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 diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, Abrin A, modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantensis inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin , fenomycin, enomycin and tricothecenes.

In another aspect, the immunoconjugate comprises an antibody of the invention conjugated to a radioactive atom to form a radioconjugate. A number of radioactive isotopes are available to produce radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , 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, I ¹³¹ , In ¹¹¹ , F ¹⁹ , C ¹³ , N ¹⁵ , O ¹⁷ , gadolinium, manganese or iron.

Antibody-cytotoxic agent conjugates can be created 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 imidoester derivatives (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 radionuclide-antibody conjugation. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug into 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); US Pat. No. 5,208,020).

As used herein, immunoconjugates or ADCs expressly include, but are not limited to, conjugates produced using cross-linking reagents, but are not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MRVH, 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 (eg, from Pierce Biotechnology, Inc., Rockford, IL., U.S.A.).

7. Multispecific antibodies

In certain aspects, an antibody provided herein is a multispecific antibody, particularly a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two antigenic determinants (eg, two different proteins or two different epitopes on the same 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 CD3 epitopes. Multispecific (eg, 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 creating multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), and knob-to-trench design " (see, for example, US Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be created by design using the effect of electrostatic interaction to create Fc heterodimeric antibody molecules (see, for example, WO 2009/089004); cross-linking two or more antibodies or fragments (see, for example, US Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); the use of leucine zippers for the production of bispecific antibodies (see, for example, 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, for example, WO 98/50431); the use of diabody technology to produce bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single chain Fv dimers (sFv) (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, for example, in 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-action FAbs" or "DAFs" containing an antigen-binding site that binds to CD3 as well as another different antigen, or two different CD3 epitopes (see, for example, US 2008/0069820 and WO 2015/095539).

Multispecific antibodies can be provided in an asymmetric form with crossover domains in one or more binding arms with the same antigen specificity (so-called “CrossMab” technology), i.e. through the exchange of VH/VL domains (see, for example, WO 2009/080252 and WO 2015/150447), CH1/CL domains (see, for example, WO 2009/080253) or full Fab arms (see, for example, WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191 and Klein at al., MAbs 8 (2016) 1010-20). Asymmetric Fab arms can also be constructed 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, for example, Spiess et al., Mol Immunol 67 (2015) 95-106).

A particular type of multispecific antibodies 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 target cells. Therefore, in preferred aspects, the antibody provided herein is a multispecific antibody, particularly a bispecific antibody, wherein one of the binding specificities is directed to CD3 and the other is directed to a target cell antigen.

Examples of bispecific antibody formats that can be used for these purposes include, but are not limited to, so-called “BiTE” (bispecific T cell-attracting activator) molecules, in which two scFv molecules are linked by a flexible linker (see, for example, 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 so-called triomabs , which are fully hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). The 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 CD3 as described herein, and comprising a second and optionally a third antigen binding domain that binds a second antigen.

In accordance with preferred aspects of the invention, the antigen binding domains contained in the antibodies are Fab molecules (ie, antigen binding domains consisting of a heavy and a light chain, each of which contains a variable and a constant domain). In one aspect, the first, second and/or, if present, 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 human heavy and light chain constant domains.

Preferably, at least one of the antigen binding domains is a Fab crossover molecule. Such 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 Fab crossover molecule useful for the (multispecific) antibody of the invention, the Fab light chain and Fab heavy chain variable domains (VL and VH, respectively) are exchanged. However, even with such domain swapping, the (multispecific) antibody preparation may contain some by-products due to the so-called Bene-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 increase the purity and yield of the desired (multispecific) antibody, charged amino acids with 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 Fab molecule(s) binding to a second antigen (eg, a target cell antigen such as TYRP-1 or EGFRvIII), as further described herein. Charge modifications are carried out in standard Fab molecules contained in a (multispecific) antibody (as shown, for example, in Fig. 1 A-B, G-J), or in VH/VL crossover Fab molecules contained in a (multispecific) antibody (as shown, for example, in Fig. 1 G-E, L-O) (but not both). In preferred aspects, charge modifications are carried out on standard Fab molecules contained in a (multispecific) antibody (which in preferred aspects binds to a second antigen, eg, a target cell antigen such as TYRP-1 or EGFRvIII).

In a preferred aspect of the invention, the (multispecific) antibody is capable of simultaneously binding to a first antigen (ie CD3) and a second antigen (eg 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 a target cell antigen. In an even more preferred aspect, such simultaneous binding results in lysis of the target cell, in particular a target cell antigen (eg, TYRP-1 or EGFRvIII) expressing tumor cell. In one aspect, such simultaneous binding results in T cell activation. In other aspects, such simultaneous binding results in a cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte selected from the group of proliferation, differentiation, cytokine secretion, 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 T cell specificity.

Preferably, the T cell in accordance with any aspects of the invention is a cytotoxic T cell. In some aspects, the T cell is a CD4 ⁺ or CD8 ⁺ T cell, particularly a CD8 ⁺ T cell.

a) First antigen binding domain

The (multispecific) antibody of the invention contains at least one antigen binding domain (first antigen binding domain) that binds 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 against (ie, specifically binds to) human and cynomolgus CD3. In some aspects, CD3 is an epsilon subunit of CD3 (CD3 epsilon).

In a preferred aspect, the (multispecific) antibody contains at most 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 CD3 is an antibody fragment selected from the group of an Fv molecule, a scFv molecule, a Fab molecule, and an F(ab') _{2 molecule} . In a preferred aspect, the antigen binding domain that binds CD3 is a Fab molecule.

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

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

In preferred aspects, the first antigen binding domain is a Fab molecule, wherein the VL and VH variable domains or the CL and CH1 constant domains, in particular the VL and VH variable domains of the Fab light chain and the Fab heavy chain are substituted for each other (i.e., in accordance with such In 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, third) antigen binding domain is a standard Fab molecule.

In one aspect, the (multispecific) antibody has at most one antigen binding domain that binds to CD3 (ie, the antibody provides monovalent binding to CD3).

b) Second (and third) antigen-binding domain

In certain aspects, a (multispecific) antibody of the invention contains at least one antigen binding domain, in particular a Fab molecule, that 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 (ie, 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 specific 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 specific type of tumor cell that expresses the second antigen.

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

In certain aspects, the (multispecific) antibody contains two antigen binding domains, in particular two Fab molecules that 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 (eg, a standard or crossover Fab molecule) and contain the same amino acid sequences containing the same amino acid substitutions in the CH1 and CL domains as described herein (if any). In one aspect, the (multispecific) antibody contains no more than two antigen binding domains, in particular two Fab molecules that bind to a second antigen.

In preferred aspects, the antigen binding domain(s) that binds the second antigen is a standard Fab molecule. In such aspects, the antigen binding domain(s) that binds CD3 is a Fab crossover molecule described herein, i.e. a Fab molecule in which the VH and VL variable domains or the CH1 and CL constant domains of the Fab heavy and light chains are replaced/replaced with each other.

In alternative aspects, the antigen binding domain(s) that binds the second antigen is a Fab crossover molecule described herein, i.e. a Fab molecule in which the VH and VL variable domains or the CH1 and CL constant domains of the Fab heavy and light chains are replaced/replaced with each other. In such aspects, the antigen binding domain(s) that binds CD3 is a standard Fab molecule.

In one aspect, the second (and, if present, third) antigen binding domain comprises a human constant region. In one aspect, the second (and, if present, third) antigen binding domain is a Fab molecule containing a human constant region, in particular a human CH1 and/or CL domain. Representative sequences of human constant domains are shown in SEQ ID NO: 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, 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 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 contain amino acid mutations described herein as “charge modifications”, and/or may contain 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, 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 the CH1 domain sequence contained in the amino acid sequence SEQ ID NO: 122. In particular, the heavy chain constant region (in particular the CH1 domain) may contain 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, 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 with SEQ ID NO: 17, and a light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 19, LCDR 2 with SEQ ID NO: 20 and LCDR 3 with SEQ ID NO : 21.

In one aspect, the second (and, if present, third) antigen binding domain is (or is derived from) a humanized antibody. In one aspect, the second (and, if present, third) antigen binding domain is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, VH and/or VL of the second (and, if present, 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, for example, 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 VH contains 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, VH contains a sequence that is at least about 95% identical amino acid sequence of SEQ ID NO: 18. In one aspect, 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 that is at least 95%, 96%, 97 %, 98%, or 99% identity, contains substitutions (eg, conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody containing the sequence retains the ability to bind TYRP-1. In certain aspects, a substitution, insertion, and/or deletion of a total of 1-10 amino acids has been made in the amino acid sequence of SEQ ID NO: 18. In certain aspects, the substitutions, insertions, or deletions are in regions outside the CDRs (i.e., in the FR) . 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 to 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 VL contains 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, VL contains a sequence that is at least about 95% identical to amino acid sequence of SEQ ID NO: 22. In one aspect, 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 that is at least 95%, 96%, 97 %, 98%, or 99% identity, contains substitutions (eg, conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody containing the sequence retains the ability to bind TYRP-1. In certain aspects, a substitution, insertion, and/or deletion has been made of a total of 1-10 amino acids 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 FR) . In one aspect, VL comprises the amino acid sequence of SEQ ID NO: 22. Optionally, VL comprises the amino acid sequence of SEQ ID NO: 22, including post-translational modifications to that sequence.

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

In one aspect, the VH of the second (and, if present, third) antigen binding domain comprises VH heavy chain CDR sequences of SEQ ID NO: 18 and a framework region with at least 95%, 96%, 97%, 98%, or 99% identity sequences with 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 with the VH framework sequence of SEQ ID NO: 18. In another aspect, VH comprises 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, a third) antigen binding domain comprises VL light chain CDR sequences of SEQ ID NO: 22 and a framework region with at least 95%, 96%, 97%, 98%, or 99% identity sequences with the VL framework sequence of SEQ ID NO: 22. In one aspect, VL comprises VL light chain CDR sequences of SEQ ID NO: 22 and a framework region with at least 95% sequence identity with the VL framework sequence of SEQ ID NO: 22. In another aspect, VL comprises 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, 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 with SEQ ID NO: 87, and a light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 89, LCDR 2 with SEQ ID NO: 90 and LCDR 3 with SEQ ID NO : 91.

In one aspect, the second (and, if present, third) antigen binding domain is (or is derived from) a humanized antibody. In one aspect, the second (and, if present, third) antigen binding domain is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, VH and/or VL of the second (and, if present, 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, for example, 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 VH contains 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, VH contains a sequence that is at least about 95% identical to amino acid sequence of SEQ ID NO: 88. In one aspect, 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 that is at least 95%, 96%, 97 %, 98% or 99% identity, contains substitutions (eg, conservative substitutions), insertions or deletions compared to the reference sequence, but the antibody containing this sequence retains the ability to bind EGFRvIII. In certain aspects, a substitution, insertion, and/or deletion of a total of 1 to 10 amino acids has been made in the amino acid sequence of SEQ ID NO: 88. In certain aspects, the substitutions, insertions, or deletions are in regions outside the CDRs (ie, in the FR). 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 VL contains 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, VL contains a sequence that is at least about 95% identical to amino acid sequence of SEQ ID NO: 92. In one aspect, 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 that is at least 95%, 96%, 97 %, 98% or 99% identity, contains substitutions (eg, conservative substitutions), insertions or deletions compared to the reference sequence, but the antibody containing this sequence retains the ability to bind EGFRvIII. In certain aspects, a substitution, insertion, and/or deletion has been made of a total of 1 to 10 amino acids in the amino acid sequence of SEQ ID NO: 92. In certain aspects, the substitutions, insertions, or deletions are in regions outside the CDRs (ie, in the FR). In one aspect, VL comprises the amino acid sequence of SEQ ID NO: 92. Optionally, 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, 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, third) antigen binding domain contains 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 contains the amino acid sequence of SEQ ID NO: 88, and VL contains 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 a VH sequence of SEQ ID NO: 88 and a 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 HCDR1, HCDR2 and HCDR3 VH amino acid sequences of SEQ ID NO: 88 and the LCDR1, LCDR2 and LCDR3 VL amino acid sequences of SEQ ID NO: 92.

In one aspect, the VH of the second (and, if present, third) antigen binding domain comprises VH heavy chain CDR sequences of SEQ ID NO: 88 and a framework region with at least 95%, 96%, 97%, 98%, or 99% identity sequences with 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 with the VH framework sequence of SEQ ID NO: 88. In another aspect, VH comprises 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 VL light chain CDR sequences of SEQ ID NO: 92 and a framework region with at least 95%, 96%, 97%, 98%, or 99% identity sequences with the VL framework sequence of SEQ ID NO: 92. In one aspect, the VL comprises VL light chain CDR sequences of SEQ ID NO: 92 and a framework region with at least 95% sequence identity with the VL framework sequence of SEQ ID NO: 92. In another aspect, VL comprises 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, third) antigen binding domain comprises a VH sequence as defined above in this section in connection with EGFRvIII, and a VL sequence as defined in any one of the aspects given above in this section in connection with EGFRvIII , but based on the following sequences (ordered by strings) instead of SEQ ID NOs 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 as defined in any one of the aspects given above in this section, and a VL sequence as defined in any one of the aspects given above in this section.

Anti-TYRP-1 and anti-EGFRvIII antibodies

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

The invention also provides an antibody that binds to EGFRvIII, comprising a VH sequence as defined above in connection with EGFRvIII and a VL sequence as defined above in connection with EGFRvIII (e.g., an antibody , which binds to EGFRvIII, containing a heavy chain variable region (VH), containing 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 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 to TYRP-1, containing VH containing the sequence SEQ ID NO: 88, and VL containing the sequence SEQ ID NO: 92).

In a further aspect, antibodies that bind to TYRP-1 or EGFRvIII, in accordance with any of the above aspects, may have other characteristics, alone or in combination, described in connection with an antibody that binds CD3 (unless they are clearly anti- CD3 antibody, e.g. binding sequences).

c) Charge modifications

The (multispecific) antibody of the invention may contain amino acid substitutions in the Fab molecules contained therein, which are particularly effective in reducing the mispairing of light chains with mismatched heavy chains (Bence-Jones type by-products) that can occur during the production of multispecific antibodies based on Fabs with a VH/VL exchange in one (or more in the case of molecules containing more than two antigen-binding Fab molecules) of their binding arms (also see PCT Publication No. WO 2015/150447, incorporated herein by reference in its entirety, in particular examples in it). The ratio of desired (multispecific) antibody versus unwanted byproducts, particularly Bence-Jones-type byproducts arising 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 into certain amino acid positions in the CH1 and CL domains (sometimes referred to herein as “charge modifications”).

Accordingly, in some aspects in which 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 (particularly the first antigen binding domain) variable the VL and VH domains of the Fab light chain and the Fab heavy chain are replaced by 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 replaced by a positively charged amino acid (numbering in accordance with Kabat), and at the same time in the CH1 constant domain of the second (and, if present, third ) of the antigen binding domain, the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (numbering according to the Kabat EU index); or

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

The (multispecific) antibody does not contain both modifications specified 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 (ie, 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 replaced by 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 or the amino acid at position 213 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index); or

ii) in the CL constant domain of the first antigen binding domain, the amino acid at position 124 is independently replaced by 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) (Kabat EU numbering).

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 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 replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In an additional 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 constant In the CH1 domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In a preferred 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 the amino acid at position 123 is independently substituted by 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 by glutamic acid (E) or aspartic acid (D) (Kabat EU numbering) and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU numbering).

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 replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by lysine (K) (numbering according to from Kabat), and in the CH1 constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 147 is replaced by glutamic acid (E) (numbering in accordance with the EU index according to Kabat) and the amino acid at position 213 is replaced by glutamic acid ( E) (numbering in accordance with 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 replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is replaced by arginine (R) (numbering according to 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 replaced by glutamic acid (E) (numbering in accordance with the EU index according to Kabat) and the amino acid at position 213 is replaced by glutamic acid (E) (numbering in accordance with the Kabat EU index).

In preferred aspects, if amino acid substitutions in accordance with 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 to the kappa isotype.

Alternatively, amino acid substitutions in accordance with 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, a 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) (Kabat EU index numbering).

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 independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

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 replaced by 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) (Kabat EU numbering).

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

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

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

(a) a first antigen binding domain that binds CD3, wherein the first antigen binding domain is a Fab molecule in which the VL and VH variable domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region (VH) containing 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) containing the 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, and

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

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

d) Multispecific antibody formats

The (multispecific) antibody of the present invention may 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, first, second, third, etc. the antigen binding domain may be referred to herein as 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 through 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 wherein (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 heavy chain The Fab 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 through a peptide linker.

A (multispecific) antibody with a single 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 Figure 1A, D, G, H , L, M), is applicable in particular in cases where, after binding to a high-affinity antigen-binding domain, internalization of a second antigen can be expected. In such cases, the presence of more than one antigen-binding domain specific for a second antigen may enhance the internalization of the second antigen, thereby reducing its availability.

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

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

In one aspect, the third antigen binding domain binds to a second antigen, for example, 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 contain the same heavy and light chain amino acid sequences and have the same domain arrangement (ie, standard or crossover). Additionally, in these aspects, the third antigen binding domain contains 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, these amino acid substitutions can 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. It is also contemplated that 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 VH and VL variable domains or the CL and CH1 constant domains of the Fab heavy and light chains are replaced/replaced 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 first and second subunits. 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 third) antigen binding domain can be fused to each other and to the Fc domain in various ways. The components can be fused to each other directly or, preferably, through one or more suitable peptide linkers. If a Fab molecule is fused to the N-terminus of an Fc domain subunit, typically the fusion occurs through the immunoglobulin hinge region.

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 a first or second subunit of the Fc domain. In such aspects, the second 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 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/replaced with 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 a Fab heavy chain to the N terminus of a first or second Fc domain subunit, and the second antigen binding domain is fused at the C terminus of a Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding domain. In a particular aspect, the (multispecific) antibody consists primarily of first and second Fab molecules, an Fc domain consisting of 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 shown schematically in FIG. 1G and 1L (in these examples, the first antigen binding domain is a VH/VL crossover molecule Fab). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can 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 primarily of first and second Fab molecules, an Fc domain consisting of first and second subunits, and optionally one or more peptide linkers, each of the first and second Fab molecules being fused at the C-terminus heavy chain Fab with the N-terminus of one of the subunits of the Fc domain. Such a configuration is shown schematically in FIG. 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 through a peptide linker. In a preferred aspect, each of the first and second Fab molecules is fused to an Fc domain via an immunoglobulin hinge region. In a particular aspect, the immunoglobulin hinge region is a human IgG ₁ hinge region, particularly when the Fc domain is a human IgG ₁ Fc domain.

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 a first or second subunit of the Fc domain. 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 VH and VL variable domains or the CL and CH1 constant domains of the Fab heavy and light chains are replaced/replaced with each other. In other such aspects, said 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 second antigen binding domain is fused at the C terminus of a Fab heavy chain to the N terminus of a first or second Fc domain subunit, and the first antigen binding domain is fused at the C terminus of a Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen-binding domain. In a particular aspect, the (multispecific) antibody consists primarily of first and second Fab molecules, an Fc domain consisting of 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 a second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of a first or second subunit of the Fc domain. Such a configuration is shown schematically 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 can be further fused to each other.

In some aspects, a third antigen binding domain, in particular a third Fab molecule, is fused at the C terminus of the Fab heavy chain to the N terminus of a first or second subunit of the Fc domain. In such 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 VH and VL variable domains or the CL and CH1 constant domains of the Fab heavy and light chains are replaced/replaced 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 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 first Fab heavy chain Fab molecules. In a particular aspect, the (multispecific) antibody consists primarily of first, second and third Fab molecules, an Fc domain consisting of first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the heavy chain Fab with 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 with 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 with the N-terminus of the second subunits of the Fc domain. Such a configuration is shown schematically in FIG. 1B and 1D (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 FIG. 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 through a peptide linker. In a preferred aspect, each of the first and third Fab molecules is fused to an Fc domain via an immunoglobulin hinge region. In a particular aspect, the immunoglobulin hinge region is a human IgG ₁ hinge region, particularly 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 can 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 a 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 a Fab heavy chain to the N terminus of a second Fab heavy chain antigen binding domain. In a particular aspect, the (multispecific) antibody consists primarily of first, second and third Fab molecules, an Fc domain consisting of first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the heavy chain Fab with 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 with 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 with the N-terminus of the second subunits of the Fc domain. Such a configuration is shown schematically in FIG. 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 FIG. 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 can be fused to the Fc domain directly or through a peptide linker. In a preferred aspect, each of the second and third Fab molecules is fused to an Fc domain via an immunoglobulin hinge region. In a particular aspect, the immunoglobulin hinge region is a human IgG ₁ hinge region, particularly 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 can be further fused to each other.

In (multispecific) antibody configurations in which a Fab molecule is fused at the C-terminus of the 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 predominantly form an immunoglobulin molecule. In a preferred aspect, the immunoglobulin molecule is an IgG immunoglobulin. 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 IgG4 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 light chain Fab of the first Fab molecule. Fusion of the Fab light chains of the first and second Fab molecules further reduces the mispairing of mismatched Fab heavy and light chains and also reduces the number of plasmids required for the expression of some of the (multispecific) antibodies of the invention.

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

In certain aspects, a (multispecific) antibody of the invention comprises a polypeptide 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 (i.e., the first Fab molecule contains a crossover heavy chain Fab, 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 the Fc domain subunit (VL ₍₁₎ -CH1 ₍₁₎ -CH2-CH3(-CH4 )), and a polypeptide in which the Fab heavy chain of the second Fab molecule shares a 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 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 Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In certain aspects, the polypeptides are covalently linked, for example, by a disulfide bond.

In certain aspects, a (multispecific) antibody of the invention comprises a polypeptide 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 (i.e., the first Fab molecule contains a crossover heavy chain Fab, 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 the Fc domain subunit (VH ₍₁₎ -CL ₍₁₎ -CH2-CH3(-CH4 )), and a polypeptide in which the Fab heavy chain of the second Fab molecule shares a 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 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 Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In certain aspects, the polypeptides are covalently linked, for example, by a disulfide bond.

In some aspects, the (multispecific) antibody comprises a polypeptide 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 (i.e., the first Fab molecule contains 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 the Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a subunit of the Fc domain (VL ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)). In other aspects, the (multispecific) antibody comprises a polypeptide wherein a Fab heavy chain of a second Fab molecule 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 heavy chain constant region. the Fab chain of the first Fab molecule (i.e., the first Fab molecule contains 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 subunit of the Fc domain (VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ -CH2-CH3(-CH4)). In some of these aspects, the (multispecific) antibody further comprises a Fab light chain crossover polypeptide of the 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 the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In other aspects, the (multispecific) antibody further comprises a polypeptide 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, 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 a second Fab molecule shares a carboxy-terminal peptide bond with a variable the Fab heavy chain 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₎ ). depending on the situation. The (multispecific) antibody in accordance with these aspects may further comprise (i) an Fc domain subunit polypeptide (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 the Fc domain subunit (VH ₍₃₎ -CH1 ₍₃₎ -CH2-CH3(-CH4)), and the Fab light chain polypeptide of the third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ). In certain aspects, the polypeptides are covalently linked, for example, by a disulfide bond.

In some aspects, the (multispecific) antibody comprises a polypeptide 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 (i.e., the first Fab molecule contains 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 the Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a subunit of the Fc domain (VH ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)). In other aspects, the (multispecific) antibody comprises a polypeptide wherein a Fab heavy chain of a second Fab molecule 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 light chain constant region. the Fab chain of the first Fab molecule (i.e., the first Fab molecule contains 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 subunit of the Fc domain (VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ -CH2-CH3(-CH4)). In some of these aspects, the (multispecific) antibody further comprises a Fab light chain crossover polypeptide of the 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 the Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ). In other aspects, the (multispecific) antibody further comprises a polypeptide 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, which in turn shares a carboxy-terminal peptide bond with a Fab light chain polypeptide of a second Fab molecule (VL ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CL ₍₂₎ ), or a polypeptide in which the Fab light chain polypeptide of a second Fab molecule shares a carboxy-terminal peptide bond with a variable the Fab heavy chain 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₎ ). depending on the situation. A (multispecific) antibody in accordance with these aspects may further comprise (i) an Fc domain subunit polypeptide (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 the Fc domain subunit (VH ₍₃₎ -CH1 ₍₃₎ -CH2-CH3(-CH4)), and the Fab light chain polypeptide of the third Fab molecule (VL ₍₃₎ -CL ₍₃₎ ). In certain aspects, the polypeptides are covalently linked, for example, by a disulfide bond.

In certain aspects, the (multispecific) antibody does not contain an Fc domain. In such preferred aspects, each of the 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 VH and VL variable domains or the CL and CH1 constant domains of the Fab heavy and light chains are replaced/replaced with each other. In other such aspects, each of the 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 advantageously consists 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 antibody. Fab chains with the N-terminus of the heavy chain of the first antigen-binding domain. Such a configuration is shown schematically in FIG. 1P and 1U (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 advantageously consists 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 heavy Fab chains with the N-terminus of the heavy chain of the second antigen-binding domain. Such a configuration is shown schematically 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, a 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 C -end of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second Fab molecule. In certain such aspects, the (multispecific) antibody consists primarily of 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 shown schematically in FIG. 1C and 1X (in these examples, the first antigen binding domain is a VH/VL crossover Fab molecule and the second and third antigen binding domains are each a standard Fab molecule) and FIG. 1III and 1H (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, a first Fab molecule is fused at the C terminus of a Fab heavy chain to the N terminus of a 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 N -end of the Fab heavy chain with the C-terminus of the Fab heavy chain of the second Fab molecule. In certain such aspects, the (multispecific) antibody consists primarily of 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 shown schematically in FIG. 1T and 1C (in these examples, the first antigen binding domain is a VH/VL crossover Fab molecule and the second and third antigen binding domains are each a standard Fab molecule) and FIG. 14 and 1Sh (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 of the invention comprises a polypeptide wherein a Fab heavy chain of a second Fab molecule 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 the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule contains a crossover Fab heavy chain in which the heavy chain variable region is replaced by the light chain variable region) (VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide 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 Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ).

In certain aspects, a (multispecific) antibody of the invention comprises a polypeptide 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 (i.e., the first Fab molecule contains a crossover a 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 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 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 Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ).

In certain aspects, a (multispecific) antibody of the invention comprises a polypeptide wherein a Fab heavy chain of a second Fab molecule 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 the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule contains a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region) (VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ). In some aspects, the (multispecific) antibody further comprises a polypeptide 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 Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ).

In certain aspects, a (multispecific) antibody of the invention comprises a polypeptide 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 (i.e., the first Fab molecule contains a crossover a 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 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 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 Fab light chain polypeptide of the second Fab molecule (VL ₍₂₎ -CL ₍₂₎ ).

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

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

In one 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 VL and VH variable domains or the CL and CH1 constant domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region (VH) containing the 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) containing the determining region light chain complementarity 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 specifically TYRP-1 or EGFRvIII, the second antigen binding domain being a (standard) Fab molecule;

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

and

(i) the first antigen binding domain of a) is fused at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen binding domain of b), and the second antigen binding domain of b) is fused at the C-terminus of the Fab heavy chain with the N-terminus one of the subunits of the Fc domain in 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) is fused at the C-terminus of the Fab heavy chain to the N-terminus one of the subunits of the Fc domain in 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 VL and VH variable domains or the CL and CH1 constant domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region (VH) containing the 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) containing the determining region light chain complementarity region (LCDR) 1 with SEQ ID NO: 8, LCDR 2 with SEQ ID NO: 9 and LCDR 3 with SEQ ID NO: 10;

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

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

and

(i) the first antigen binding domain of a) is fused at the C-terminus of the Fab heavy chain with 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 to C-terminus of the Fab heavy chain with the N-terminus of one of the Fc-domain subunits in 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 to The C-terminus of the Fab heavy chain with the N-terminus of one of the Fc-domain subunits in c).

In another 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 VL and VH variable domains or the CL and CH1 constant domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region (VH) containing the 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) containing the determining region light chain complementarity 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 specifically TYRP-1 or EGFRvIII, the second antigen binding domain being 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), each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain in c).

In all of the different configurations of a (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 located in the CH1 and CL domains of the second and (if present) third antigen binding domain/Fab molecule. In accordance with 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 those described in Herein, amino acid substitutions 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. Amino acid substitutions are preferably carried out in (multispecific) antibodies containing a Fab molecule, in in which the variable domains VL and VH1 of the Fab light chain and the Fab heavy chain are replaced with each other.

In preferred aspects of the (multispecific) antibody of the invention, particularly in which 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) antigen binding domain/Fab molecule presence, third) of the Fab molecule belongs to the kappa isotype. In other aspects of the (multispecific) antibody of the invention, particularly in which 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, a 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 VL and VH variable domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region (VH) containing a defining heavy chain complementarity 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) containing the 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 specifically TYRP-1 or EGFRvIII, the second antigen binding domain being 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 in b) the amino acid at position 124 is replaced by lysine (K) (numbering in accordance with Kabat) and the amino acid in position 123 is replaced by lysine (K) or arginine (R) (numbering in accordance with Kabat) ( most preferably arginine (R)), and in the CH1 constant domain of the second antigen-binding domain in b) the amino acid at position 147 is replaced by glutamic acid (E) (numbering in accordance with the Kabat EU index) and the amino acid at position 213 is replaced by glutamic acid ( E) (numbering in accordance with the EU index according to Kabat); And

and

(i) the first antigen binding domain of a) is fused at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the second antigen binding domain of b), and the second antigen binding domain of b) is fused at the C-terminus of the Fab heavy chain with the N-terminus one of the subunits of the Fc domain in 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) is fused at the C-terminus of the Fab heavy chain to the N-terminus one of the subunits of the Fc domain in 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 VL and VH variable domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region (VH) containing a defining heavy chain complementarity 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) containing the 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) second and third antigen binding domains that bind to a second antigen, in particular a target cell antigen, more specifically TYRP-1 or EGFRvIII, each of the second and third antigen binding domains being a (standard) Fab molecule; And

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

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

and

(i) the first antigen binding domain of a) is fused at the C-terminus of the Fab heavy chain with 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 to C-terminus of the Fab heavy chain with the N-terminus of one of the Fc-domain subunits in 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 to The C-terminus of the Fab heavy chain with the N-terminus of one of the Fc-domain subunits in 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 VL and VH variable domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region (VH) containing a defining heavy chain complementarity 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) containing the 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 specifically TYRP-1 or EGFRvIII, the second antigen binding domain being 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 in b) the amino acid at position 124 is replaced by lysine (K) (numbering in accordance with Kabat) and the amino acid in position 123 is replaced by lysine (K) or arginine (R) (numbering in accordance with Kabat) ( most preferably arginine (R)), and in the CH1 constant domain of the second antigen-binding domain in b) the amino acid at position 147 is replaced by glutamic acid (E) (numbering in accordance with the Kabat EU index) and the amino acid at position 213 is replaced by glutamic acid ( E) (numbering in accordance with the EU index according to Kabat); And

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

In accordance with any of the above aspects, components of a (multispecific) antibody (e.g. Fab molecules, Fc domain) can be fused directly or through various linkers, in particular peptide linkers containing one or more amino acids, typically about 2-20 amino acids that 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 , as a rule, 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 VL and VH variable domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region (VH) containing a complementarity determining region heavy chain 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) containing 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) 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 contains a heavy chain variable region (VH) containing a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO: 15, HCDR 2 with SEQ ID NO: 16 and HCDR 3 with SEQ ID NO: 17, and a light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 19 , LCDR 2 with SEQ ID NO: 20 and LCDR 3 with SEQ ID NO: 21;

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

and

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

and at the same time additionally

the second antigen-binding domain of b) is fused at the C-terminus of the Fab heavy chain with 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 heavy chain Fab with the N-terminus of one of the subunits of the Fc domain in c).

In a further 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 VL and VH variable domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 11;

b) 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 contains a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18, and a light chain variable region a chain containing the amino acid sequence SEQ ID NO: 22;

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

and

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

and at the same time additionally

the second antigen-binding domain of b) is fused at the C-terminus of the Fab heavy chain with 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 heavy chain Fab with the N-terminus of one of the subunits of the Fc domain in 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 VL and VH variable domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region (VH) containing a complementarity determining region heavy chain 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) containing 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) 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 contains a heavy chain variable region (VH) containing a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 85, HCDR 2 with SEQ ID NO: 86 and HCDR 3 with SEQ ID NO: 87, and a light chain variable region (VL) containing light chain complementarity determining region (LCDR) 1 with SEQ ID NO: 89, LCDR 2 with SEQ ID NO: 90 and LCDR 3 with SEQ ID NO: 91;

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

and

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

and at the same time additionally

the second antigen-binding domain of b) is fused at the C-terminus of the Fab heavy chain with 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 heavy chain Fab with the N-terminus of one of the subunits of the Fc domain in c).

In a further 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 VL and VH variable domains of the Fab light chain and the Fab heavy chain are replaced with each other, and comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 11;

b) 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 contains a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 88, and a light chain variable region, containing the amino acid sequence SEQ ID NO: 92;

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

and

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

and at the same time additionally

the second antigen-binding domain of b) is fused at the C-terminus of the Fab heavy chain with 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 heavy chain Fab with the N-terminus of one of the subunits of the Fc domain in c).

In one aspect, in accordance with these aspects of the invention, in the first Fc domain subunit, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second Fc domain subunit, the tyrosine residue at position 407 is replaced by a valine residue (Y407V) and, optionally, a threonine 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 in accordance with the EU index according to Kabat).

In a further aspect, in accordance with these aspects of the invention, in the first subunit of the Fc domain, further 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) (in particular, the serine residue at position 354 is replaced by a cysteine residue cysteine), and in the second subunit of the Fc domain, an additional tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numbering in accordance with the EU index according to Kabat).

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

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

In a preferred specific 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 is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 24, a polypeptide (especially two polypeptides) containing an amino acid sequence that is at least 95%, 96%, 97% identical , 98% or 99% identical to the sequence of SEQ ID NO: 25, and a polypeptide containing an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 27. In addition In a preferred particular aspect, the (multispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 23, a polypeptide containing the amino acid sequence of SEQ ID NO: 24, a polypeptide (specifically two polypeptides) containing the amino acid sequence of SEQ ID NO: 25, and a polypeptide, containing the amino acid sequence 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 SEQ ID sequence NO: 23, a polypeptide containing 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) containing 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 containing 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 containing the amino acid sequence of SEQ ID NO: 23, a polypeptide containing the amino acid sequence of SEQ ID NO: : 24, a polypeptide (specifically two polypeptides) containing the amino acid sequence of SEQ ID NO: 25, and a polypeptide containing the amino acid sequence of SEQ ID NO: 27.

In a further preferred specific 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: 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 (especially two polypeptides) containing 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 containing an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 27. B 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 (specifically two polypeptides) comprising the amino acid sequence of SEQ ID NO: 111, and a polypeptide , containing 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 containing 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) containing an amino acid sequence that is at least is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 111, and a polypeptide containing an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical 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 containing the amino acid sequence of SEQ ID NO: 110, a polypeptide (specifically two polypeptides) containing the amino acid sequence of SEQ ID NO: 111, and a polypeptide containing 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 first and second subunits.

The Fc domain of a (multispecific) antibody consists of a pair of polypeptide chains containing the heavy chain domains of the immunoglobulin molecule. For example, the Fc domain of the immunoglobulin G (IgG) molecule is a dimer, each subunit of which contains the CH2 and CH3 heavy chain constant domains of IgG. The two subunits of the Fc domain are capable of stable association with each other. In one aspect, the (multispecific) antibody of the invention contains at most 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 ₁ Fc domain. In a more specific aspect, the Fc domain is an IgG ₄ Fc domain containing an amino acid substitution at position S228 (Kabat EU numbering), in particular the amino acid substitution S228P. This amino acid substitution reduces the in vivo turnover of 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. An exemplary human IgG ₁ Fc region sequence is shown in SEQ ID NO: 117.

a) Modifications of the Fc domain promoting heterodimerization

The (multispecific) antibodies of the invention contain different antigen binding domains that can be fused to one or the other of two Fc domain subunits, such that the two Fc domain subunits are typically contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Therefore, to improve the yield and purity of (multispecific) antibodies when produced recombinantly, it would be advisable to introduce a modification into the Fc domain of the (multispecific) antibody to facilitate the association of the necessary polypeptides.

Accordingly, in preferred aspects, the Fc domain of an antibody of the invention comprises a modification that promotes 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 in the CH3 domain of the Fc domain in order to stimulate 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. Typically, in all such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3 domain exchange of the second subunit of the Fc domain are constructed 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 a complementarily designed other CH3 domain (so that heterodimerization of the first and second CH3 domains occurs) domains and there was no formation of homodimers between the first two or two second CH3 domains). These different approaches to improve heavy chain heterodimerization are provided as different alternatives in combination with heavy-light chain modifications (e.g. exchange/replacement of VH and VL in the same binding arm and introducing substitutions of charged amino acids with opposite charges at the CH1/CL interface) in a (multispecific) antibody, which reduces heavy/light chain mispairing and Bence-Jones by-products.

In a specific aspect, said modifications that promote association of the first and second Fc domain subunits are a so-called knob-to-groove modification, including a knob modification in one of the two Fc domain subunits and a notch modification in the other. two subunits of the Fc domain.

The projection-to-recess technology is described, for example, in US 5731168; US 7695936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a bulge ("bump") on the interface of the first polypeptide and a corresponding cavity ("dimp") on the interface of the second polypeptide, such that the bulge can be positioned in the cavity to promote heterodimer formation and inhibit homodimer formation. Bumps are constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (eg tyrosine or tryptophan). Compensating cavities of identical or similar size with 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, an 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 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, in which the bulge in CH3- can be located 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) first subunit of the Fc domain (the knob subunit), the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the (CH3 domain) the second subunit of the Fc domain (the trench subunit) the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one aspect, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced by a serine residue (T366S) and the leucine residue at position 368 is replaced by an alanine residue (L368A) (Kabat EU numbering).

In an additional aspect, 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) (in particular, the serine residue at position 354 is replaced by a cysteine residue), and in the second In the Fc domain subunit, an additional tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numbering in accordance with the EU index according to Kabat). The addition of two cysteine residues results in 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 contains amino acid substitutions S354C and T366W, and the second Fc domain subunit contains amino acid substitutions Y349C, T366S, L368A and Y407V (Kabat EU index numbering).

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

Other CH3 modification techniques for promoting heterodimerization are provided as alternatives in accordance with 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 1870459 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 contact surface of the CH3/CH3 domains between two subunits of the Fc domain. A specific aspect for the (multispecific) antibody of the invention are the amino acid mutations R409D; K370E in one of the two CH3 domains (Fc domain) and amino acid mutation D399K; E357K in the other of the two CH3 domains of the Fc domain (numbering according to the Kabat EU index).

In another aspect, the (multispecific) antibody of the invention contains 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 mutation R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutation 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 amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said (multispecific) antibody contains amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numbering is 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 contains the T366K amino acid mutation and the second CH3 domain contains the L351D amino acid mutation (Kabat EU numbering). In an additional aspect, the first CH3 domain contains an additional amino acid mutation L351K. In a further aspect, the second CH3 domain contains an additional amino acid mutation selected from Y349E, Y349D and L368E (in particular L368E) (Kabat EU index numbering).

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

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 (numbered according to the Kabat EU index).

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

In one aspect, the (multispecific) antibody or Fc domain thereof is of the IgG ₂ subclass, and alternatively the heterodimerization approach described in WO 2010/129304 is used.

In an alternative aspect, the modification that promotes association of the first and second subunits of the Fc domain includes a modification that mediates the effects of electrostatic interaction, for example, as described in PCT publication WO 2009/089004. In general, this method involves replacing one of the amino acid residues at the interface of the Fc domain subunits with a charged amino acid residue so 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), particularly K392D or N392D), and the second CH3 domain comprises an amino acid substitution D399, E356 , D356 or E357 with a positively charged amino acid (for example, 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 of K409 or R409 with a negatively charged amino acid (eg, glutamic acid (E) or aspartic acid (D), particularly 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 Kabat's EU index).

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

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

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

b) Modifications of the Fc domain that reduce Fc receptor binding and/or effector function

The Fc domain imparts favorable pharmacokinetic properties to the (multispecific) antibody, including a long serum half-life that promotes good accumulation in the target tissue and a favorable distribution ratio in tissue and blood. However, at the same time, this 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 can lead to the release of cytokines, which, in combination with the T-cell activating properties and long half-life of the (multispecific) antibody, leads to excessive activation of cytokine receptors 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 an antibody of the invention exhibits reduced Fc receptor binding affinity and/or reduced effector function compared to the native IgG ₁ Fc domain. In one such aspect, an Fc domain (or a (multispecific) antibody comprising said Fc domain) exhibits less than 50%, particularly less than 20%, more specifically less than 10%, and most particularly less than 5% Fc receptor binding affinity compared to native IgG ₁ Fc domain (or (multispecific) antibody containing native IgG ₁ Fc domain), and/or less than 50%, in particular less than 20%, more specifically less than 10% and most particularly less than 5% effector function compared to a native IgG ₁ Fc domain (or a (multispecific) antibody containing a native IgG ₁ Fc domain). In one aspect, the Fc domain (or a (multispecific) antibody containing said Fc domain) substantially does not bind to the Fc receptor and/or does not induce 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 particular aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. In one aspect, the effector function is one or more functions selected from the group 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 IgG ₁ Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or a (multispecific) antibody containing said Fc domain) exhibits greater than about 70%, in particular greater than about 80%, more particularly greater than about 90% binding affinity of the native Fc- IgG ₁ domain (or (multispecific) antibody containing the native IgG ₁ Fc domain) with FcRn.

In certain aspects, the Fc domain is engineered to have reduced Fc receptor binding affinity and/or reduced effector function compared to a non-engineered Fc domain. In preferred aspects, the Fc domain of the (multispecific) antibody contains one or more amino acid mutations that reduce the Fc domain's Fc receptor binding affinity 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 to the Fc receptor. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to the 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 times or even at least 50 times. In one aspect, a (multispecific) antibody containing an engineered Fc domain exhibits less than 20%, in particular less than 10%, more specifically less than 5% Fc receptor binding affinity compared to a (multispecific) antibody containing 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 an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some aspects, complement component binding affinity, particularly Clq binding affinity, is also reduced. In one aspect, binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Almost similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to the specified receptor is achieved when the Fc domain (or (multispecific) antibody containing the specified Fc domain) exhibits more than about 70% binding affinity of the non-engineered form of the Fc domain (or (multispecific) antibody, containing the specified non-designed form of the Fc domain) with FcRn. The Fc domain or (multispecific) antibodies of the invention containing said Fc domain may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain aspects, the Fc domain of a (multispecific) antibody is engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: decreased complement-dependent cytotoxicity (CDC), decreased antibody-dependent cell-mediated cytotoxicity (ADCC), decreased antibody-dependent cellular phagocytosis (ADCP), decreased cytokine secretion, decreased immune complex-mediated antigen uptake antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced forward apoptosis-inducing signaling, reduced cross-linking of target-bound antibodies, reduced maturation of dendritic cells, or reduced T-cell priming cells. In one aspect, the reduced effector function is one or more functions selected from the group of reduced ADCC, reduced ADCC, reduced ADCC, 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 containing a non-engineered Fc domain).

In one aspect, an amino acid mutation that reduces the Fc domain's Fc receptor binding affinity 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 (Kabat EU numbering). In a more specific aspect, the Fc domain contains an amino acid substitution at a position selected from the group of L234, L235 and P329 (Kabat EU numbering). In some aspects, the Fc domain contains amino acid mutations L234A and L235A (Kabat EU numbering). 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 contains an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, in particular P329G (Kabat EU numbering). In one aspect, the Fc domain comprises an amino acid substitution at position P329 and an additional amino acid substitution at position selected from E233, L234, L235, N297, and P331 (Kabat EU numbering). In a more specific aspect, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In preferred aspects, the Fc domain contains amino acid substitutions at positions P329, L234 and L235 (Kabat EU numbering). In more preferred aspects, the Fc domain contains the amino acid mutations L234A, L235A, and P329G (“P329G LALA,” “PGLALA,” or “LALAPG”). In particular, in preferred aspects, each subunit of the Fc domain contains amino acid substitutions L234A, L235A and P329G (numbered according to the Kabat EU index), i.e. in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering in accordance with 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 combination of amino acid substitutions "P329G LALA" almost completely eliminates Fcγ receptor (as well as complement) binding of the Fc domain of human IgG ₁ , 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 their properties, such as Fc receptor binding or effector functions.

IgG ₄ antibodies exhibit reduced binding affinity to 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 an IgG ₄ Fc domain, in particular a human IgG ₄ Fc domain. In one aspect, the Fc domain of IgG ₄ contains an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU numbering). To further reduce Fc receptor binding affinity and/or effector function, in one aspect, the Fc domain of IgG ₄ contains an amino acid substitution at position L235, particularly the amino acid substitution L235E (Kabat EU numbering). In another aspect, the Fc domain of IgG ₄ contains an amino acid substitution at position P329, in particular the amino acid substitution P329G (Kabat EU numbering). In a preferred aspect, the Fc domain of IgG ₄ contains amino acid substitutions at positions S228, L235 and P329, in particular amino acid substitutions S228P, L235E and P329G (numbering according to Kabat's EU index). Such mutant IgG ₄ Fc domains and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012/130831, incorporated herein by reference in its entirety.

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

In certain aspects, N-glycosylation of the Fc domain has been eliminated. In one such aspect, the Fc domain contains an amino acid mutation at position N297, specifically an amino acid substitution substituting alanine (N297A) or aspartic acid (N297D) for asparagine (Kabat EU numbering).

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 substitution of one or more residues of the Fc domain 238, 265, 269, 270, 297, 327 and 329 (US Pat. No. 6,737,056) (Kabat EU numbering). 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 residues 265 and 297 replaced with alanine (US Pat. No. 7,332,581).

Mutant Fc domains can be generated by deletion, substitution, insertion or modification of amino acids using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the DNA coding sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

Binding to Fc receptors can be easily 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 FcγIIIa receptor.

The effector function of an Fc domain or a (multispecific) Fc domain-containing antibody can be determined by methods known in the art. Examples of in vitro assays for assessing the ADCC activity of a molecule of interest are described in US Pat. No. 5,500,362; Hellström et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); US patent No. 5821337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays may be used (see, for example, the ACTI™ Non-Radioactive Flow Cytometry Assay (CellTechnology, Inc. Mountain View, CA); and the CytoTox ^96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WD). Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest 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 Clq, is reduced. Accordingly, in some aspects in which the Fc domain is designed to have reduced effector function, said reduced effector function includes reduced CCC. Clq binding assays can be performed to determine whether an Fc domain or a (multispecific) Fc domain containing antibody is capable of binding Clq, and therefore has CDC activity. See, for example, Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. A CDC assay can be used to assess complement activation (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, for example, 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 an antibody of the invention. Said isolated polynucleotide may be a single polynucleotide or a plurality of polynucleotides.

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

In some aspects, the isolated polynucleotide encodes the 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, for example, in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

B. Recombinant methods

Antibodies of the invention can be produced, for example, by solid-phase peptide synthesis (eg, solid-phase Merrifield synthesis) or recombinant production. For recombinant production, one or more antibody-encoding polynucleotides, such as those 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 easily isolated and sequenced using conventional procedures. In one aspect, there is provided a vector, in particular an expression vector, containing a polynucleotide (among other things, one polynucleotide or multiple polynucleotides) according to the invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequence along with appropriate transcriptional/translational regulatory signals. These methods include in vitro recombinant DNA technologies, synthesis technologies and in vivo recombination/genetic recombination. See, for example, the procedures 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 (ie, a coding region) is cloned in operative association with a promoter and/or other transcriptional or translational regulatory elements. As used herein, a "coding region" is a portion of a nucleic acid that consists of codons translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it can 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 vectors. In addition, any vector may contain one coding region or may contain two or more coding regions, for example, 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, a vector, polynucleotide or nucleic acid of the invention may encode heterologous coding regions, fused or unfused 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. Functional linkage occurs when the coding region of a genetic 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 those regulatory sequences. Two fragments of DNA (such as a polypeptide-coding region and its associated promoter) are "operably linked" if induction of the promoter results in transcription of the 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 expression control sequences to direct expression gene product or does not interfere with the ability to transcribe the 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 that nucleic acid. The promoter may be a cell-specific promoter that causes sufficient DNA transcription only in certain cells. In addition to the promoter, other transcriptional regulatory elements, such as enhancers, operators, repressors and transcription termination signals, can be operably linked to the polynucleotide to control 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., immediate early promoter in combination with intron-A), simian virus 40 (e.g., 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 β-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 (eg, 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 called the CITE sequence). The expression cassette may also include other elements such as an origin of replication and/or chromosomal integration elements such as retroviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

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 control 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 can be placed upstream of a nucleic acid encoding an antibody of the invention or a fragment thereof. According to the signaling hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from mature protein after initiation of export of the growing 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, which is cleaved from the translated polypeptide to form the secreted or “mature” form of the polypeptide. In certain aspects, a native signal peptide is used, 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 thereto. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be replaced by a human tissue plasminogen activator (TPA) or murine β-glucuronidase leader sequence.

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

In an additional aspect, a host cell is provided comprising a polynucleotide (ie, a single polynucleotide or multiple polynucleotides) of the invention. In certain aspects, a host cell containing a vector of the invention is provided. Polynucleotides and vectors may include any of the elements, alone or in combination, described herein in connection with polynucleotides and vectors, respectively. In one such aspect, the host cell contains (eg, has been transformed or transfected with) one or more vectors containing 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 replication and for maintaining antibody expression are well known in the art. Such cells can, if necessary, be transfected or transduced with a specific expression vector, 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, and the like. For example, polypeptides can be produced in bacteria, particularly if glycosylation is not required. Once expressed, 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 the cloning or expression of polypeptide-encoding vectors, including fungal and yeast strains 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 the expression of (glycosylated) polypeptides are also obtained 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, US Patent Nos. 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 for growth in suspension can be used. Other examples of useful mammalian host cell lines include the monkey kidney cell line CV1 transformed with SV40 (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)), neonatal hamster kidney (BHK) cells, mouse Sertoli cells (TM4 cells, described, for example, in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green kidney cells marmoset (VERO-76), human cervical carcinoma cells (HELA), dog kidney cells (MDCK), gray rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), breast tumor cells mouse glands (MMT 060562), TRI cells described, for example, in Mather et al., Annals NY 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, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few, but also cells found in the body of a transgenic animal, transgenic plant, or 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 (eg, a YO, NSO, Sp20 cell). In one aspect, the host cell is not a cell in a human body.

Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide containing either a heavy or a light chain of an antigen binding domain, such as an antibody, can be engineered to also express the other of the antibody chains such 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 of the invention is provided, comprising culturing a host cell containing a polynucleotide encoding an antibody as provided herein under conditions suitable for expression of the antibody, and optionally isolating the antibody from the host cell (or culture medium host cells).

The components of the (multispecific) antibody of the invention may be genetically fused to each other. A (multispecific) antibody can 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 can be determined in accordance with methods well known in the art, and can be tested for effectiveness. Examples of linker sequences between different components of (multispecific) antibodies are proposed 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, etc., and will be apparent to those skilled in the art. In the case of affinity chromatography purification, 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 purify 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, and the like.

D. Methods of analysis

Antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity using a variety of assays known in the art.

1. Binding Assays

Binding (affinity) to the 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 obtained from using recombinant expression. Alternatively, the 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 exemplary and exemplary aspect for determining CD3 binding activity is described below.

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

PPR is performed using a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibody (GE Healthcare, #28958325) was immobilized onto 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% P20 surfactant) was used as running and dilution buffer. Anti-CD3 antibody at a concentration of 2 μg/ml (in 20 mM His, 140 mM NaCl, pH 6.0) was infused over approximately 60 s at a flow rate of 5 μl/min. The CD3 antigen used is a heterodimer of CD3 delta and CD3 epsilon ectodomains fused to a human Fc domain with knob-to-groove modifications and a C-terminal Avi tag (see SEQ ID NOs 28 and 29). CD3 antigen is administered at a concentration of 10 μg/ml over 120 s and dissociation is monitored at a flow rate of 5 μl/min for approximately 120 s. The chip surface is restored by two successive injections of 10 mM glycine, pH 2.1, for approximately 60 s each. The volume refractive index difference is corrected by subtracting the blank injections and by subtracting the response obtained from the flow cell blank control. For evaluation, the binding response is determined 5 seconds after the end of administration. To normalize the binding signal, CD3 binding is divided by the anti-Fab response (the signal (PE) 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 relative active concentration (OAK)) is calculated by comparing the binding activity of an antibody sample after a particular treatment with the binding activity of a corresponding antibody sample after another treatment.

2. Activity tests

The biological activity of the (multispecific) antibodies of the invention can be measured using various assays, as described in the examples. Biological activities may, 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 regression tumors and/or improved survival.

D. Compositions, formulations and routes of administration

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

Additionally provided is a method of producing an antibody of the invention in a form suitable for administration in vivo, comprising (a) preparing 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 administration in vivo .

The pharmaceutical compositions of the present invention contain an effective amount of an antibody dissolved or dispersed in a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable" refers to molecular substances and compositions that are generally non-toxic to recipients at the dosages and concentrations used, i.e. do not cause an adverse, allergic or other undesirable reaction when administered to an animal such as, for example, a human, as appropriate. The method of preparing a pharmaceutical composition that contains an antibody and, optionally, an additional active ingredient will be understood by one skilled 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. In addition, if administered to an animal (e.g., human), it should be understood that the products must meet the standards of sterility, non-pyrogenicity, general safety, and purity as required by the FDA's Division of Biological Standards or appropriate authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, a "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption retarding agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavors, colors, similar materials and combinations thereof, as would be known to one skilled in the art (see eg, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated.

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 required, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The dosage can be administered in any convenient manner, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or continuous.

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 Hank's solution, Ringer's solution or physiological saline buffer. The solution may contain auxiliary agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibodies may be in powder form for mixing with a suitable carrier, such as sterile pyrogen-free water, prior to use. Sterile injection solutions are prepared by adding the antibodies of the invention in the required amount to a suitable diluent with various other ingredients listed below, as necessary. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes. In general, dispersions are prepared by adding various sterilized active ingredients to a sterile vehicle that 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 preparation methods are vacuum drying or freeze drying technologies, which produce a powder of the active ingredient plus any additional required ingredient from a pre-sterile filtered liquid medium. If necessary, the liquid medium should be properly buffered and the liquid vehicle first made isotonic with sufficient saline or glucose before injection. The composition must be stable under the conditions of production and storage and protected from 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, such as 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 octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (containing 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 complexes (eg Zn-protein complexes) and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that 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 that 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.

Active ingredients can also be encapsulated in microcapsules produced, for example, by coacervation technologies or by interfacial polymerization, e.g. hydroxymethylcellulose or gelatin microcapsules and polymethyl methacrylate 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). Slow-release formulations can be obtained. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing a polypeptide, the matrices being in the form of molded articles such as films or microcapsules. In certain aspects, prolonged absorption of the injectable composition can be achieved by the use of absorption delaying agents in the compositions, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

In addition to the previously described compositions, antibodies can also be formulated as depot preparations. Such long-acting formulations can be administered by implantation (eg, subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, antibodies can be formulated with suitable polymeric or hydrophobic materials (eg, as an emulsion in a suitable oil) or ion exchange resins, or as slightly soluble derivatives, eg, as a slightly soluble salt.

Pharmaceutical compositions containing the antibodies of the invention can be prepared through conventional processes of mixing, dissolution, emulsification, encapsulation, entrapment or lyophilization. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients that facilitate the processing of proteins into preparations that can be used for pharmaceutical purposes. The appropriate formulation depends on the route of administration chosen.

Antibodies can be formulated 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 those formed with the free amino groups of the protein composition or formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric or mandelic acid. Salts formed with carboxyl groups can also be obtained from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides; or organic bases such 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 cancer.

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 the practitioner.

In one aspect, the antibodies of the invention are provided for use as a medicine. In additional aspects, the 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 being treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In certain aspects, the method further includes administering to the individual an effective amount of at least one additional therapeutic agent, for example, an anticancer agent if the disease being treated is cancer. In additional 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 of inducing lysis of a target cell, particularly a tumor cell, in an individual, comprising administering to the individual an effective amount of the antibody to induce lysis of the target cell. "Individual" in accordance with 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 formulation of a medicinal product. In one aspect, the medicament is for treating a disease in an individual in need thereof. In a further aspect, the drug is for use in a method of treating a disease, comprising administering to an individual having the disease an effective amount of the drug. In certain aspects, the disease being treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In one aspect, the method further includes administering to the individual an effective amount of at least one additional therapeutic agent, for example, an anticancer agent, if the disease being treated is cancer. In a further aspect, the drug is intended to induce lysis of a target cell, in particular a tumor cell. In a further aspect, the drug is for use in a method of inducing lysis of a target cell, particularly a tumor cell, in an individual, comprising administering to the individual an effective amount of the drug to induce lysis of the target cell. The "individual" in accordance with any of the above aspects may be a mammal, preferably a human.

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

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

In certain aspects, the disease being treated is a proliferative disorder, particularly 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 may be treated with the antibody of the present invention include, but are not limited to, neoplasms located in: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal gland) , parathyroid gland, pituitary gland, testes, ovaries, thymus, thyroid gland), 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 lesions or lesions and cancerous 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, in which the antibody is a multispecific antibody that binds to TYRP-1 as a second antigen, the cancer is a cancer that expresses TYRP-1. In one aspect in particular, in which the antibody is a multispecific antibody that binds to TYRP-1 as a second antigen, the cancer is skin cancer, particularly melanoma. In one aspect in particular, in which the antibody is a multispecific antibody that binds to EGFRvIII as a second antigen, the cancer is a cancer expressing EGFRvIII. In one aspect in particular, in which the antibody is a multispecific antibody that binds to EGFRvIII as a second antigen, the cancer is a brain cancer, particularly glioblastoma. One skilled in the art will appreciate that in many cases an 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 antibody that produces 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 being administered prophylactically or therapeutically, previous or concurrent therapeutic interventions, the patient's medical history and response to the antibody, and the treating physician's judgment. The practitioner responsible for administration will in any case determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for each individual subject. Various dosing regimens are provided herein, including, but not limited to, single or multiple dosing at different times, bolus dosing, and pulse infusion.

The antibody is conveniently administered to the patient once or over a series of courses of treatment. Depending on the type and severity of the disease, the initial candidate dosage for administration to a patient may be from about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg - 10 mg/kg) of antibody, e.g., in one or more individual injections or as a continuous infusion. One typical daily dosage may range from about 1 mcg/kg to 100 mg/kg or more, depending on the factors mentioned above. In case of repeated administrations over several days or more, depending on the condition, treatment is usually carried out until the required degree of suppression of the symptoms of the disease is achieved. One typical antibody dosage ranges from about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, the dose may also range 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 also any range obtained from these values. In non-limiting examples of the range obtained from the values listed herein, a dose may be administered in the range of about 5 mg/kg body weight to about 100 mg/kg body weight, from about 5 micrograms/kg body weight to about 500 milligrams/kg body weight, and etc. taking into account the above numerical values. Thus, the patient may be administered 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). Such doses may be administered at intervals, for example, once a week or every three weeks (eg, 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 may be administered followed by one or more lower doses. At the same time, other dosage schedules can be used. The effectiveness of such therapy can be easily monitored using traditional techniques and analytical methods.

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.

In the case of systemic administration, the effective dose can first be estimated from an in vitro assay, such as a cell culture assay. The dosage 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 applicable to humans.

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

The dosage amount and interval can be individually adjusted to ensure plasma antibody levels sufficient to maintain therapeutic effect. Typical dosages for patients when administered by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses every 24 hours. Plasma levels can be measured, for example, using HPLC.

An effective dose of the antibodies of the invention will generally provide therapeutic benefits without significant toxicity. The toxicity and therapeutic efficacy of an antibody can be determined through 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 that is lethal in 50% of the population) and the ED ₅₀ (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD ₅₀ /ED ₅₀ . Antibodies that demonstrate high therapeutic indices are preferred. In one aspect, the antibody of the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating dosage ranges suitable for human use. The dosage is preferably within a circulating concentration range that includes the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on a number of factors, for example, the dosage form used, the route of administration used, the condition of the subject, and the like. The exact composition, route of administration and dosage can be selected by the personal physician taking into account the patient's condition (see, for example, Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein in its entirety document by reference).

The attending physician of patients being treated with antibodies of the invention must know how and when to stop, interrupt, or adjust administration due to toxicity, organ dysfunction, or the like. Conversely, the treating physician should also know how to adjust treatment to higher levels if clinical response has been insufficient (avoiding toxicity). The amount of dosage administered in treating the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, and the like. The severity of the condition can, for example, be assessed in part using 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 therapy. For example, an antibody of the invention may be co-administered 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 additional therapeutic agent may include any active ingredients suitable for the particular disease being treated, preferably those that have complementary activity and do not adversely 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 sensitizes cells to inducers of apoptosis. In a preferred aspect, the additional therapeutic agent is an anticancer agent, for example, a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.

Such other agents are suitably present in 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 at the same dosages and routes as described herein, or at dosages ranging from about 1% to 99% of the dosages described herein, or at any dosage and route as empirically/clinically determined as suitable.

Such combination therapies noted above include combined administration (where 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 additional therapeutic agent and/or adjuvant. The antibodies of the invention can also be used in combination with radiation therapy.

G. Finished products

In another aspect of the invention, a finished product is provided containing materials intended for the treatment, prevention and/or diagnosis of the above-described disorders. The finished product includes a container and a label or package insert located on or associated with it. Suitable containers include, for example, bottles, vials, syringes, IV bags, etc. Containers can be made from a number of materials, such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective for the treatment, prevention and/or diagnosis of a pathological condition, and may have a sterile inlet (for example, the container may be an intravenous solution bag or a vial with a stopper pierced by a hypodermic injection needle). At least one active agent in the composition is an antibody according to the invention. The label or package insert states that the composition is used to treat the selected pathological condition. In addition, the finished product may comprise (a) a first container containing a composition therein, said composition comprising an antibody of the invention; and (b) a second container containing a composition therein, wherein said composition contains an additional cytotoxic or other therapeutic agent. The finished product in this aspect may further comprise a package insert stating that the compositions can be used to treat a particular disease condition. Alternatively or additionally, the finished product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also contain other materials required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.

3. 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 (eg, 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 an additional aspect, a method is provided for detecting the presence of CD3, TYRP-1, or EGFRvIII in a biological sample. In certain aspects, the method includes contacting a biological sample with an antibody of the present invention under conditions allowing the antibody to bind to CD3, TYRP-1, or EGFRvIII, and detecting whether a complex has 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 to be treated with an antibody that binds CD3, TYRP-1 and/or EGFRvIII, for example, if 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 of the present invention is provided, wherein the antibody is labeled. Labels include, but are not limited to, directly detectable tags or moieties (e.g., fluorescent, chromophore, electron dense, chemiluminescent, and radioactive tags), as well as indirectly detectable moieties, such as enzymes or ligands, such as through an enzymatic reaction or molecular interaction. Typical labels include, but are not limited to, the 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, e.g. , firefly luciferase and bacterial luciferase (US Pat. 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 tags, bacteriophage tags, stable free radicals, etc. .

II. SEQUENCES

IV. EXAMPLES

Below are examples of methods and compositions according to the invention. It should be understood that a number of other aspects may be implemented in practice, taking into account the general description given above.

Example 1 - Generation of an Optimized CD3 Binding Fragment

Starting with the previously described (see, for example, WO 2014/131712, incorporated herein by reference) CD3 binding fragment, referred to herein as the “CD3 _orig ” and containing the VH and VL sequences of SEQ ID NOs 6 and 11, respectively, we set The goal is to optimize the properties of this binding fragment by removing two aspartic deamidation sequence motifs at positions 97 and 100 of the heavy chain Kabat CDR3.

To this end, we generated a phage display-friendly heavy chain antibody library with both asparagines at Kabat positions 97 and 100 deleted and additionally randomized H1, H2, and H3 CDRs to compensate for the loss of affinity caused by Asn97 and Asn100 substitution through the affinity maturation process.

This library was applied to filamentous phage by 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 as Fab fragments (produced by E. coli).

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

The selected clone, referred to herein as “CD3 _opt ” and containing the VH and VL sequences of SEQ ID NOs 7 and 11, respectively, was further evaluated and converted into 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 human IgG1 format with mutations P329G L234A L235A ("PGLALA", EU numbering) in Fc regions (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 examined after temperature stress for 14 days at 37°C or 40°C. Samples that were stored at -80°C were used as reference. Reference samples and samples stressed at 40°C were in 20 mM His, 140 mM NaCl, pH 6.0, and samples stressed at 37°C were in PBS, pH 7.4, all at a concentration of 1. 2-1.3 mg/ml. After the stress period (14 days), the samples were reverse dialyzed in PBS in 20 mM His, 140 mM NaCl, pH 6.0 for additional analysis.

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

SPR was performed on a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibody (GE Healthcare, #28958325) was immobilized onto a CM5 S-series sensor chip (GE Healthcare) using a standard amine conjugation 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% surfactant P20) 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 administered at a concentration of 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 successive injections of 10 mM glycine, pH 2.1, for 60 s each. Volume refractive index differences were corrected by subtracting the blank injections and by subtracting the response obtained from the flow cell blank control. For evaluation, the binding response was determined 5 seconds after the end of administration. To normalize the binding signal, CD3 binding was divided by the anti-Fab response (signal (PE) obtained after capture of anti-CD3 antibody on 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 CD3 delta and CD3 epsilon ectodomains fused to a human Fc domain with knob-to-groove 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 showed 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 removal of the deamidation site was successful and produced an antibody with excellent stability properties related to its in vivo half-life as well as the composition of the antibody at neutral pH.

Binding to CD3 on Jurkat cells

Binding to CD3 on the human T cell reporter line Jurkat NFAT was determined by FACS for the optimized CD3 binding fragment “CD3 _opt ” and the original CD3 binding fragment “CD3 _orig ”, both in human IgG ₁ format with mutations P329G L234A L235A (“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 #CS 176501) 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 NaHCO ₃ , 10% FCS, 25 mM GEPES, 2 mM L-glutamine, 1 x NEAA, 1 x sodium pyruvate at 0.1-0.5 million cells per ml. Each time cells were passaged, a final concentration of 200 μg per ml of hygromycin B was added.

For binding assays, 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 per well were seeded. The plates were centrifuged for 4 min at 400 x g and the supernatant was removed. The test antibodies were diluted in FACS buffer and 20 μl of antibody solution was added to the wells for 30 min at 4°C. To remove unbound antibody, cells were washed twice with FACS buffer before adding diluted secondary antibody (PE-conjugated AffiniPure P(ab')2-fragment goat anti-human IgG Fcg-specific; Jackson Immuno Research #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 P device.

As can be seen 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 the activity of the original CD3 binding fragment "CD3 orig". To examine functional IgG activity, anti-PGLALA-expressing CHO cells were co-incubated with Jurkat NFAT reporter cells in the presence of increasing concentrations of human IgG1 PGLALA anti-CD3 _opt or human IgG ₁ PGLALA anti-CD3 _orig . Activation of CD3 on Jurkat NFAT reporter cells after T cell cross-linking induces luciferase production, and luminescence can be measured as a marker of activation. Human anti-CD3 _orig wt IgG1 was included as a negative control that 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 medium containing 5% FBS+1% GluMax. Jurkat NFAT reporter cells correspond to those described in example 2.

Upon simultaneous binding of CD3 hulgG1 PGLALA to anti-PGLALA expressed on CHO and CD3 expressed on Jurkat-NFAT reporter cells, the NFAT promoter is activated and leads to the expression of active firefly luciferase. The intensity of the luminescence signal (obtained after addition of the 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 NaHCO3, 10% FBS, 25 mM GEPES, 2 mM L-glutamine, 1 x NEAA, 1 x sodium pyruvate at 0. 1 0.5 million cells per ml, 200 μg per ml hygromycin. For analysis, CHO cells were collected and viability determined using ViCell. 30,000 target cells/well were seeded into a flat-bottomed 96-well plate with white walls (Greiner bio-one No. 655098) in 100 μl of medium, and 50 μl/well of diluted antibodies or medium (for controls) was added to the CHO cells. Jurkat-NFAT reporter cells were then harvested and viability 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 obtain a final effector to target (E:M) ratio of 2:1 and a final volume 200 µl per well. Then 4 μl of GloSensor (Promega No. E1291) was added to each well (2% of the final volume). 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 NOs 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 generated by humanizing the TYRP1 binding fragment "TA99" (see GenBank entries AXQ57811 and AXQ57813 for heavy and light chain, respectively) and contained the heavy and light chain variable region sequences set forth in SEQ ID NO 18 and 22, respectively.

A schematic illustration of the TCB 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 was also prepared (SEQ ID NOs 23, 24, 25 and 26).

Bispecific molecules were generated by transiently transfecting HEK293 cells with EBNA. Cells were transfected with the corresponding expression vectors in a ratio of 1:2:1:1 (“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)"). The cells were centrifuged and the medium was replaced with prewarmed CD CHO medium (Thermo Fisher, #10743029). Expression vectors were mixed in CD CHO medium, PEI (polyethylenimine, Polysciences, No. 23966-1) was added, the solution was vortexed and incubated for 10 minutes at room temperature. After this, 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 shaker-incubator with an atmosphere of 5% CO ₂ . After incubation, Excell medium with additives was added (80% of the total volume). One day after transfection, supplements were added (Feed, 12% of the total volume). Cell supernatants were collected after 7 days by centrifugation followed by 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 carried out at pH 3.0, followed by immediate neutralization of the sample pH. Protein was concentrated by centrifugation (Millipore Amicon® ULTRA-15 (#UFC903096)) and aggregated protein was separated from monomeric protein using 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 absorbance at 280 nm using a mass extinction coefficient calculated from the amino acid sequence according to Race et al. (1995), Protein Science 4, 2411-23. Protein purity and molecular weight were analyzed by CE-SDS in the presence or absence of a reducing agent using LabChipGXII (Perkin Elmer) (Table 1). The content of aggregates was determined by HPLC chromatography at 25°C using an analytical size exclusion column (TSKgel G3000 SW XL or _UP -SW3000) equilibrated in a running buffer (25 mM _K2HPO4 , 125 mM NaCl, 200 mM L-monohydrochloride arginine, 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

The binding of TYRP1 TCB to recombinant CD3 was assessed by SPR using TCB 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). 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 reaction kit (GE Healthcare). TSV molecules were captured for 30 s at 5 nM with a flow rate of 10 μL/min.

Human and cynomolgus antigens were carried out 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 restored 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 CD3 delta and CD3 epsilon ectodomains fused to a human Fc domain with knob-to-valve modifications and a C-terminal Avi tag (see SEQ ID NOs 28 and 29) (human CD3 ) and SEQ ID NOs 30 and 31 (cynomolgus CD3)).

Volume refractive index differences were corrected by subtracting the response obtained on the reference flow cell (without captured TSV). Affinity constants were obtained from kinetic rate constants by a 1:1 fit to the Langmuir binding model using BIAeval software (GE Healthcare).

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

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

The binding of TYRP1 TCB to recombinant human CD3 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 carried out 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, TSVs 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 TSVs 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 reaction 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 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 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-to-valve modifications (and PG LALA) and a C-terminal Avi tag (see SEQ ID NO 32 and 35) (human TYRP1), SEQ ID NOs 33 and 35 (cynomolgus TYRP1) or SEQ ID NOs 34 and 35 (mouse TYRP1)).

Volume 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 a 1:1 fit to the Langmuir binding model using BIAeval software (GE Healthcare).

_KD values for binding to human, cynomolgus 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).

The 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. They confirm that binding to human TYRP1 in the case of both TSVs (as well as in the case of the corresponding TYRP1 binding fragment in IgG format) is not affected by stress conditions.

Binding to CD3 on Jurkat cells

Binding to CD3 on the human Jurkat NFAT reporter T cell line was determined by FACS for TYRP1 TSVs 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, TSVs containing the optimized CD3 binding fragment "CD3 _opt " bind to CD3 on Jurkat cells at least comparable well to TSVs 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 Ml50543 (a primary melanoma cell line derived from the Dermatological 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 leads to the expression of active firefly luciferase. The intensity of the luminescence signal (obtained after addition of the luciferase substrate) is proportional to the intensity of CD3 activation and signaling. This assay was performed as described in Example 3 using M150543 instead of anti-PGLALA expressing CHO cells.

As seen for IgG (Example 3), both TCBs containing CD3 _opt or CD3 _orig had similar functional activity on NFAT reporter cells and induced CD3 activation in a concentration-dependent manner (Figure 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 hours and 48 hours. CD4 and CD8 T cell activation was analyzed by upregulation of CD69 and CD25 in both cell subsets at 48 hours.

Briefly, 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 left overnight for attachment. 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 layered in a Histopaque gradient (Sigma no. H8889). After centrifugation (450 x g, 30 minutes, room temperature), the plasma above the PBMC-containing intermediate phase was drained and the PBMCs were transferred into a new Falcon tube, which was then filled with 50 ml of PBS. The mixture was centrifuged (400 x g, 10 minutes, room temperature), the supernatant was discarded, and the pelleted PBMCs were washed twice with sterile PBS (centrifugation steps 350 x g, 10 minutes). The resulting PBMC population was automatically enumerated (ViCell) and stored in RPMI1640 medium containing 10% FBS and 1% L-alanyl-L-glutamine (Biochrom #K0302), at 37°C, 5% CO ₂ in a cell incubator until further use (no longer than 24 hours). For the killing assay, antibodies were added at the indicated concentrations in triplicate. PBMCs were added to target cells at a final effector to target (E:M) ratio of 10:1. Target cell killing was analyzed after 24 hours of incubation at 37°C, 5% CO ₂ by quantifying LDH released into cell supernatants by apoptotic/necrotic cells (LDH Detection Kit, Roche Applied Science #11 644 793 001). Maximum lysis of target cells (=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.

CD8 and CD4 T cell activation following TSV-mediated T cell killing of target cells was analyzed by flow cytometry using antibodies recognizing the T cell activation markers CD25 (late activation marker) CD69 (early activation marker). After 48 h of incubation, PBMCs were transferred to a round-bottomed 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) were performed according to the supplier's instructions. Cells were washed twice with 150 μl/well of FACS buffer and fixed for 15 min at 4°C using 100 μl/well of 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 TSVs containing the optimized or original CD3 binding fragment induced T cell activation and tumor cell lysis in a comparable manner (Figure 11). The EC50 values for tumor cell lysis for all three donors after 48 hours are shown in Table 3.

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

The pharmacokinetics (PK) of TYRP1 TSV with different CD3 binding fragments (CD3 _orig and CD3 _opt ) were studied after intravenous bolus administration at 1 mg/kg in human FcRn transgenic (line 32, homozygous) and FcRn knockout mice (Jackson Laboratory line numbers 003982 and 014565) (n=3/line/test compound). Serial blood microsamples were collected from human FcRn transgenic (tg) mice up to 672 h (9 samples per mouse from 5 min to 672 h postdose) and up to 96 h from FcRn knockout (co) mice (8 samples per mouse from 5 min to 672 h postdose). min to 96 h after dose). Serum was obtained and stored frozen until analysis. Mouse serum samples were analyzed by the general human Ig/Fab CH1/kappa domain specific ECLIA method using the cobas® e411 instrument (Roche) under GLP-free conditions. Pharmacokinetic evaluation was performed using standard non-compartmental analysis.

The results of this study are illustrated in Table 4. They show that the design of the CDR does not result in other 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 - Generation 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 NOs 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 TSV (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 TCB 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 was also prepared (SEQ ID NOs 109, 110, 111 and 26).

Bispecific molecules were created by transiently transfecting 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 IgG ₁ format in a similar manner (transfection of HEK293 EBNA cells with IgG heavy and light chain expression vectors in a 1:1 ratio) for use as described below.

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

Selection of antibodies to EGFRvIII

Antibodies to EGFRvIII were obtained from phage display and allowed for affinity maturation. Antibodies demonstrating 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 EGFRwt binding and nontargeting DP47 IgG as a negative control. All selected antibodies specifically bound to EGFRvIII without cross-reactivity with EGFRwt and were considered for additional study.

At the next stage, the functional activity of these antibodies to EGFRvIII in the form of IgG1 PGLALA (human IgG1 format with mutations P329G L234A L235A (“PGLALA”, EU numbering) in the Fc region) was analyzed on DK-MG cells, which were incubated together 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 IgG1 PGLALA was included as a negative control. All EGFRvIII antibodies tested induced strong activation of CAR-expressing Jurkat NFAT reporter cells (Figure 13). All EGFRvIII antibodies tested, with the exception of P047.019, which showed the weakest binding and activation, were selected for conversion to TCB format (with CD _orig as the CD3 binding fragment).

Binding of selected TCB-converted anti-EGFRvIII antibodies to CHO-EGFRvIII cells was compared with that of the corresponding IgGs (Figure 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 the 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 TCB was then examined in a Jurkat NFAT reporter cell assay on EGFRvIII-positive DK-MG cells (Figure 15). All EGFRvIII TCBs 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 lowest activity. EGFRvIII TSV was then tested in a tumor cell lysis assay with PBMCs that were co-cultured with DK-MG or MKN-45 cells to exclude cross-reactivity of EGFRvIII TSV with EGFRwt (Figure 16). In this assay, in addition to tumor cell lysis, T cell activation (Figure 17) and cytokine release (Figure 18) were measured as additional outcomes. As seen in the previous reporter cell assay, EGFRvIII TCB P056.021 had the greatest activity on EGFRvIII-positive cells without any activity on EGFRwt-positive cells. EGFRvIII TCB P057.011 showed nonspecific activity on EGFRwt cells and was therefore excluded. EGFRvIII TCBs P056.027, P056.052 and P057.012 had comparable activity. Based on these results, the EGFRvIII binding fragments P056.021 and P057.012 were selected for an additional round of affinity maturation.

Good binding fragments could not be obtained from P057.012 (results not shown). The EGFRvIII affinity and specificity of selected EGFRvIII binding fragments derived from P056.021, determined by SPR, are shown in Table 6.

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 to the parent binding fragment in regarding specific binding to EGFRvIII on U87MG-EGFRvIII 5 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 CD3 _orig or CD3 _opt as the CD3 binding fragment.

The functional activity of EGFRvIII TCB P063.056 (with CD3 _opt or 10 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 TSVs induced specific activation of Jurkat NFAT only in the presence of EGFRvIII-positive cells. EGFRvIII TSV P063.056 had slightly greater activity than the parent EGFRvIII TSV 15 P056.021.

Methods

Surface plasmon resonance

The affinity of the anti-EGFRvIII antibody to 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) surface -active substance P20; GE Healthcare) at 25°C. Anti-EGFRvIII PGLALA IgG was captured for 30 s at 25 nM with an antibody that specifically binds human IgG ₁ Fc(PGLALA) (see WO 2017/072210, incorporated herein by reference) immobilized on the CM5 chip. EGFRvIII-ECD avi his antigen (see below, example 9) was carried out at a concentration of 12.4-1000 nM with 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 sample solution to HBS-EP. The chip surface was restored after each cycle using two injections of 10 mM glycine, pH 2.0, over 30 s. Volume refractive index differences were corrected by subtracting the response obtained from the reference flow cell. Affinity constants were obtained from kinetic rate constants by a 1:1 fit to the Langmuir binding model using BIAeval software (GE Healthcare).

To determine specificity, EGFRvIII and EGFRwt ECD antigens were captured using anti-his (Penta His, Qiagen) immobilized on a CM5 chip for 40 s at 100 nM. One injection of anti-EGFRvIII antibody was performed at 500 nM for 60 s before reducing with 10 mM glycine, pH 2.0, for 60 s. Response unit values above 50 for EGFRvIII binding were observed. Responses above 5 response units (RU) were considered positive for EGFRwt binding, and IgG was categorized as specific for responses below 5 RU for EGFRwt.

Cell lines

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS 176501) 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 NaHCO ₃ , 10% FCS, 25 mM GEPES, 1% GlutaMAX, 1 x NEAA, 1 x sodium pyruvate at 0.1-0.5 million cells per ml . Each time cells were passaged, a final concentration of 200 μg per ml of hygromycin B was added.

Jurkat NFAT cells with PGLALA CAR were created 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. During culture, cells were grown in suspension in RPMI1640 supplemented with 10% FCS 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 No. ACC 277) is a human glioblastoma cell line. DK-MG cells were enriched by cell sorting for EGFRvIII expression. Cells were cultured in RPMI 1860, 10% FCS and 1% GlutaMAX.

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

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

Target binding 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 collected, counted, and plated at 100,000 to 200,000 cells per well. The plates were centrifuged for 4 min at 400 x g and the supernatant was removed. The antibodies under study were diluted in FACS buffer and 20 μl of antibody solution was added to the wells for 30 min at 4°C. To remove unbound antibody, cells were washed twice with FACS buffer before adding diluted secondary antibody, PE-conjugated AffiniPure F(ab') ₂ -fragment goat anti-human IgG Fcg-specific (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 a CAR J NFAT reporter cell assay. The principle of this assay is to co-culture engineered Jurkat-NFAT effector cells with cancer cells expressing tumor antigen. Only after simultaneous binding of IgG to CAR due to the PGLALA mutation and the target antigen EGFRvIII, the NFAT promoter is activated and leads to increased luciferase expression in Jurkat effector cells. After adding sufficient substrate, active firefly luciferase results in the emission of luminescence, which can be measured as a CAR-mediated activation signal. Briefly, target cells were collected and viability determined. 30,000 target cells/well were seeded in a flat-bottomed 96-well plate with white walls (Greiner bio-one No. 655098) in 100 μl of medium one day before the start of the assay. The next day, the medium was removed and 25 μl/well of diluted antibodies or medium (for controls) was added to the target cells. Jurkat-NFAT reporter cells were then harvested and viability 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 obtain a final effector to target (E:M) ratio of 2.5:1 and final volume 75 µl per well. Then 4 μl of GloSensor (Promega #E1291) was added to each well (2% of the final volume). Cells were incubated for 24 h at 37°C in a humidified incubator. At the end of the incubation time, the plates were adjusted to room temperature (about 15 min). Then 25 ml/well of One-Glo luciferase (Promega, No. E6120) was added and the plate was incubated for 15 min in the dark before recording luminescence using TEC AN Spark.

Jurkat NFAT reporter cell assay with EGFRvIII TSV

The ability of EGFRvIII TCB with improved CD3 binding 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 TCB to EGFRvIII-positive target cells and CD3 antigen (expressed on Jurkat-NFAT reporter cells), the NFAT promoter is activated and leads to the expression of active firefly luciferase. The intensity of the luminescence signal (obtained after addition of the luciferase substrate) is proportional to the intensity of CD3 activation and signaling. For analysis, target cells were collected and viability determined. 30,000 target cells/well were seeded in a flat-bottomed 96-well plate with white walls (Greiner bio-one No. 655098) in 100 μl of medium, and 50 μl/well of diluted antibodies or medium (for controls) was added to the target cells. Jurkat-NFAT reporter cells were then harvested and viability 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 obtain a final effector to target (E:M) ratio of 2:1 and a final volume 200 µl per well. Then 4 μl of GloSensor (Promega No. E1291) was added to each well (2% of the final volume). Cells were incubated for 24 h at 37°C in a humidified incubator. At the end of the incubation time, luminescence was recorded using 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 left overnight for attachment. 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 layered in a Histopaque gradient (Sigma, no. H8889). After centrifugation (450 x g, 30 minutes, room temperature), the plasma above the PBMC-containing intermediate phase was drained and the PBMCs were transferred into a new Falcon tube, which was then filled with 50 ml of PBS. The mixture was centrifuged (400 x g, 10 minutes, room temperature), the supernatant was discarded, and the pelleted PBMCs were washed twice with sterile PBS (centrifugation steps 350 x g, 10 minutes). The resulting PBMC population was automatically enumerated (ViCell) and stored in RPMI1640 medium containing 10% FBS and 1% GlutaMAX at 37°C, 5% CO ₂ in a cell incubator until further use (no longer than 24 hours). For the killing assay, antibody was added at the indicated concentrations in triplicate. PBMCs were added to target cells at a final effector to target (E:M) ratio of 10:1. Target cell killing was analyzed after 24 hours of incubation at 37°C, 5% CO ₂ by quantifying LDH released into cell supernatants by apoptotic/necrotic cells (LDH Detection Kit, Roche Applied Science, no. 11 644 793 001). Maximum lysis of target cells (=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.

CD8 and CD4 T cell activation following TSV-mediated T cell killing of target cells was analyzed by flow cytometry using antibodies recognizing the T cell activation markers CD25 (late activation marker) CD69 (early activation marker). After 48 h of incubation, PBMCs were transferred to a round-bottomed 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) were performed according to the supplier's instructions. Cells were washed twice with 150 μl/well of FACS buffer and fixed for 15 min at 4°C using 100 μl/well of 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 (IFNγ), Flex Set CBA with human granzyme B, and Flex Set CBA with human TNF. Sample measurements were performed using BD FACS Canto II or BD FACS Fortessa, and analysis was performed using Diva software (BD Biosciences).

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

Binding to recombinant CD3

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

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

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

The binding of EGFRvIII TCB to recombinant human CD3 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 carried out 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, TSVs 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 TSVs 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

The binding of EGFRvIII TCB 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 reaction kit (GE Healthcare). EGFRvIII TSV uptake (5 nM) was performed at a flow rate of 10 μL/min for 30 s. 3-fold serial dilutions of EGFRvIII antigen were performed through 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 restored after each cycle using one injection of 3 M MgCL ₂ 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).

Volume refractive index differences were corrected by subtracting the response obtained on the reference flow cell (without captured TSW). Affinity constants (K _D ) were obtained from kinetic rate constants by a 1:1 fit to the Langmuir binding model using BIAeval software (GE Healthcare). The apparent avidity constant K _D was approximated by kinetic analysis in terms of rate constants using a 1:1 binding approximation 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 .

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

The results of this experiment are illustrated in Fig. 22. They confirm that binding to EGFRvIII in the case of both TSVs is not affected by stress conditions.

Binding to CD3 on Jurkat cells

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

As can be seen in FIG. 23, TCB 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 TSVs containing the EGFRvIII binding fragment P063.056 with CD3 _opt or CD3 _orig , or the EGFRvIII clone P056.021 with CD3 _orig . 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 the 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 a selected EGFRvIII binding fragment (P063.056) and an optimized CD3 binding fragment CD3 _opt or an original CD3 binding fragment CD3 _orig were tested in an assay with Jurkat NFAT reporter cells in the presence of EGFRvIII-positive glioblastoma cells DK-MG, U87MG-huEGFRvIII and EGFRwt-positive MKN45 cells as described above in Example 8.

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

Destruction of target cells

EGFRvIII TSVs containing the selected EGFRvIII binding fragment (P063.056) and an optimized CD3 binding fragment CD3 _opt or the original CD3 binding fragment CD3 _orig were compared with EGFRvIII TSVs containing the parent EGFRvIII binding fragment P056.021 and the CD3 binding fragment CD3 _orig in a killing of tumor cells in the presence of the glioblastoma cell line U87MG-EGFRvIII and PBMCs, as described above in example 8. As seen in Jurkat NFAT reporter cells, the functional activity of TCB with CD3 _opt or CD3 _orig is similar in terms of induction of tumor cell lysis and activation of CD4 and CD8 T cells as measured by CD69 upregulation (Figure 25).

In addition, the functional activity of EGFRvIII TSV 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 TSV with the same EGFRvIII and CD3 binding fragments in “ "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 for both CD3 activation measured at assay with Jurkat NFAT reporter cells (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 EGFRvIII TSV

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

Lysis of tumor cells EGFRvIII TSV

Subsequently, EGFRvIII TCB containing a selected EGFR binding fragment (P063.056) and an optimized CD3 binding fragment CD3 _opt , and EGFRvIII TCB containing the parent EGFRvIII binding fragment P056.021 and a CD3 binding fragment CD3 _orig , were tested in a tumor cell lysis assay with PBMCs. which were 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 secondary outcomes. As can be seen earlier, P063.056 EGFRvIII TCB with CD3 _opt had higher activity than P056.021 EGFRvIII TSV with CD3 _orig in relation to tumor cell lysis, T cell activation and IFNγ and TNFα release.

Activation of T cells and lysis of tumor cells TYRP1 TSV

The functional property of TYRP1 TCB to induce the release of cytokines was studied by co-cultivating the primary melanoma cell line M150543 with PBMCs isolated from a healthy donor. Tumor cell lysis mediated by T cells through TCB TYRP1 was analyzed at 24 hours and 48 hours after treatment (Figure 30). The release of IFNγ and TNFα into the supernatant, as well as the activation of CD4 and CD8 T cells, were analyzed 48 h after treatment. TYRP1 TCB was able to induce active tumor cell lysis within 24 hours. This was accompanied by strong activation of CD4 and CD8 T cells, as measured by CD25 upregulation, as well as significant release of IFNγ and TNFα.

Methods

Isolation of ICPC

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 layered in a Histopaque gradient (Sigma, no. H8889). After centrifugation (450 x g, 30 minutes, room temperature), the plasma above the PBMC-containing intermediate phase was drained and the PBMCs were transferred into a new Falcon tube, which was then filled with 50 ml of PBS. The mixture was centrifuged (400 x g, 10 minutes, room temperature), the supernatant was discarded, and the pelleted PBMCs were washed twice with sterile PBS (centrifugation steps 350 x g, 10 minutes). The resulting PBMC population was automatically enumerated (ViCell) and stored in RPMI1640 medium containing 10% FCS and 1% GlutaMAX at 37°C, 5% CO ₂ in a cell incubator until further use (no longer than 24 hours) or frozen and stored in liquid nitrogen for further use. One day before use, frozen PBMCs were thawed and cultured overnight at 37°C.

T cell proliferation

Briefly, target cells were collected, counted, and washed twice with PBS. Cells were resuspended at 5 million cells per ml in PBS. Cells were stained with cell proliferation assay dye eFluor 670 (eBioscience, #65-0840-85) with a final concentration of 5 μM for 10 min at 37°C. To stop the staining reaction, 4 volumes of complete cell culture medium were added to the cells and incubated for 5 min at 4°C, and then washed three times with medium. Labeled target cells were counted and adjusted to 0.1 million cells per ml in RPMI1640, 10% FCS, and 1% GlutaMax. 10,000 target cells per well were seeded in a 96-well plate. Treatment was 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, no. 563548), CD4 PE (BioLegend, no. 300508), CD8 APC (BioLegend, no. 344722), CD25 PE/Cy7 (BioLegend, No. 302612). Proliferation was determined by dilution of eFluor 670 dye in CD4 T cells and CD8 T cells measured by flow cytometry (FACS Fortessa, BD Bioscience), and activation of CD4 T cells and CD8 T cells 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 left overnight for attachment. For the killing assay, antibodies were added at the indicated concentrations in triplicate. PBMCs were added to target cells at a final effector to target (E:M) ratio of 10:1. Target cell killing was analyzed after 24 hours of incubation at 37°C, 5% CO ₂ by quantifying LDH released into cell supernatants by apoptotic/necrotic cells (LDH Detection Kit, Roche Applied Science, no. 11 644 793 001). Maximum lysis of target cells (=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

CD8 and CD4 T cell activation following TSV-mediated T cell killing of target cells was analyzed by flow cytometry using antibodies recognizing the T cell activation markers CD25 (late activation marker) CD69 (early activation marker). After 48 h of incubation, PBMCs were transferred to a round-bottomed 96-well plate, centrifuged at 350 x g for 5 min, and washed twice with FACS buffer. Surface staining for CD4 APC (BioLegend, no. 300514), CD8 FITC (no. 344704, BioLegend), CD25 BV421 (BioLegend, no. 302630), and CD69 PE (BioLegend, no. 310906) were performed according to the supplier's instructions. Cells were washed twice with 150 μl/well of FACS buffer and fixed for 15 min at 4°C using 100 μl/well of 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

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 (IFNγ) and Flex Set CBA with human TNF. Sample measurements were performed using 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 TCB (containing the optimized CD3 binding fragment identified in Example 1) was examined for its antitumor efficacy in a human tumor cell line xenograft mouse model, the IGR-1 melanoma xenograft model.

IGR-1 (human melanoma) cells were cultured in DMEM containing 10% FBS (Sigma). Cells were cultured at 37°C in a humidified atmosphere at 5% CO ₂ . Passage 6 was used for transplantation. Cell viability was 96.7%. 2x10 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 kept in special pathogen-free conditions with circadian cycles of 12 h day/12 h night in accordance with mandatory guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local authority (ZH223/2017). Continuous monitoring of health status was carried out on an ongoing basis.

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

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

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

U87 (human glioblastoma) cells were initially obtained from ATCC (Manassas, USA) and stably transfected to express human EGFRvIII protein (Roche Glycart AG, Switzerland). After expansion, cells were deposited into the Roche Glycart internal cell bank. The U87-EGFRvIII cell line was cultured in DMEM containing 10% FCS (Sigma) and 0.5 μg/ml puromycin (Invitrogen). Cells were cultured at 37°C in a humidified atmosphere with 5% CO ₂ . Passage 8 was used for transplantation. Cell viability was 94.7%. 5×10 ⁵ 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 kept in special pathogen-free conditions with circadian cycles of 12 h day/12 h night in accordance with mandatory guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local authority (ZH223/2017). Continuous monitoring of health status was carried out on an ongoing basis.

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

In FIG. 32 showed 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 was studied after intravenous bolus administration at 1 mg/kg in human FcRn transgenic mice (line 32, homozygous) and NOD-SCID mice. Serial microblood samples were collected from human FcRn transgenic (tg) mice and NOD-SCID mice up to 672 h (9 samples per mouse from 5 min to 672 h postdose). Mouse serum samples treated with EGFRvIII TCB were analyzed using a specific enzyme-linked immunosorbent assay (ELISA) under GLP-free conditions. EGFRvIII TSV capture was performed with biotinylated EGFRvIII antigen (huEGFRvIII his biotin) on streptavidin-coated microtiter plates (SA-MTP). Detection of EGFRvIII-bound TSV was performed using digoxigenin-labeled anti-human IgG ₁ Fc(PGLALA) monoclonal antibody (see Example 3) followed by the addition of an anti-digoxigenin-POD secondary antibody for detection. The signal was generated by adding peroxidase substrate (ABTS). The calibration range was from 2.35 ng/ml to 150 ng/ml, and 2.5 ng/ml corresponded to the lower limit of quantitative detection (LOQD).

The results of this study are illustrated in Table 7. The PK profile of EGFRvIII TSV is within the expected range for both mouse strains studied. This shows that the construction of a CDR or CD3 binding fragment does not result in other sequence properties that would affect antibody clearance.

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

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LIST OF SEQUENCES

<110> F. HOFFMANN-LA ROCHE AG

<120> ANTIBODIES BINDING TO CD3

<130> P35214

<150>EP18214994.8

<151> 2018-12-21

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<170> Patent In version 3.5

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

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

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

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

15

<210> 21

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> TYRP1 LCDR3

<400> 21

Gln His Phe Trp Ser Leu Pro Phe Thr

15

<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 (groove, 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 barrel - Fc(bump) - 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 Trunk - Fc (Fall) - 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 monkey CD3 epsilon barrel - 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> Barrel CD3 delta - Fc (valley) - 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 ECD TYRP1 - Fc (protrusion) - 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> Cynomolgus monkey TYRP1 ECD - 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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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

15

<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 (protrusion,

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 (groove, 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 (46)

1. An antibody that binds to CD3 and EGFRvIII, 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 with SEQ ID NO: 3 and HCDR 3 with SEQ ID NO: 5, and a light chain variable region (VL) containing 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; and (b) a second and optionally third antigen binding domain that binds EGFRvIII, comprising a VH containing HCDR1 of SEQ ID NO: 85, HCDR 2 of SEQ ID NO: 86, and HCDR 3 of SEQ ID NO: 87, and VL containing LCDR 1 with SEQ ID NO: 89, LCDR 2 with SEQ ID NO: 90 and LCDR 3 with SEQ ID NO: 91. 2. The antibody of claim 1, wherein the VH of the first antigen binding domain contains 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 contains 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, where present, third antigen binding domain contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence of SEQ ID NO: 88, and/or VL of the second and, where present, third antigen binding domain contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence SEQ ID NO: 92. 4. An antibody that binds to CD3 and EGFRvIII, the antibody comprising (a) a first antigen binding domain that binds to CD3 comprising a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 11; and (b) a second and optionally third antigen binding domain that binds EGFRvIII, comprising a VH sequence of SEQ ID NO: 88 and a VL sequence of SEQ ID NO: 92. 5. Antibody according to any one of paragraphs. 1-4, wherein the first antigen binding domain is a Fab molecule. 6. Antibody according to any one of paragraphs. 1-5, containing an Fc domain consisting of the first and second subunits. 7. Antibody according to any one of paragraphs. 1-6, wherein the second and/or, if present, third antigen binding domain is a Fab molecule. 8. Antibody according to any one of paragraphs. 1-7, in which 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 replaced with each other. 9. Antibody according to any one of paragraphs. 1-8, wherein the second and, if present, third antigen binding domain is a standard Fab molecule. 10. Antibody according to any one of paragraphs. 1-9, in which the second and, if present, third antigen-binding domain is a Fab molecule, and in the constant domain CL, the amino acid at position 124 is independently replaced by lysine (K), arginine (R) or histidine (H), numbered according to with 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), numbered according to the Kabat EU index, and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D), numbered according to the Kabat EU index. 11. Antibody according to any one of paragraphs. 1-10, in which the first and second antigen-binding domains are fused to each other, optionally through a peptide linker. 12. Antibody according to any one of paragraphs. 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 the domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second antigen-binding domain. 13. Antibody according to any one of paragraphs. 1-12, in which each of the first, second and, if present, third antigen binding domains is a Fab molecule and the antibody contains an Fc domain consisting of first and second subunits; 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 a 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 Fab 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 Fc domain subunit, and the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit. 15. Antibody according to any one of paragraphs. 6-14, in which the Fc domain is an IgG Fc domain, in particular IgG1. 16. Antibody according to any one of paragraphs. 6-15, in which the Fc domain is a human Fc domain. 17. Antibody according to any one of paragraphs. 6-16, in which the Fc domain is an IgG1 Fc domain. 18. Antibody according to any one of paragraphs. 6-17, in which the Fc domain contains a modification that promotes the association of the first and second subunits of the Fc domain. 19. Antibody according to any one of paragraphs. 6-18, in which in the CH3 domain of the first subunit of the Fc domain, an amino acid residue is replaced by an amino acid residue having a larger side chain volume, thus creating a bulge in the CH3 domain of the first subunit, which 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, which can accommodate a bulge in the CH3 domain of the first subunit. 20. Antibody according to any one of paragraphs. 6-19, in which in the first subunit of the Fc domain, an additional threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, a tyrosine residue at position 407 is replaced by a valine residue (Y407V) and where optional (a) in the second subunit of the Fc domain, the threonine residue at position 366 is additionally replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A), numbering in accordance with the EU index according to Kabat, and/or (b) in the first subunit of the Fc domain, an additional serine residue at position 354 is replaced by a cysteine residue (S354C) or a glutamic acid residue at position 356 is replaced by a cysteine residue (E356C), and in the second subunit of the Fc domain, an additional tyrosine residue at position 349 is replaced by a residue cysteine (Y349C), numbered according to the EU index according to Kabat. 21. Antibody according to any one of paragraphs. 6-20, in which the first subunit of the Fc domain contains amino acid substitutions S354C and T366W, and the second subunit of the Fc domain contains amino acid substitutions Y349C, T366S, L368A and Y407V, numbered in accordance with the Kabat EU index. 22. Antibody according to any one of paragraphs. 6-21, wherein the Fc domain contains one or more amino acid substitutions that reduce Fc receptor binding and/or effector function. 23. Antibody according to any one of paragraphs. 6-22, where the Fc domain contains an amino acid substitution at positions selected from the group E233, L234, L235, N297, P331 and P329, in particular at a position selected from the group L234, L235 and P329, numbering in accordance with the EU index according to Kabat. 24. Antibody according to any one of paragraphs. 6-23, in which each subunit of the Fc domain contains amino acid substitutions L234A, L235A and P329G, numbered according to the Kabat EU index. 25. Antibody according to any one of paragraphs. 1-24, wherein the antibody comprises a polypeptide containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 109, a polypeptide containing an amino acid sequence that is at least is 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 110, a polypeptide, particularly two polypeptides, containing an amino acid sequence that is at least 95%, 96%, 97%, 98% identical or 99% identical to the sequence of SEQ ID NO: 111, and a polypeptide containing 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 of producing an antibody that binds to CD3 and EGFRvIII, comprising the steps of (a) culturing the host cell of claim 27 under conditions suitable for expressing the antibody, and optionally (b) isolating the antibody. 29. A pharmaceutical composition for the treatment of cancer containing an antibody according to any one of paragraphs. 1-25 and a pharmaceutically acceptable carrier. 30. Use of an antibody according to any one of paragraphs. 1-25 as a drug for the treatment of cancer. 31. Use according to claim 30, wherein the cancer is a cancer expressing EGFRvIII and/or where the cancer is a brain cancer, in particular glioblastoma. 32. Use of the pharmaceutical composition according to claim 29 as a drug for the treatment of cancer. 33. Use according to claim 32, wherein the cancer is a cancer expressing EGFRvIII and/or where the cancer is a brain cancer, in particular glioblastoma. 34. Use of an antibody according to any one of paragraphs. 1-25 for cancer treatment. 35. Use according to claim 34, wherein the cancer is a cancer expressing EGFRvIII and/or where the cancer is a brain cancer, in particular glioblastoma. 36. Use of the pharmaceutical composition according to claim 29 for the treatment of cancer. 37. Use according to claim 36, wherein the cancer is a cancer expressing EGFRvIII and/or where the cancer is a brain cancer, in particular glioblastoma. 38. Use of an antibody according to any one of paragraphs. 1-25 for obtaining a drug for the treatment of cancer. 39. Use according to claim 38, wherein the cancer is a cancer expressing EGFRvIII and/or where the cancer is a brain cancer, in particular glioblastoma. 40. Use of the pharmaceutical composition according to claim 29 for the production of a medicinal product for the treatment of cancer. 41. Use according to claim 40, wherein the cancer is a cancer expressing EGFRvIII and/or where the cancer is a brain cancer, in particular glioblastoma. 42. 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 the pharmaceutical composition according to claim 29. 43. The method of claim 42, wherein the cancer is a cancer expressing EGFRvIII and/or where the cancer is a brain cancer, in particular a glioblastoma.