HK40091593A - Antibodies binding to cd3 and folr1 - Google Patents

Description

Antibodies that bind to CD3 and FolR1

Technical Field

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

Background Technology

In various clinical settings, it is often desirable to selectively destroy individual cells or specific cell types. For example, a primary goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues intact.

One attractive approach to achieving this goal is to induce an immune response against the tumor, enabling immune effector cells, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs), to attack and destroy tumor cells. CTLs are the most effective effector cells of the immune system, but they cannot be activated by the effector mechanisms mediated by the Fc domain of conventional therapeutic antibodies.

In this regard, bispecific antibodies designed with one "arm" binding to surface antigens on target cells and a second "arm" binding to the activated, non-variant component of the T-cell receptor (TCR) complex have attracted attention in recent years. The simultaneous binding of such an antibody to both of its targets forces a transient interaction between the target cell and T cells, thereby activating any cytotoxic T cells and subsequently lysing the target cell. Thus, the immune response is redirected to the target cell and is independent of peptide antigen presentation by the target cell or T-cell specificity, relating to the normal MHC-restricted activation of CTLs. Crucially, in this context, CTLs are activated only when target cells present them with bispecific antibodies, mimicking an immune synapse. Bispecific antibodies that do not require lymphocyte pretreatment or co-stimulation to induce effective lysis of target cells are particularly desirable.

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

The most recent application of CD3 antibodies is in the form of bispecific antibodies, which bind to CD3 on one hand and tumor cell antigens on the other. The simultaneous binding of such antibodies to both of their targets forces a temporary interaction between the target cell and T cells, thereby activating any cytotoxic T cells and subsequently lysing the target cell.

FOLR1 is expressed on epithelial tumor cells from various sources, such as ovarian cancer, lung cancer, breast cancer, renal cancer, colorectal cancer, and endometrial cancer. Several methods for targeting FOLR1 with therapeutic antibodies such as falizumab, antibody-drug conjugates, or adoptive T-cell therapies for tumor imaging have been described (Kandalaft et al., J Transl Med. 2012 Aug 3; 10:157. doi:10.1186/1479-5876-10-157; van Dam et al., Nat Med. 2011 Sep 18; 17(10):1315-9. doi:10.1038/nm.2472; Clifton et al., Hum Vac cin.2011 Feb;7(2):183-90.Epub 2011 Feb 1; Kelemen et al., Int J Cancer.2006 Jul 15;119(2):243-50; Vaitilingam et al. , J Nucl Med.2012 Jul;53(7);Teng et al., 2012 Aug;9(8):901-8.doi:10.1517/17425247.2012.694863.Epub 2012 Jun 5). Several attempts have been made to target folate receptor-positive tumors with constructs targeting folate receptors and CD3 (Kranz et al., Proc Natl Acad Sci U S A. Sep 26, 1995; 92(20):9057–9061; Roy et al., Adv Drug Deliv Rev. 2004 Apr 29; 56(8):1219-31; Huiting Cui et al., Biol Chem Aug 17, 2012; 287(34):28206–28214; Lamers et al., Int. J. Cancer. 60(4):450 (1995); Thompson et al., MAbs. 2009 Jul-Aug; 1(4):348-56. Epub 2009 Jul 19; Mezzanzanca et al., Int. J. Cancer, 41, 609–615 (1988)). However, the methods used to date have many drawbacks. The molecules used to date cannot be produced easily and reliably because they require chemical cross-linking. Similarly, hybrid molecules cannot be produced on a large scale as human proteins and instead require the use of rat, mouse, or other proteins that are highly immunogenic when administered to humans, thus limiting their therapeutic value. Furthermore, many existing molecules retain FcgR binding capacity.

Recently, WO2016/079076 described a bispecific antigen-binding molecule that targets CD3 and FolR1 for T cell activation.

For therapeutic purposes, an important requirement that antibodies must meet is sufficient stability both in vitro (for drug storage) and in vivo (after administration to patients).

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

Given the enormous therapeutic potential of antibodies, especially bispecific antibodies for activating T cells, there is a need for bispecific CD3/FolR1 antibodies with optimized properties.

Summary of the Invention

This invention provides antibodies that bind to CD3, including multispecific (e.g., bispecific) antibodies that are resistant to, for example, asparagine deamidation degradation, and are therefore particularly stable, meeting the requirements for therapeutic purposes. These (multispecific) antibodies further combine good efficacy and manufacturability with low toxicity and favorable pharmacokinetic properties.

As shown herein, the CD3-binding antibodies (including multispecific antibodies) provided by the present invention retain more than about 90% of their binding activity after 2 weeks at pH 7.4 and 37°C, relative to their binding activity to CD3 after 2 weeks at pH 6 and -80°C, as determined by surface plasmon resonance (SPR).

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

In one aspect, a bispecific antibody is provided that binds to CD3 and folate receptor 1 (FolR1), wherein the bispecific antibody comprises

(i) a first antigen-binding domain capable of specifically binding to CD3, the first antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising 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 the light chain variable region comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8, LCDR 2 of SEQ ID NO:9, and LCDR 3 of SEQ ID NO:10; and

(ii) A second antigen-binding domain that can specifically bind to FolR1.

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

In one respect, bispecific antibodies bind to CD3 and FolR1, wherein the bispecific antibody contains

(i) a first antigen-binding domain capable of specifically binding to CD3, the first antigen-binding domain comprising the VH sequence of SEQ ID NO:7 and the VL sequence of SEQ ID NO:11; and

(ii) A second antigen-binding domain that can specifically bind to FolR1.

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

In one respect, bispecific antibodies contain an Fc domain consisting of a first subunit and a second subunit.

In one respect, bispecific antibodies contain a third antigen-binding domain capable of specifically binding to FolR1.

In one respect, the second antigen-binding domain and/or, in the presence of the third antigen-binding domain, are Fab molecules.

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

In one respect, the second antigen-binding domain and, in their presence, the third antigen-binding domain are typical of Fab molecules.

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

In one aspect, the first antigen-binding domain and the second antigen-binding domain are fused together, optionally via a peptide linker.

In one aspect, the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, and (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain.

In one aspect, the first antigen-binding domain, the second antigen-binding domain, and, in the presence, the third antigen-binding domain are each Fab molecules, and the bispecific antibody comprises an Fc domain consisting of a first subunit and a second subunit; wherein (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; and the third antigen-binding domain, in the presence, 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 respect, the Fc domain is IgG, specifically the _IgG1 Fc domain.

In one respect, the Fc domain is the human Fc domain.

In one respect, Fc contains modifications that promote the association of the first and second subunits of the Fc domain.

In one respect, the Fc domain contains one or more amino acid substitutions that reduce binding to the Fc receptor and/or effector function.

In one aspect, the second antigen-binding domain and, in the presence, the third antigen-binding domain comprise VH and VL, wherein the VH comprises HCDR 1 of SEQ ID NO:124, HCDR 2 of SEQ ID NO:125, and HCDR 3 of SEQ ID NO:126; and the VL comprises LCDR 1 of SEQ ID NO:8, LCDR 2 of SEQ ID NO:9, and LCDR 3 of SEQ ID NO:10.

In one aspect, a bispecific antibody as described above is provided, wherein the second antigen-binding domain and the third antigen-binding domain, in the presence thereof, comprise: VH, which comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:123; and/or VL, which comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:11.

In one aspect, a separated polynucleotide is provided that encodes the bispecific antibody of the present invention.

In one aspect, a host cell is provided that contains isolated polynucleotides.

In one aspect, a method for producing a bispecific antibody that binds to CD3 and FolR1 is provided, comprising the steps of: (a) culturing host cells under conditions suitable for expressing the bispecific antibody, and optionally (b) recovering the bispecific antibody.

In one aspect, a bispecific antibody binding to CD3 and FolR1, produced by the method described above, is provided.

In one aspect, a pharmaceutical composition is provided comprising: the bispecific antibody of the present invention, and a pharmaceutical carrier.

In one aspect, the bispecific antibody of the present invention or the pharmaceutical composition of the present invention is provided for use as a medicine.

In one aspect, the present invention provides a bispecific antibody or a pharmaceutical composition thereof for use in the treatment of cancer.

In one aspect, the use of the bispecific antibody or pharmaceutical composition of the present invention in the preparation of a medicament is provided.

In one aspect, the use of the bispecific antibody or pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer is provided.

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

In one respect, the disease is cancer.

Attached Figure Description

Figure 1. Exemplary configurations of the (multispecific) antibody of the present invention. (A,D) Schematic diagram of a “1+1 CrossMab” molecule. (B,E) Schematic diagram of a “2+1 IgG Crossfab” molecule, wherein the order of the Crossfab and Fab components alternates (“inverted”). (C,F) Schematic diagram of a “2+1 IgG Crossfab” molecule. (G,K) Schematic diagram of a “1+1 IgG Crossfab” molecule, wherein the order of the Crossfab and Fab components alternates (“inverted”). (H,L) Schematic diagram of a “1+1 IgG Crossfab” molecule. (H,L) Schematic diagram of a “2+1 IgG Crossfab” molecule having two CrossFabs. (J,N) Schematic diagram of a “2+1 IgG Crossfab” molecule having two CrossFabs, wherein the order of the Crossfab and Fab components alternates (“inverted”). (O,S) Schematic diagram of a “Fab-Crossfab” molecule. (P,T) Schematic diagram of a “Crossfab-Fab” molecule. (Q,U) Schematic diagram of the "(Fab) ₂ -Crossfab" molecule. (R,V) Schematic diagram of the "Crossfab-(Fab) ₂ " molecule. (W,Y) Schematic diagram of the "Fab-(Crossfab) ₂ " molecule. (X,Z) Schematic diagram of the "(Crossfab) ₂ -Fab" molecule. Black dots: Optional modifications in the Fc domain to promote heterodimerization. ++, --: Optional introduction of amino acids with opposite charges in the CH1 and CL domains. Crossfab molecules are described as containing the exchange of VH and VL regions, but can alternatively include the exchange of CH1 and CL domains—in aspects where no charge modification is introduced in the CH1 and CL domains.

Figure 2. Relative binding activity of the original and optimized CD3 binders (CD3 _orig and CD3 _opt ) to recombinant CD3, which was determined by SPR after 14 days of storage at 40°C and pH 6 under stress-free conditions or after 14 days of storage at 37°C and pH 7.4 (IgG form).

Figure 3. Binding of the original and optimized CD3 binding agents (CD3 _orig and CD3 _opt ) to Jurkat NFAT cells, as measured by flow cytometry (IgG form). Antibodies binding to Jurkat NFAT cells were detected using fluorescently labeled anti-human Fc-specific secondary antibodies.

Figure 4. Schematic diagram of CD3 activation assay used in Example 3.

Figure 5. Jurkat NFAT activation (IgG form) of the original and optimized CD3 binding agents (CD3 _orig and CD3 _opt ). Jurkat NFAT reporter cells were co-incubated with CHO cells expressing anti-PGLALA (CHO-PGLALA) in the presence of CD3 _orig or CD3 _opt IgG PGLALA, or with CD3 _opt IgG wt as a negative control. After 24 h, CD3 activation was quantitatively analyzed by measuring luminescence.

Figure 6. Schematic diagram of the T-cell bispecific antibody (TCB) molecules prepared in the example. All tested TCB antibody molecules were produced as charged-modified “2+1 IgG CrossFab, inverted” (VH/VL exchange in the CD3 conjugate, charge modification in the target antigen conjugate, EE = 147E, 213E; RK = 123R, 124K).

Figure 7. Relative binding activity of TYRP1 TCB containing original and optimized CD3 binders (CD3 _orig or CD3 _opt ) to recombinant CD3, which was determined by SPR after 14 days of storage under stress-free conditions at 40°C and pH 6 or after 14 days of storage at 37°C and pH 7.4.

Figure 8. Relative binding activity of TYRP1 TCB with recombinant TYRP1 containing original and optimized CD3 binders (CD3 _orig or CD3 _opt ) or corresponding TYRP1 IgG, which was determined by SPR after 14 days of storage at 40°C and pH 6 under stress-free conditions or after 14 days of storage at 37°C and pH 7.4.

Figure 9. Binding of TYRP1 TCBs containing the original and optimized CD3 binding agents (CD3 _orig or CD3 _opt ) to Jurkat NFAT cells, as measured by flow cytometry. TCB binding to Jurkat NFAT cells was detected using fluorescently labeled anti-human Fc-specific secondary antibody.

Figure 10. Jurkat NFAT activation with TYRP1 TCB containing the original and optimized CD3 binding agents. Jurkat NFAT reporter cells were co-incubated with the melanoma cell line M150543 in the presence of TYRP1 TCB CD3 _orig or TYRP1 TCB CD3 _opt . After 24 h, CD3 activation in the presence of TCB was quantitatively analyzed by measuring luminescence.

Figure 11. Tumor cell killing and T cell activation effects of TYRP1 TCB containing the original and optimized CD3 binding agents. The killing effect of PBMCs from three different healthy donors (AF: donor 1, GL: donor 2, MR: donor 3) on the melanoma cell line M150543 was determined using LDH released after 24 h (A, G, M) and 48 h (B, H, N) after treatment with TYRP1 TCB CD3 _orig and TYRP1 TCB CD3 _opt . In parallel, the downregulation of CD25 (C, E, I, K, O, Q) and CD69 (D, F, J, L, P, R) in CD8 (E, F, K, L, Q, R) and CD4 (C, D, I, J, O, P) T cells within PBMCs was measured by flow cytometry as a marker of T cell activation after 48 h.

Figure 12. Specific binding of EGFRvIII IgG PGLALA. Flow cytometry was used to detect the specific binding of EGFRvIII IgG PGLALA antibodies to EGFRvIII (which showed no cross-reactivity with EGFRwt) on CHO-EGFRvIII (A), EGFRvIII-positive DK-MG (B), and EGFRwt-expressing MKN-45 (C). Cetuximab was used as a positive control for EGFRwt expression.

Figure 13. CAR J activation of EGFRvIII IgG PGLALA. Jurkat NFAT reporter cells expressing anti-PGLALA CAR were co-incubated with DK-MG cells expressing EGFRvIII, and EGFRvIII IgG PGLALA antibody or DP47 IgG PGLALA was used as a negative control. The activation of Jurkat NFAT cells was quantitatively analyzed by measuring luminescence after 22 h.

Figure 14. Binding of EGFRvIII, IgG PGLALA, and the corresponding TCB to EGFRvIII. Flow cytometry was used to measure the specific binding of IgG PGLALA and EGFRvIII-converted TCB to CHO-EGFRvIII (A) and MKN-45 (B) cells.

Figure 15. Jurkat NFAT activation of EGFRvIII TCB. Jurkat NFAT activation was measured and used as a marker of CD3 binding to EGFRvIII TCB in the presence of EGFRvIII-positive DK-MG cells. DP47 TCB was used as a negative control.

Figure 16. Tumor cell lysis effect of EGFRvIII TCB. Specific tumor cell lysis induced by EGFRvIII TCB was measured after culturing 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).

Figure 17. T cell activation by EGFRvIII TCB. T cell activation induced by EGFRvIII TCB was measured after co-culturing with freshly isolated PBMCs and EGFRvIII-positive DK-MG cells (A, B, E, F) or EGFRwt-positive MKN-45 cells (C, D, G, H), using activation markers CD25 (A, C, E, G) or CD69 (B, D, F, H) on CD4 T cells (A-D) or CD8 T cells (E-H).

Figure 18. Cytokine release by EGFRvIII TCB. After co-culturing with freshly isolated PBMCs and EGFRvIII-positive DK-MG cells (A-C) or EGFRwt-positive MKN-45 cells (D-F), the release of IFNγ (A,D), TNFα (B,E), and granzyme B (C,F) induced by EGFRvIII TCB was measured.

Figure 19. Specific binding of affinity-matured EGFRvIII IgG PGLALA. The specific binding of affinity-matured EGFRvIII antibodies and parental EGFRvIII binders to EGFRvIII was compared on U87MG-EGFRvIII cells (A) and the EGFRwt-positive cell line MKN-45 (B).

Figure 20. Jurkat NFAT activation of EGFRvIII TCB. Jurkat NFAT activation was measured as a marker of CD3 binding to EGFRvIII TCB in the presence of EGFRvIII-positive DK-MG cells (A), U87MG-EGFRvIII cells (B), and MKN-45 cells (C). DP47 TCB was used as a negative control.

Figure 21. Relative binding activity of EGFRvIII TCB containing original and optimized CD3 binders (CD3 _orig or CD3 _opt ) to recombinant CD3, which was determined by SPR after 14 days of storage under stress-free conditions at 40°C and pH 6 or after 14 days of storage at 37°C and pH 7.4.

Figure 22. Relative binding activity of EGFRvIII TCB containing original and optimized CD3 binders (CD3 _orig or CD3 _opt ) to recombinant EGFRvIII, which was determined by SPR after 14 days of storage under stress-free conditions at 40°C and pH 6 or after 14 days of storage at 37°C and pH 7.4.

Figure 23. Binding of EGFRvIII TCBs containing the original and optimized CD3 binding agents (CD3 _orig or CD3 _opt ) to Jurkat NFAT cells, the binding being measured by flow cytometry. TCB binding to Jurkat NFAT cells was detected using fluorescently labeled anti-human Fc-specific secondary antibody.

Figure 24. Binding of EGFRvIII TCBs containing P063.056 or P056.021 EGFRvIII binding agents to U87MG-EGFRvIII cells, as measured by flow cytometry. TCBs binding to U87MG-EGFRvIII cells were detected using fluorescently labeled anti-human Fc-specific secondary antibodies.

Figure 25. Tumor cell lysis and T cell activation effects of EGFRvIII TCB. Specific tumor cell lysis (A, B) and T cell activation (C, D) induced by EGFRvIII TCB were measured after culturing with freshly isolated PBMCs and U87MG-EGFRvIII cells for 24 h (A, C) or 48 h (B, D). DP47 TCB was used as a negative control.

Figure 26. Comparison of Jurkat NFAT activation in EGFRvIII TCB 2+1 and 1+1 forms. Jurkat NFAT activation was measured as a marker of CD3 binding to EGFRvIII TCB in 2+1 inverted form and 1+1 head-to-tail form in the presence of EGFRvIII-positive U87MG-EGFRvIII cells.

Figure 27. Comparison of tumor cell lysis and T cell activation by EGFRvIII TCB in 2+1 and 1+1 forms. Specific tumor cell lysis (A, B) and T cell activation (C, D) induced by EGFRvIII TCB in the 2+1 inverted form and the 1+1 head-to-tail form were measured after culturing with freshly isolated PBMCs and U87MG-EGFRvIII cells for 24 h (A, C) or 48 h (B, D).

Figure 28. T cell activation and proliferation effects of EGFRvIII TCB. After co-culturing with U87MG-EGFRvIII and PBMCs isolated from healthy donors, T cell proliferation (A,C) and T cell activation of CD4 T cells (A,B) and CD8 T cells (C,D) induced by EGFRvIII TCB were measured.

Figure 29. Tumor cell lysis, T cell activation, and cytokine release induced by EGFRvIII TCB. After co-culturing with U87MG-EGFRvIII cells and PBMCs, the effects of EGFRvIII TCB on tumor cell lysis (A, B), T cell activation (C, D), and the release of IFNγ and TNFα (E, F) were measured. Tumor cell lysis was measured after 24 h and 48 h of treatment; T cell activation and cytokine release were measured after 48 h.

Figure 30. Tumor cell lysis, T cell activation, and cytokine release induced by TYRP-1 TCB. Tumor cell lysis (A, B), T cell activation (C, D), and IFNγ and TNFα release (E, F) induced by TYRP-1 TCB after co-culturing with patient-derived melanoma cell lines M150543 and PBMCs were measured. Tumor cell lysis was measured at 24 h and 48 h after treatment; T cell activation and cytokine release were measured at 48 h.

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

Figure 32. In vivo efficacy of EGFRvIII TCB. In humanized NSG mice, the U87-hu EGFRvIII human glioblastoma cell line was subcutaneously injected to investigate tumor growth inhibition in a subcutaneous xenograft model of glioblastoma. Significant tumor control was observed in the EGFRvIII TCB group, with all mice achieving complete remission.

Figure 33. Forms of FolR1 TCB molecules. Figure 33A: Classical 2+1 TCB molecule, containing a CD3 Fab fused to the inner FOLR1 Fab via a (G4S)2 linker at VH. Heterodimerization via a "mortar and pestle" technique, with a PGLALA mutation in the Fc. Figure 33B: Inverted 2+1 FOLR1 TCB, containing a CD3 _{opt Fab} within the Fc mortar chain. Figure 33C: Classical 1+1 head-to-tail FOLR1 TCB molecule, containing a CD3 _opt Fab fused to the inner FOLR1 Fab via a (G4S)2 linker at VH. Heterodimerization via a "mortar and pestle" technique, with a PGLALA mutation in the Fc. Figure 33D: Inverted 1+1 head-to-tail FOLR1 TCB, containing a CD3 _{opt Fab} within the Fc mortar chain. Figure 33E: 1+1 IgG-like FOLR1 TCB molecule, containing a CD3 _opt Fab on the Fc mortar chain and a FOLR1 Fab on the Fc mortar chain. The PGLALA mutation in Fc is achieved through heterodimerization using the "mortar and pestle" technique.

Figure 34. Jurkat NFAT activation mediated by FOLR1-TCB (CD3 _opt ). This shows Jurkat NFAT activation mediated by FOLR1-TCB containing CD3 _opt . FOLR1-TCB was incubated with huFOLR1-coated beads and Jurkat NFAT effector cells at 37°C for 5.5 h. Dashed lines represent beads and JurkatT cells without TCB. Each point represents the mean of three technical replicates. Standard deviation is indicated by error bars (n=1).

Figure 35. Tumor cell lysis and T cell activation using FOLR1(pro-)TCB (CD3 _opt ). Dose-dependent tumor cell lysis and T cell activation were analyzed after co-incubation of human PBMCs (effective cells) and FOLR1-positive target cells (Ovcar-3) with FOLR1 TCB for 24 h and 48 h (E:T = 10:1, effector cells were human PBMCs). Induction of tumor cell lysis at 24 h and 48 h (A). T cell activation after 48 h of treatment was measured by quantitative analysis of CD69 in CD4 and CD8 T cells using FACS. CD69-positive CD4 T cells (C) and CD8 T cells (D) are shown in the upper figure. In the lower figure, the median of CD69{PE} for CD4(E) and CD8(F) positive T cells is plotted. Each point represents the mean of three technical replicates. Standard deviation is indicated by error bars.

Detailed Implementation

I. Definition

Unless otherwise defined below, the terminology used herein is generally as it is used in the art.

As used herein, the terms “first,” “second,” or “third” relating to antigen-binding domains, etc., are used for ease of distinction when there are more than one of each type of part. Unless explicitly stated otherwise, the use of these terms is not intended to assign a particular order or orientation to the parts.

The terms "anti-CD3 antibody" and "CD3-binding antibody" refer to antibodies that bind to CD3 with sufficient affinity, making them usable as diagnostic and/or therapeutic agents targeting CD3. In one aspect, anti-CD3 antibodies bind to less than about 10% of unrelated, non-CD3 proteins, as measured, for example by surface plasmon resonance (SPR). In other aspects, CD3-binding antibodies have dissociation constants (K_{_D}) ≤1 μM, ≤500 nM, ≤200 nM, or ≤100 nM. When the K_{_D} of an antibody is 1 μM or less, as measured, for example by SPR, the antibody is said to "specifically bind" to CD3. In some aspects, anti-CD3 antibodies bind to epitopes of CD3 that are conserved in CD3 from different species.

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

An "antibody fragment" is a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies; and multispecific antibodies formed from antibody fragments. For a review of some antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably in this document to refer to antibodies that have a structure substantially similar to that of natural antibodies.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that, apart from possible variant antibodies, the individual antibodies in this population are identical and/or bind to the same epitopes, such as those containing naturally occurring mutations or generated during the production of the monoclonal antibody preparation. These variants are typically present in trace amounts. In contrast to polyclonal antibody preparations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially 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 prepared using a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. Such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

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

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

A “humanized” antibody is a chimeric antibody that comprises amino acid residues from a nonhuman CDR and amino acid residues from a human FR. In some respects, a humanized antibody will substantially contain all of at least one, typically two, variable domains, wherein all or substantially all CDRs correspond to the CDRs of the nonhuman antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Such variable domains are referred to herein as “humanized variable regions.” A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. In some respects, some FR residues in a humanized antibody are replaced by corresponding residues from a nonhuman antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity. The “humanized form” of an antibody (e.g., a nonhuman antibody) refers to an antibody that has undergone humanization.

A "human antibody" is an antibody whose amino acid sequence corresponds to that of antibodies produced by humans or human cells, or to a non-human antibody derived from a complete human antibody library or other human antibody-coding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. In some respects, human antibodies are derived from non-human transgenic mammals, such as mice, rats, or rabbits. In some respects, human antibodies are derived from hybridoma cell lines. Antibodies or antibody fragments isolated from human antibody libraries in this paper are also considered human antibodies or human antibody fragments.

The term "antigen-binding domain" refers to a portion of an antibody that contains a region that binds partially or entirely to and is complementary to an antigen. Antigen-binding domains can be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In a preferred aspect, 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 a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and complementarity-determining regions (CDRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using either the VH or VL domain from the antibody binding to that antigen to screen libraries of complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As used in this paper, the “Kabat numbering” associated with variable region sequences refers to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

As used herein, the amino acid positions of all constant regions and constant domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as “according to Kabat numbering” or “Kabat numbering”. Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647–660) is used for the light chain constant domains CL of the κ and λ isoforms, and the Kabat EU index numbering system (see pp. 661–723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), which is further explained in this paper by referring to it as “according to Kabat EU index numbering” or “Kabat EU index numbering” in this case.

As used herein, the term "hypervariant region" or "HVR" refers to the regions within the variable domains of an antibody that are sequence-highly variable and determine antigen-binding specificity, such as "complementarity-determining regions" ("CDRs"). Generally, an antibody contains six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs in this document include:

(a) Hypervariable rings present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia and Lesk, J.Mol.Biol.196:901-917(1987));

(b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

(c) Antigen contact sites present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) (MacCallum et al., J.Mol.Biol.262:732-745 (1996)).

Unless otherwise specified, the CDR is determined according to the method described by Kabat et al., citing above. Those skilled in the art will understand that the CDR name may also be determined according to the method described by Chothia, citing above, the method described by McCallum, citing above, or any other scientifically accepted naming system.

"Frame" or "FR" refers to the variable domain residues other than the complementarity-determining region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR sequences and FR sequences usually appear in VH (or VL) in the following order: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

Unless otherwise specified, CDR residues and other residues (e.g., FR residues) in the variable domain are referenced herein by Kabat et al., ibid.

For the purposes of this document, a “recipient human frame” is a frame that comprises an amino acid sequence of a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame derived from the human immunoglobulin frame or the human common frame as defined below. A recipient human frame “derived from” the human immunoglobulin frame or the human common frame may contain the same amino acid sequence as said human immunoglobulin frame or human common frame, or it may contain amino acid sequence variations. In some aspects, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some aspects, the VL recipient human frame is sequenceally identical to the VL human immunoglobulin frame sequence or the human common frame sequence.

The “human common framework” is a framework that represents the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences comes from a subgroup of variable domain sequences. Typically, these subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3.

The term "immunoglobulin molecule" in this article refers to a protein with the structure of naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable domain (VH) (also called a variable heavy chain domain or heavy chain variable region) from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions). Similarly, each light chain has a variable domain (VL) (also called a variable light chain domain or light chain variable region) from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain (also called a light chain constant region). The heavy chains of immunoglobulins can be classified into one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subtypes such as _γ1 ( _IgG1 ), _γ2 ( _IgG2 ), _γ3 ( _IgG3 ), _γ4 ( _IgG4 ), _α1 ( _IgA1 ), and _α2 ( _IgA2 ). The light chains of immunoglobulins can be classified into one of two types based on the amino acid sequence of their constant domains: kappa (κ) and lambda (λ). Immunoglobulins are essentially composed of two Fab molecules linked by an immunoglobulin hinge region and an Fc domain.

The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 . The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

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

The term "crossfab" refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., replaced). Specifically, a crossfab molecule comprises a peptide chain consisting of a light chain variable domain VL and a heavy chain constant domain 1CH1 (VL-CH1, N-terminal to C-terminal direction), and a peptide chain consisting of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, N-terminal to C-terminal direction). For clarity, in crossfab molecules where the variable domains of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (cross)fab molecule. Conversely, in crossfab molecules where the constant domains of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (cross)fab molecule.

In contrast, the so-called "conventional" Fab molecule refers to the Fab molecule in its natural form, that is, the heavy chain (VH-CH1, in the direction from the N end to the C end) consisting of a heavy chain with variable and constant structural domains, and the light chain (VL-CL, in the direction from the N end to the C end) consisting of a light chain with variable and constant structural domains.

In some embodiments, the present invention relates to bispecific molecules in which at least two binding moieties have the same light chain and a corresponding remodeled heavy chain, which confers specific binding to the corresponding antigen (e.g., CD3 and FolR1). This so-called "common light chain" principle, i.e., combining two or more binders that share a single light chain but still possess individual specificity, prevents light chain mismatch. Therefore, fewer byproducts are generated during production, which facilitates the homogeneous preparation of bispecific molecules.

The term "Fc domain" or "Fc region" used herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain may comprise the full-length heavy chain, or the antibody may comprise a cleaved variant of the full-length heavy chain. This could be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to Kabat's EU index number). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the amino acid sequence of a heavy chain containing an Fc region (or a subunit of an Fc domain as defined herein) is represented herein as lacking a C-terminal glycine-lysine dipeptide. In one aspect, a heavy chain including an Fc region (subunit) as specified herein is included in an antibody according to the invention, the heavy chain containing an additional C-terminal glycine-lysine dipeptide (G446 and K447, according to Kabat EU index numbers). In another aspect, a heavy chain including an Fc region (subunit) as specified herein is included in an antibody according to the invention, the heavy chain containing an additional C-terminal glycine residue (G446, according to Kabat EU index number). Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, the term "subunit" of the Fc domain refers to one of two polypeptides forming the dimer Fc domain, namely a polypeptide containing the C-terminal constant region of the immunoglobulin heavy chain that is stably self-associating. For example, the subunit of the IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains.

The term "fusion" refers to the connection of components (such as Fab molecules and Fc domain subunits) directly or via one or more peptide linkers through peptide bonds.

The term "multispecific" means that the antibody can specifically bind to at least two different antigenic determinants. A multispecific antibody can be, for example, a bispecific antibody. Typically, a bispecific antibody contains two antigen-binding sites, each of which is specific to a different antigenic determinant. In some respects, the multispecific (e.g., bispecific) antibody can bind to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.

As used herein, the term "valence" indicates the presence of a specified number of antigen-binding sites in an antigen-binding molecule. Therefore, the term "monovalently bound to antigen" indicates the presence of one (and no more than one) antigen-binding site in an antigen-binding molecule that is specific to the antigen.

An "antigen binding site" is a site on an antigen-binding molecule that provides an interaction with an antigen; it is typically one or more amino acid residues. For example, the antigen binding site of an antibody contains amino acid residues from the complementarity-determining region (CDR). Natural immunoglobulin molecules usually have two antigen binding sites, while Fab molecules usually have a single antigen binding site.

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

Unless otherwise specified, “CD3” means any naturally occurring CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term covers “full-length” unprocessed CD3, as well as any form of CD3 produced by cellular processing. The term also covers naturally occurring variants of CD3, such as splice variants or allelic variants. In one aspect, CD3 is human CD3, particularly the ε subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in SEQ ID NO:112 (no signal peptide). See also UniProt (www.uniprot.org) accession number P07766 (version 189), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. In another aspect, CD3 is cynomolgus monkey (Macacafascicularis) CD3, particularly cynomolgus monkey CD3ε. The amino acid sequence of cynomolgus CD3ε is shown in SEQ ID NO:113 (no signal peptide). See also NCBI GenBank number BAB71849.1. In some aspects, the antibody of the present invention binds to epitopes of CD3 (particularly human and cynomolgus CD3) that are conserved in CD3 antigens from different species. In a preferred aspect, 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, such as a cell in a tumor (e.g., a cancer cell or a cell in the tumor stroma) (in this case, “tumor cell antigen”). Preferably, the target cell antigen is not CD3, and/or is expressed on cells different from CD3. In one aspect, the target cell antigen is TYRP-1, particularly human TYRP-1. In another aspect, the target cell antigen is EGFRvIII, particularly human EGFRvIII. In a preferred embodiment, the target antigen is folic acid receptor 1 (FolR1).

“FolR1” stands for folate receptor 1 (synonyms include, but are not limited to, folate receptor α (FRA), folate-binding protein (FBP), MOv18, P15328, FRA1, FRAI), a protein receptor that mediates the renewal of folate and reduced folate derivatives into the cell. The sequence of human FolR1 is shown in SEQ ID NO:137. See also UniProt accession number P15328. Unless otherwise specified, as used herein, “FolR1” means any naturally occurring FolR1 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term covers “full-length” unprocessed FolR1, as well as any form of FolR11 produced by cellular processing. The term also covers naturally occurring variants of FolR1, such as splice variants or allelic variants. In one respect, FolR1 is human FolR1.

“TYRP1” or “TYRP-1” stands for Tyrosine-associated protein 1, an enzyme involved in melanin synthesis. The mature form of TYRP1, originally also known as gp75, is a 75 kDa transmembrane glycoprotein. The sequence of human TYRP1 is shown in SEQ ID NO: 114 (signal peptide-free). See also UniProt accession number P17643 (ed. 185). Unless otherwise stated, “TYRP1” as used herein refers to any naturally occurring TYRP1 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term covers “full-length” unprocessed TYRP1, as well as any form of TYRP1 produced by cellular processing. The term also covers naturally occurring variants of TYRP1, such as splice variants or allelic variants. In one respect, TYRP1 is human TYRP1.

“EGFRvIII” stands for epidermal growth factor receptor variant III, a mutant of EGFR formed by in-frame deletion of exons 2-7, resulting in a 267-amino acid deletion with a glycine substitution at the junction. The sequence of human EGFRvIII is shown in SEQ ID NO:115 (no signal peptide). The sequence of wild-type human EGFR is shown in SEQ ID NO:116 (no signal peptide). See also UniProt accession number P00533 (version 258). Unless otherwise stated, as used herein, “EGFRvIII” means any naturally occurring EGFRvIII from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term covers “full-length” unprocessed EGFRvIII (excluding wild-type EGFR) as well as any form of EGFRvIII produced through cellular processing (e.g., EGFRvIII without a signal peptide). In one respect, EGFRvIII is human EGFRvIII.

“Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K_{_D} ). Affinity can be measured by well-established methods known in the art, including those described herein. A preferred method for measuring affinity is surface plasmon resonance (SPR).

"Affinity-mature" antibodies are those that have one or more alterations in one or more complementarity-determining regions (CDRs) that result in improved affinity of the antibody for the antigen compared to parental antibodies without such alterations.

"Reduced binding," such as reduced binding to the Fc receptor, refers to a decrease in affinity for the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes reducing the affinity to zero (or below the detection limit of the analytical method), i.e., completely eliminating the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the corresponding interaction.

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

"Modification that promotes association between the first and second subunits of the Fc domain" refers to manipulation of the peptide backbone or post-translational modification of the Fc domain subunits that reduces or prevents the association of a polypeptide containing an Fc domain subunit with the same polypeptide to form a homodimer. As used herein, "association-promoting modification" preferably includes individual modification of each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) to which association is desired, wherein said modification is complementary to each other to promote association between the two Fc domain subunits. For example, association-promoting modification may alter the structure or charge of one or both of the Fc domain subunits to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, which may differ with respect to additional components (e.g., antigen-binding domains) fused to each subunit. In some aspects, modifications that promote association between the first and second subunits of the Fc domain include amino acid mutations, specifically amino acid substitutions, in the Fc domain. In a preferred aspect, the modifications that promote association between the first and second subunits of the Fc domain include individual amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

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

"Activated Fc receptors" are Fc receptors that, upon binding to the Fc domain of an antibody, trigger signal transduction events that stimulate receptor-carrying cells to perform effector functions. Human activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells that specifically bind to an antibody or a derivative thereof containing an Fc region, typically via the N-terminal protein portion of the Fc region. As used herein, the term “reduced ADCC” is defined as a reduction in the number of target cells lysed within a given time period at a given antibody concentration in the culture medium surrounding the target cells via the ADCC mechanism defined above, and/or an increase in the antibody concentration necessary to achieve lysis of a given number of target cells within a given time period via the ADCC mechanism in the culture medium surrounding the target cells. Reduced ADCC is ADCC mediated by the same antibody produced from the same type of host cells but not engineered, using the same standard methods of production, purification, formulation, and storage (such methods are known to those skilled in the art). For example, reduced ADCC mediated by an antibody containing an amino acid substitution in the Fc domain that reduces ADCC is relative to ADCC mediated by the same antibody without that amino acid substitution in the Fc domain. Suitable methods 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 “engineering,” “engineered,” and “engineered modification” are considered to include any manipulation of the peptide backbone or post-translational modification of naturally occurring or recombinant peptides or fragments thereof. Engineering modification includes modifications to the amino acid sequence, glycosylation patterns, or side chain groups of individual amino acids, as well as combinations of these methods.

As used herein, the term "amino acid mutation" encompasses amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to obtain a final construct, provided that the final construct possesses the desired characteristics, such as reduced binding to the Fc receptor or increased association with another peptide. Amino acid sequence deletions and insertions include deletions and insertions of the amino-terminus and/or carboxyl-terminus of amino acids. Preferred amino acid mutations are amino acid substitutions. Non-conservative amino acid substitutions, i.e., replacing an amino acid with another amino acid having a different structure and/or chemical properties, are particularly preferred for the purpose of altering, for example, the binding characteristics of the Fc region. Amino acid substitutions include substitution with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for altering amino acid side chain groups by means other than genetic engineering (such as chemical modification) are also considered useful. Various names may be used herein to refer to the same amino acid mutation. For example, replacing proline at position 329 of the Fc domain with glycine can be represented as 329G, G329, G ₃₂₉ , P329G, or Pro329Gly.

The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate the percentage of identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, and the source code has been submitted with the user documentation to the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registry No. TXU510087 and as described in WO 2001/007611.

Unless otherwise specified, for the purposes of this paper, amino acid sequence identity % values were generated using the ggsearch program of FASTA package version 36.3.8c or later with the BLOSUM50 comparison matrix. The FASTA package was created by W.R. Pearson and D.J. Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988) 2444-2448); W.R. Pearson (“Effective protein sequence comparison” Meth. Enzymol. 266 (1996) 227-258); and Pearson et al. (Genomics 46 (1997) 24-36) and is publicly available at www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/fasta. Alternatively, sequences can be compared using a public server accessible at fasta.bioch.virginia.edu/fasta_www2/index.cgi, using the ggsearch (global protein: protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup=2) to ensure a global rather than local alignment. The percentage of amino acid identity is given in the output alignment header.

The terms "polynucleotide" or "nucleic acid molecule" include any compound and/or substance comprising 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 ester group. Typically, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses 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. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of antibodies used in this invention in vitro and/or in vivo (e.g., in a host or patient). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoding molecule, making it possible to inject mRNA into a subject to generate in vivo antibodies (see, for example, Stadler et al., (2017) Nature Medicine 23:815-817, or EP 2 101823 B1).

"Isolated" nucleic acid molecules refer to nucleic acid molecules that have been isolated from components of their natural environment. Isolated nucleic acid molecules include those contained in cells that normally contain said nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

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

As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are operatively 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 in which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and progeny derived from said primary transformed cells, regardless of passage number. Progeny may not be identical to the nucleic acid contents of the parent cells and may contain mutations. This includes mutant progeny with the same function or biological activity as screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to produce the antibodies of the present invention. Host cells include cultured cells, such as cultured mammalian cells, such as, to name just a few, HEK cells, CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In one aspect, the host cells of the present invention are eukaryotic cells, particularly mammalian cells. In one respect, the host cell is not a cell within the human body.

The terms “pharmaceutical composition” or “pharmaceutical formulation” refer to a formulation in which the bioactive ingredient contained therein is in a form in which the composition is bioactively effective and which does not contain any additional components that would have unacceptable toxicity to a subject to whom the composition will be administered.

"Pharmaceutical carrier" refers to a component in a pharmaceutical composition or formulation other than the active ingredient, which is non-toxic to the subject. Pharmaceutical carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, “treatment” (and its grammatical variations) refers to an attempt to alter the natural course of a disease in the treated individual and is a clinical intervention that can be performed for prevention or may be performed during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some aspects, the antibodies of this invention are used to delay the development of disease or slow its progression.

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

The “effective amount” of a pharmaceutical agent (such as a pharmaceutical composition) refers to the amount that is sufficient to effectively achieve the desired therapeutic or preventative outcome at the necessary dose for the necessary period of time.

The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information concerning the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings related to the use of such therapeutic products.

II. Compositions and Methods

This invention provides bispecific antibodies that bind CD3 and FolR1. These antibodies exhibit excellent stability and combine with other advantageous properties for therapeutic applications, such as efficacy and safety, pharmacokinetics, and manufacturability. The antibodies of this invention can be used, for example, to treat diseases such as cancer.

A. Bispecific anti-CD3 and anti-FolR1 antibody

In one aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1. In another aspect, a separate bispecific antibody that binds to CD3 and FolR1 is provided. In yet another aspect, the present invention provides a bispecific antibody that specifically binds to CD3 and FolR1. In some aspects, the bispecific anti-CD3 anti-FolR1 antibody retains more than about 90% of its binding activity to CD3 after 2 weeks at pH 7.4 and 37°C, as determined by surface plasmon resonance (SPR), relative to its binding activity to CD3 after 2 weeks at pH 6 and -80°C.

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

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

In one respect, VH and/or VL contain a receptor human framework, such as a human immunoglobulin framework or a human common framework.

In one aspect, VH comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and/or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO:7. In one aspect, VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:7. In one aspect, VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:7. In one aspect, VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:7. In some aspects, the VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the antibody containing this sequence retains its ability to bind to CD3. In some aspects, a total of 1 to 10 amino acids in the amino acid sequence of SEQ ID NO:7 have been substituted, inserted, and/or deleted. In some respects, substitutions, insertions, or deletions occur in regions outside the CDR (i.e., in the FR). In one respect, VH comprises the amino acid sequence of SEQ ID NO:7. Optionally, VH comprises the amino acid sequence of SEQ ID NO:7, including post-translational modifications of that sequence.

In one aspect, the VL comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and/or FR4 sequences) of the light chain variable region sequence of SEQ ID NO:11. In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:11. In one aspect, the VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:11. In one aspect, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:11. In some aspects, the VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the antibody containing this sequence retains its ability to bind to CD3. In some aspects, a total of 1 to 10 amino acids in the amino acid sequence of SEQ ID NO:11 have been substituted, inserted, and/or deleted. In some respects, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). In one respect, the VL comprises the amino acid sequence of SEQ ID NO:11. Optionally, the VL comprises the amino acid sequence of SEQ ID NO:11, including post-translational modifications of that sequence.

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

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

In another aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1, wherein the antibody comprises a first antigen-binding domain comprising the VH sequence of SEQ ID NO:7 and the VL sequence of SEQ ID NO:11.

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

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

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

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

In one aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1, wherein the antibody comprises a first antigen-binding domain comprising a VH sequence and a VL sequence, the VH sequence being as described in any of the aspects provided above, and the VL sequence being as described in any of the aspects provided above.

In one aspect, the bispecific antibody comprises a human constant region. In another aspect, the bispecific antibody is an immunoglobulin molecule comprising a human constant region, particularly an IgG-type immunoglobulin molecule comprising human CH1, CH2, CH3, and/or CL domains. Exemplary sequences of the human constant domains are given in SEQ ID NO:120 and SEQ ID NO:121 (human κ and λCL domains, respectively) and SEQ ID NO:122 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one aspect, the bispecific 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 bispecific antibody includes a heavy chain constant region containing 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. Specifically, 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 another aspect, the first antigen-binding portion is a Fab molecule comprising a human constant region, particularly a human CH1 and/or CL domain. In another 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, particularly the amino acid sequence of SEQ ID NO:120. Specifically, the light chain constant region may comprise amino acid mutations under “charged modification” as described herein and/or, if in a cross-Fab molecule, may comprise deletions or substitutions of one or more (particularly two) N-terminal amino acids. 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. Specifically, the heavy chain constant region (especially the CH1 domain) can contain amino acid mutations that are under “charged modification” as described in this paper.

In one respect, bispecific antibodies are monoclonal antibodies.

In one respect, bispecific antibodies are IgG, particularly _IgG1 antibodies. In another respect, bispecific antibodies are full-length antibodies.

In another aspect, the first antigen-binding domain and/or the second antigen-binding domain and/or one or more additional antigen-binding domains are one or more antibody fragments selected from the group consisting of: (a) one or more Fv molecules, (a) one or more scFv molecules, (a) one or more Fab molecules, and (a) one or more F(ab') ₂ molecules; particularly (a) one or more Fab molecules. In another aspect, the one or more antibody fragments are (a) one or more bisomatic antibodies, (a) one or more trisomatic antibodies, or (a) one or more tetrasomatic antibodies.

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

On the other hand, the antibody according to any one of the foregoing aspects may be combined, alone or in combination, with the features described in Part II A.1.-8 below.

In a preferred aspect, the antibody comprises an Fc domain, particularly an IgG Fc domain, and more particularly an IgG1 Fc domain. In one aspect, the Fc domain is a human Fc domain. In another aspect, the Fc domain is an _IgG1 Fc domain. The Fc domain is composed of a first subunit and a second subunit and may incorporate, alone or in combination, any of the features described below with respect to Fc domain variants (Section II. A.8.).

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

1. Antibody fragments

In some respects, the antigen-binding domains presented in this article are antibody fragments.

In one respect, antibody fragments are Fab', Fab'-SH, or F(ab') ₂ molecules, particularly Fab molecules as described herein. A "Fab'molecule" differs from a Fab molecule in that the Fab' fragment has residues added to the carboxyl terminus of the CH1 domain, including one or more cysteine residues from the antibody hinge region. Fab'-SH is a Fab' molecule in which the cysteine residues in the constant domain have free thiol groups. Pepsin treatment produces F(ab') ₂ molecules, which have two antigen-binding sites (two Fab molecules) and a portion of the Fc region.

On the other hand, antibody fragments are bisomatic, trisomatic, or tetrasomatic antibodies. A “bisomatic antibody” is an antibody fragment having 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). Trisomatic and tetrasomatic antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

In another aspect, the antibody fragment is a single-chain Fab molecule. A “single-chain Fab molecule” or “scFab” is a polypeptide composed 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 the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: 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. Specifically, the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab molecule is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab molecules can be further stabilized by generating interchain disulfide bonds via the insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

On the other hand, the antibody fragment is a single-chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the antibody's heavy chain variable domain (VH) and light chain variable domain (VL), linked by a linker. Specifically, the linker is a short polypeptide of approximately 10 to 25 amino acids, typically rich in glycine for flexibility and serine or threonine for cleavage, and can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original antibody. For reviews of scFv fragments, see, for example, Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore (Springer-Verlag, New York), pp. 269–315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.

On the other hand, antibody fragments are single-domain antibodies. A "single-domain antibody" is an antibody fragment containing all or part of the variable heavy chain domain or all or part of the variable light chain domain of an antibody. In some respects, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516B1).

Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and recombinant production from recombinant host cells (e.g., E. coli), as described herein.

2. Humanized antibodies

In some respects, the antibodies (e.g., bispecific antibodies) provided herein are humanized antibodies. Typically, nonhuman antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent nonhuman antibody. Typically, humanized antibodies contain one or more variable domains, wherein the CDR (or a portion thereof) is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Humanized antibodies may also optionally contain at least a portion of a human constant region. In some respects, some FR residues in the humanized antibody are substituted with corresponding residues from the nonhuman antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

Humanized antibodies and their preparation methods are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); US patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Meth Methods 36:25-34 (2005) (describes specific determination region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “surface reshaping”); Dall’Acqua et al., Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes a “guided selection” approach for FR shuffling).

Human structural regions that can be used for humanization include, but are not limited to: structural regions selected using a “best fit” method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); structural regions of the common sequence of human antibodies derived from specific subgroups 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)). 3)); human mature (somatic mutation) architecture region or human germline architecture region (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and architecture regions derived from screened FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

3. Glycosylation variants

In some respects, the antibodies (e.g., bispecific antibodies) presented herein are designed to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

When an antibody contains an Fc region, the oligosaccharide associated with it can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are typically linked via N-bonds to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the biantennary oligosaccharide structure. In some aspects, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

On the one hand, antibody variants with unfucosylated oligosaccharides are provided, i.e., oligosaccharide structures lacking (directly or indirectly) fucose linked to the Fc region. Such unfucosylated oligosaccharides (also known as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack the fucose residues linking the first GlcNAc in the stem of the biantennary oligosaccharide structure. On the other hand, antibody variants with an increased proportion of unfucosylated oligosaccharides in the Fc region compared to natural or parental antibodies are provided. For example, the proportion of unfucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucose-linked oligosaccharides present). The percentage of non-fucosylated oligosaccharides, as described, for example, in WO2006/082515, and as measured by MALDI-TOF mass spectrometry, is the (average) amount of oligosaccharides lacking fucosylate residues relative to the sum of all oligosaccharides (e.g., complex, heterozygous, and high-mannose structures) linked to Asn 297. Asn297 refers to the asparagine residue (EU number of Fc region residues) located at approximately position 297 in the Fc region; however, due to minor sequence variations in antibodies, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may exhibit improved FcγRIIIa receptor binding and/or improved effector function, particularly improved ADCC function. See, for example, US 2003/0157108 and US 2004/0093621.

Examples of cell lines capable of producing antibodies against reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, particularly in Example 11), and knockout cell lines, such as those with the α-1,6-fucosylation gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohn). uki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107), or cells with reduced or cancelled GDP-fucose synthesis or transporter activity (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

On the other hand, antibody variants provide bipartite oligosaccharides, for example, wherein biantennary oligosaccharides linked to the Fc region of the antibody are bipartitely divided by GlcNAc. As mentioned above, such antibody variants can have reduced fucosylation and/or improved ADCC function. 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.

Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. 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 some respects, it may be necessary to produce cysteine-engineered antibodies, such as THIOMAB ^™ antibodies, in which one or more residues of the antibody are substituted with cysteine residues. In a preferred aspect, the substituted residues are located at accessible sites on the antibody. As further described herein, by substituting those residues with cysteine, a reactive thiol group is positioned at an accessible site on the antibody and can be used to conjugate the antibody with other parts, such as a pharmaceutical part or a linker-pharmaceutical part, to produce an immunoconjugate. Cysteine-engineered antibodies can be produced, for example, as described in U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO 2016040856.

5. Antibody derivatives

In some respects, the antibodies provided herein (e.g., bispecific antibodies) can be further modified to contain additional non-protein moieties known in the art and readily available. Suitable moieties 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), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, and whether the antibody derivative will be used for a specific therapeutic purpose.

6. Immunoconjugates

The present invention also provides an immunoconjugate comprising the anti-CD3/anti-FolR1 antibody described herein, which is conjugated (chemically bonded) to one or more therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant or animal-derived enzyme-active toxins or fragments thereof) or radioisotopes.

On the one hand, immunoconjugates are antibody-drug conjugates (ADCs), in which an antibody is conjugated to one or more therapeutic agents. A linker is typically used to connect the antibody to one or more therapeutic agents. An overview of ADC technology is provided in Pharmacol Review 68:3-19 (2016), which includes examples of therapeutic agents, drugs, and linkers.

On the other hand, the immunoconjugate comprises the antibody of the present invention conjugated to an enzyme-active toxin or a fragment thereof, said enzyme-active toxin or fragment thereof including but not limited to diphtheria A chain, non-conjugated active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, absinthecin A chain, sarcin, tung oil protein, dianthin protein, Phytolaca Americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelatin, mitogellin, localized aspergillin, phenolmycin, enomycin, and trichothecenes.

On the other hand, the immunoconjugate comprises the antibody of the present invention conjugated with a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce the radioconjugate. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and Lu. When the radioconjugate is used for detection, it may contain radioactive atoms for scintillation studies, such as Tc ^99m or I ¹²³ , or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as I ¹²³ , I ¹³¹ , In ¹¹¹ , F ¹⁹ , C ¹³ , N ¹⁵ , O ¹⁷ , gadolinium, manganese, or iron.

A variety of bifunctional protein conjugates, such as N-succinimino-3-(2-pyridyldithio)propionate (SPDP), succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), iminothiacyclopentane (IT), bifunctional derivatives of imino esters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimino octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazido derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene), can be used to prepare conjugates of antibodies and cytotoxic agents. For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See WO 94/11026. The adapter can be a “cleavable adapter” that promotes the release of cytotoxic drugs from cells. For example, acid-labile adapters, peptidase-sensitive adapters, light-labile adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., Cancer Res. 52:127-131 (1992); US Patent No. 5,208,020).

The immunoconjugates or ADCs described herein are explicitly considered, but not limited to, such conjugates prepared with crosslinking agents, including but not limited to commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC, sulfon-SMPB, and SVSB (succinimino-(4-vinyl sulfone)benzoate).

7. Multispecific antibodies

The antibodies described herein are multispecific antibodies, particularly bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different antigenic determinants (e.g., two different proteins, or two different epitopes on the same protein). In some respects, multispecific antibodies have three or more binding specificities. In some respects, one binding specificity is against CD3, and another is against FolR1. In some respects, multispecific antibodies can bind to two (or more) different epitopes of CD3. Multispecific (e.g., bispecific) antibodies can also be used to target cytotoxic agents or cells to cells expressing CD3. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and engineered “mortar and pestle structures” (see, for example, U.S. Patent 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997)). Multispecific antibodies can also be prepared by: engineering electrostatic manipulation effects used to prepare antibody Fc-heterodimer molecules (see, for example, WO 2009/089004); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011/034605); making Common light chain techniques used to avoid light chain mismatch problems (see, for example, WO 98/50431); "dimer antibody" techniques used to prepare 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 (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and the preparation of trispecific antibodies as described in Tutt et al., J. Immunol. 147:60 (1991).

This document also includes engineered antibodies having three or more antigen-binding sites, including, for example, “octopus antibodies” or DVD-Ig (see, for example, WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies having 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 their antigen-binding fragments also include “dual-acting FAbs” or “DAFs” that contain antigen-binding sites that bind to CD3 and another different antigen or to two different epitopes of CD3 (see, for example, US 2008/0069820 and WO 2015/095539).

Multispecific antibodies can also be provided asymmetrically, wherein there is domain crossing in one or more binding arms having the same antigen specificity (so-called "CrossMab" technology), i.e., by exchanging VH/VL domains (see, for example, WO 2009/080252 and WO 2015/150447), CH1/CL domains (see, for example, WO 2009/080253), or the complete Fab arm (see, for example, WO 2009/080251, WO 2016/016299, also see Schaefer et al., PNAS, 108(2011) 1187-1191, and Klein et al., MAbs 8(2016) 1010-20). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interfaces to guide the correct Fab pairing. See, for example, WO 2016/172485.

Also provided are multispecific antibodies in which binding arms with different specificities share a common light chain. The inventors of this invention have generated a bispecific antibody in which the binding portions share a common light chain that retains the specificity and efficacy of a parental monospecific antibody against CD3, and the same light chain can be used to bind a second antigen (e.g., FolR1). Generating a bispecific molecule comprising a common light chain that retains the binding properties of the parental antibody is not straightforward because the common CDR of the hybrid light chains must achieve binding specificity for both targets. In one aspect, the present invention provides a T-cell activation bispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion, wherein one antigen-binding portion is a Fab molecule capable of specifically binding to CD3 and the other antigen-binding portion is a Fab molecule capable of specifically binding to FolR1, wherein the first Fab molecule and the second Fab molecule have the same VLCL light chain. In one embodiment, the same light chain (VLCL) comprises the light chain CDR of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. In one embodiment, the same light chain (VLCL) comprises SEQ ID NO:129.

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

This document also includes a specific type of multispecific antibody, which is a bispecific antibody designed to simultaneously bind to a surface antigen (such as FolR1) on a target cell (e.g., tumor cells) and an activation-invariant component of the T cell receptor (TCR) complex (such as CD3) for retargeting T cells to kill the target cells. Therefore, in a preferred aspect, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, wherein one binding specificity targets CD3 and the other targets FolR1.

Examples of bispecific antibody forms that can be used for this purpose include, but are not limited to: so-called “BiTE” (bispecific T-cell recruiter) molecules in which two scFv molecules are fused via a flexible linker (see, for example, WO 2004/106381; WO2005/061547; WO 2007/042261; WO 2008/119567; Nagorsen and Exp Cell Res 317, 1255-1260 (2011)); bisomatic antibodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem antibodies. Bispecific antibodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (bi-affinity retargeting) molecules, which are based on bispecific antibody forms but have C-terminal disulfide bonds for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)); and so-called trifunctional antibodies, which are complete hybrid mouse/rat IgG molecules (as reviewed by Seimetz et al.: Cancer Treat Rev 36, 458-467 (2010)). The specific T-cell bispecific antibody forms included in this article are described in the following references: WO 2013/026833; WO 2013/026839; WO 2016/020309; Bacac et al., Oncoimmunology 5(8)(2016)e1203498.

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

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

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

In a preferred aspect of the invention, the (multispecific) antibody is capable of simultaneously binding to a first antigen (i.e., CD3) and a second antigen (i.e., FolR1). In one aspect, the (multispecific) antibody is capable of cross-linking T cells and target cells by simultaneously binding to CD3 and FolR1. In an even more preferred aspect, this simultaneous binding leads to the lysis of target cells, particularly tumor cells expressing the target cell antigen (i.e., FolR1). In one aspect, this simultaneous binding leads to the activation of T cells. In other aspects, this simultaneous binding leads to a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one aspect, binding of the (multispecific) antibody to CD3 without simultaneously binding to FolR1 does not lead to T cell activation.

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

Preferably, the T cells according to any aspect of the invention are cytotoxic T cells. In some aspects, the T cells are CD4 ⁺ or CD8 ⁺ T cells, particularly CD8 ⁺ T cells.

a) First antigen-binding domain

The (multispecific) antibody of the present invention comprises at least one antigen-binding domain (first antigen-binding domain) that binds to CD3. In a preferred aspect, CD3 is human CD3 (SEQ ID NO: 112) or cynomolgus monkey CD3 (SEQ ID NO: 113), most particularly human CD3. In one aspect, the first antigen-binding domain cross-reacts (i.e., specifically binds) to human and cynomolgus monkey CD3. In some aspects, CD3 is the ε subunit of CD3 (CD3ε).

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

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

b) Second (and third) antigen-binding domains

In some aspects, the (multispecific) antibody of the present invention comprises at least one antigen-binding domain, particularly a Fab molecule, that binds to a second antigen. The second antigen is preferably not CD3, i.e., different from CD3. In one aspect, the second antigen is an antigen expressed on cells different from CD3 (e.g., expressed on cells other than T cells). In another aspect, the second antigen is a target cell antigen, particularly a tumor cell antigen. In a preferred embodiment, the second antigen is FolR1. The second antigen-binding domain is capable of directing the (multispecific) antibody to a target site, for example, to a specific type of tumor cell expressing the second antigen.

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

In some respects, (multispecific) antibodies comprise two antigen-binding domains, particularly Fab molecules, that bind to a second antigen. In a preferred embodiment of this, each of these antigen-binding domains binds to the same antigenic determinant. In an even more preferred embodiment, all these antigen-binding domains are preferably identical, i.e., they have the same molecular form (e.g., conventional Fab molecules) and contain the same amino acid sequence (including the same amino acid substitutions in the CH1 and CL domains, as described herein (if any)). In one embodiment, (multispecific) antibodies comprise no more than two antigen-binding domains, particularly Fab molecules, that bind to a second antigen.

In one aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) comprises a human constant region. In another aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) is a Fab molecule comprising a human constant region, particularly a human CH1 and/or CL domain. Exemplary sequences of the human constant domain are given in SEQ ID NO:120 and SEQ ID NO:121 (human κ and λCL domains, respectively) and SEQ ID NO:122 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) comprises a light chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:120 or SEQ ID NO:121, particularly the amino acid sequence of SEQ ID NO:120. Specifically, the light chain constant region may contain amino acid mutations under “charged modification” as described herein and/or, in the case of a cross-Fab molecule, may contain deletions or substitutions of one or more (particularly two) N-terminal amino acids. In some aspects, the second antigen-binding domain (and, where present, the third antigen-binding domain) comprises a heavy chain constant region containing 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. Specifically, the heavy chain constant region (particularly the CH1 domain) may contain amino acid mutations under “charged modification” as described herein.

TYRP-1

In some aspects of this disclosure, the second antigen is TYRP-1, particularly human TYRP-1 (SEQ ID NO:114).

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

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

In one aspect, the VH and/or VL of the second antigen-binding domain (and, where present, the third antigen-binding domain) contain a receptor human frame, such as the human immunoglobulin frame or the human common frame.

In one aspect, the VH of the second antigen-binding domain (and, where present, the third antigen-binding domain) comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and/or FR4 sequences) of SEQ ID NO:18. In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:18. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:18. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:18. In some aspects, the VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the antibody containing this sequence retains its ability to bind to TYRP-1. In some aspects, a total of 1 to 10 amino acids in the amino acid sequence of SEQ ID NO:18 have been substituted, inserted, and/or deleted. In some respects, substitutions, insertions, or deletions occur in regions outside the CDR (i.e., in the FR). In one respect, VH comprises the amino acid sequence of SEQ ID NO:18. Optionally, VH comprises the amino acid sequence of SEQ ID NO:18, including post-translational modifications of that sequence.

In one aspect, the VL of the second antigen-binding domain (and, in its presence, the third antigen-binding domain) comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and/or FR4 sequences) of SEQ ID NO:22. In one aspect, the VL comprises 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 VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:22. In one aspect, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, the VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the antibody containing this sequence retains its ability to bind to TYRP-1. In some aspects, a total of 1 to 10 amino acids in the amino acid sequence of SEQ ID NO:22 have been substituted, inserted, and/or deleted. In some respects, substitutions, insertions, or deletions occur in regions outside the CDR (i.e., in the FR). In one respect, the VL comprises the amino acid sequence of SEQ ID NO:22. Optionally, the VL comprises the amino acid sequence of SEQ ID NO:22, including post-translational modifications of that sequence.

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

On the other hand, the second antigen-binding domain (and, in its presence, the third antigen-binding domain) comprises VH and VL, wherein the VH comprises the sequence of SEQ ID NO:18 and the VL comprises the sequence of SEQ ID NO:22.

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

On the other hand, the second antigen-binding domain (and the third antigen-binding domain, if present) comprises VH and VL, wherein the VH comprises the heavy chain CDR sequence of VH of SEQ ID NO:18; and the VL comprises the light chain CDR sequence of VL of SEQ ID NO:22.

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

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

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

EGFRvIII

In some aspects of this disclosure, the second antigen is EGFRvIII, particularly human EGFRvIII (SEQ ID NO:115).

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

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

In one aspect, the VH and/or VL of the second antigen-binding domain (and, where present, the third antigen-binding domain) contain a receptor human frame, such as the human immunoglobulin frame or the human common frame.

In one aspect, the VH of the second antigen-binding domain (and, in its presence, the third antigen-binding domain) comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and/or FR4 sequences) of SEQ ID NO:88. In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:88. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:88. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:88. In some aspects, the VH sequence having at least 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the antibody containing this sequence retains its ability to bind to EGFRvIII. In some aspects, a total of 1 to 10 amino acids in the amino acid sequence of SEQ ID NO:88 have been substituted, inserted, and/or deleted. In some respects, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). In one respect, VH comprises the amino acid sequence of SEQ ID NO:88. Optionally, VH comprises the amino acid sequence of SEQ ID NO:88, including post-translational modifications of that sequence.

In one aspect, the VL of the second antigen-binding domain (and, in its presence, the third antigen-binding domain) comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and/or FR4 sequences) of SEQ ID NO:92. In one aspect, the VL comprises 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 VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:92. In one aspect, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:92. In some aspects, the VL sequence having at least 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the antibody containing this sequence retains its ability to bind to EGFRvIII. In some aspects, a total of 1 to 10 amino acids in the amino acid sequence of SEQ ID NO:92 have been substituted, inserted, and/or deleted. In some respects, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). In one respect, the VL comprises the amino acid sequence of SEQ ID NO:92. Optionally, the VL comprises the amino acid sequence of SEQ ID NO:92, including post-translational modifications of that sequence.

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

On the other hand, the second antigen-binding domain (and, in its presence, the third antigen-binding domain) comprises VH and VL, wherein the VH comprises the sequence of SEQ ID NO:88 and the VL comprises the sequence of SEQ ID NO:92.

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

On the other hand, the second antigen-binding domain (and the third antigen-binding domain, if present) comprises VH and VL, wherein the VH comprises the heavy chain CDR sequence of VH of SEQ ID NO:88; and the VL comprises the light chain CDR sequence of VL of SEQ ID NO:92.

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

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

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

In terms of alternatives, the second antigen-binding domain (and, where present, the third antigen-binding domain) comprises: a VH sequence as described above in any aspect of EGFRvIII; and a VL sequence as described above in any aspect of EGFRvIII; but based on the following sequences (in row order), instead of SEQ ID NO:85 (HCDR1), SEQ ID NO:86 (HCDR2), SEQ ID NO:87 (HCDR3), SEQ ID NO:88 (VH), SEQ ID NO:89 (LCDR1), SEQ ID NO:90 (LCDR2), SEQ ID NO:91 (LCDR3), and SEQ ID NO:92 (VL):

FolR1

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

In one aspect, the second antigen-binding domain (and, where present, the third antigen-binding domain) comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:124, the HCDR 2 of SEQ ID NO:125, and the HCDR 3 of SEQ ID NO:126; the light chain variable region comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8, the LCDR 2 of SEQ ID NO:9, and the LCDR 3 of SEQ ID NO:10.

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

In one aspect, the VH and/or VL of the second antigen-binding domain (and, where present, the third antigen-binding domain) contain a receptor human frame, such as the human immunoglobulin frame or the human common frame.

In one aspect, the VH of the second antigen-binding domain (and, in its presence, the 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:123. In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:123. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:123. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:123. In some aspects, the VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the antibody containing this sequence retains its ability to bind to FolR1. In some respects, a total of 1 to 10 amino acids in the amino acid sequence of SEQ ID NO:123 have been substituted, inserted, and/or deleted. In some respects, the substitution, insertion, or deletion occurs in a region outside the CDR (i.e., in the FR). In one respect, VH comprises the amino acid sequence of SEQ ID NO:123. Optionally, VH comprises the amino acid sequence of SEQ ID NO:123, including post-translational modifications of that sequence.

In one aspect, the VL of the second antigen-binding domain (and, in its presence, the 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:11. In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:11. In one aspect, the VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:11. In one aspect, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:11. In some aspects, the VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the antibody containing this sequence retains its ability to bind to FolR1. In some aspects, a total of 1 to 10 amino acids in the amino acid sequence of SEQ ID NO:11 have been substituted, inserted, and/or deleted. In some respects, substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). In one respect, the VL comprises the amino acid sequence of SEQ ID NO:11. Optionally, the VL comprises the amino acid sequence of SEQ ID NO:11, including post-translational modifications of that sequence.

In one aspect, the VH of the second antigen-binding domain (and, where present, the third antigen-binding domain) comprises at least about 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 123, and the VL of the second antigen-binding domain comprises at least about 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO: 11. In another aspect, the VH comprises the amino acid sequence of SEQ ID NO: 123, and the VL comprises the amino acid sequence of SEQ ID NO: 11.

On the other hand, the second antigen-binding domain (and, in its presence, the third antigen-binding domain) comprises VH and VL, wherein the VH comprises the sequence of SEQ ID NO:123 and the VL comprises the sequence of SEQ ID NO:11.

In another aspect, the second antigen-binding domain (and the third antigen-binding domain, if present) comprises the VH sequence of SEQ ID NO:123 and the VL sequence of SEQ ID NO:11.

On the other hand, the second antigen-binding domain (and the third antigen-binding domain, if present) comprises VH and VL, wherein the VH comprises the heavy chain CDR sequence of VH of SEQ ID NO:123; and the VL comprises the light chain CDR sequence of VL of SEQ ID NO:11.

In another aspect, the second antigen-binding domain (and the third antigen-binding domain, if present) comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 of VH in SEQ ID NO:123 and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL in SEQ ID NO:11.

In one aspect, the VH of the second antigen-binding domain (and, where present, the third antigen-binding domain) comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 123, and a frame having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the frame sequence of the VH of SEQ ID NO: 123. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 123, and a frame having at least 95% sequence identity with the frame sequence of the VH of SEQ ID NO: 123. In yet another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 123, and a frame having at least 98% sequence identity with the frame sequence of the VH of SEQ ID NO: 123.

In one aspect, the VL of the second antigen-binding domain (and, where present, the third antigen-binding domain) comprises the light chain CDR sequence of the VL of SEQ ID NO: 11, and a frame having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the frame sequence of the VL of SEQ ID NO: 11. In another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 11, and a frame having at least 95% sequence identity with the frame sequence of the VL of SEQ ID NO: 11. In yet another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 11, and a frame having at least 98% sequence identity with the frame sequence of the VL of SEQ ID NO: 11.

In one aspect, the second antigen-binding domain (and, where present, the third antigen-binding domain) comprises the VH sequence as provided in any aspect of this section above and the VL sequence as provided in any aspect of this section above.

Anti-TYRP-1 and anti-EGFRvIII antibodies

This disclosure also provides antibodies that bind to TYRP-1, the antibodies comprising a VH sequence as provided in any aspect of TYRP-1 in this section above and a VL sequence as provided in any aspect of TYRP-1 in this section above (e.g., an antibody that binds to TYRP-1 comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:15, HCDR 2 of SEQ ID NO:16 and HCDR 3 of SEQ ID NO:17, the light chain variable region comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:19, LCDR 2 of SEQ ID NO:20 and LCDR 3 of SEQ ID NO:21; or an antibody that binds to TYRP-1 comprising VH and VL, the VH comprising the sequence of SEQ ID NO:18 and the VL comprising the sequence of SEQ ID NO:22).

This disclosure also provides antibodies that bind to EGFRvIII, comprising a VH sequence as provided in any aspect of EGFRvIII as described in this section above and a VL sequence as provided in any aspect of EGFRvIII as described in this section above (e.g., an antibody that binds to EGFRvIII comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the 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 the light chain variable region comprising the 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 comprising VH and VL, the VH comprising the sequence of SEQ ID NO:88, and the VL comprising the sequence of SEQ ID NO:92).

Anti-FolR1 antibody

This invention provides an antibody that binds to FolR1, the antibody comprising a VH sequence as provided in any aspect of FolR1 in this section above and a VL sequence as provided in any aspect of FolR1 in this section above (e.g., an antibody that binds to FolR1 comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:124, HCDR 2 of SEQ ID NO:125 and HCDR 3 of SEQ ID NO:126, the light chain variable region comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10; or an antibody that binds to FolR1 comprising VH and VL, the VH comprising the sequence of SEQ ID NO:123 and the VL comprising the sequence of SEQ ID NO:11).

In another aspect of the invention, the antibody that binds to FolR1 according to any of the foregoing aspects may bind, alone or in combination, any of the features of the antibody that binds to CD3 as described herein (unless explicitly referring to an anti-CD3 antibody, such as a binding sequence).

c) Charge modification

The (bispecific) antibody of the present invention may contain amino acid substitutions in the Fab molecule therein, which are particularly effective in reducing mismatches of the light chain and the mismatched heavy chain (Bence-Jones-type byproducts). These mismatches can occur in the production of Fab-based multispecific antibodies, where the bispecific/multispecific antigen-binding molecule has VH/VL exchanges in one of its binding arms (or multiple arms in the case of a molecule containing more than two antigen-binding Fab molecules) (see also PCT Publication WO 2015/150447, in particular examples therein, the entire contents of which are incorporated herein by reference). The ratio of desired (bispecific) antibody to undesirable byproducts, particularly the Bence-Jones-type byproducts appearing in multispecific antibodies with VH/VL domain exchanges in one of the binding arms, can be improved by introducing charged amino acids with opposite charges (sometimes referred to herein as "charge modification") at specific amino acid positions in the CH1 and CL domains.

Therefore, in some respects, the first and second antigen-binding domains (and, in their presence, the third antigen-binding domain) of the (bispecific) antibody are both Fab molecules, and in one of the antigen-binding domains (particularly the first antigen-binding domain), the variable domains VL and VH of the Fab light chain and Fab heavy chain are interchanged.

i) In the constant domain CL of the second antigen-binding domain (and, where present, the third antigen-binding domain), the amino acid at position 124 is replaced by a positively charged amino acid (according to Kabat numbering), and wherein in the constant domain CH1 of the second antigen-binding domain (and, where present, the third antigen-binding domain), the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (according to Kabat EU indexing); or

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

(Bispecific) antibodies do not include the two modifications mentioned in i) and ii). The constant domain CL and constant domain CH1 of the antigen-binding domain with VH/VL exchange do not substitute for each other (i.e., remain unexchanged).

In a more specific sense,

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

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

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

On the other hand, in the constant domain CL of the second antigen-binding domain (and, where present, the third antigen-binding domain), the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding domain (and, where present, the third antigen-binding domain), the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing).

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

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

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

In a preferred aspect, if the amino acid substitution according to the above aspects is carried out in the constant domains CL and CH1 of the second antigen-binding domain (and the third antigen-binding domain when present), then the constant domain CL of the second antigen-binding domain (and the third antigen-binding domain when present) is a κ isotype.

Alternatively, the amino acid substitutions according to the above aspects can be carried out in the constant domains CL and CH1 of the first antigen-binding domain, rather than in the constant domains CL and CH1 of the second antigen-binding domain (and, where present, the third antigen-binding domain). In a preferred embodiment of this aspect, the constant domain CL of the first antigen-binding domain is of the κ isotype.

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

On the other hand, in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is independently replaced by lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 is independently replaced by glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering).

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

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

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

In a preferred aspect, the (bispecific) antibody of the present invention comprises...

(a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein variable domains VL and VH of the Fab light chain and Fab heavy chain are interchanged, and includes a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region including 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, the light chain variable region including the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10; and

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

d) Multispecific antibody forms

The (bispecific and/or multispecific) antibodies according to the present invention can have various configurations. Exemplary configurations are described in Figure 1.

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

In one aspect, the first antigen-binding domain and the second antigen-binding domain of the (bispecific) antibody are fused together, optionally via a peptide linker. In a preferred aspect, both the first and second antigen-binding domains are Fab molecules. 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. 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 Fab heavy chain of the first antigen-binding domain, the Fab light chain of the first antigen-binding domain and the Fab light chain of the second antigen-binding domain may optionally be fused together via a peptide linker.

Bispecific antibodies (such as Fab molecules) possessing a single antigen-binding domain capable of specifically binding to a second antigen (e.g., a target cell antigen, such as FolR1 (e.g., as shown in Figures 1A, 1D, 1G, 1H, 1K, and 1L)) are useful, especially when the internalization of the second antigen is expected after binding to a high-affinity antigen-binding domain. In such cases, the presence of more than one antigen-binding domain specific to the second antigen may enhance the internalization of the second antigen, thereby reducing its usability.

However, in other cases, it would be advantageous to have (bispecific) antibodies (such as Fab molecules) containing two or more antigen-binding domains specific to a second antigen, such as a target cell antigen (see Figures 1B, 1C, 1E, 1F, 1I, 1J, 1M, 1N, 33A, and 33B), for example, to optimize targeting of the target site or to allow cross-linking of the target cell antigen.

Therefore, in a preferred aspect, the (multispecific, e.g., bispecific) antibody according to the invention comprises a third antigen-binding domain.

In one respect, the third antigen-binding domain binds to the second antigen (e.g., a target cell antigen, such as FolR1). In another respect, the third antigen-binding domain is a Fab molecule.

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

In some respects, the third and second antigen-binding domains are each Fab molecules, and the third antigen-binding domain is identical to the second antigen-binding domain. Therefore, in these respects, the second and third antigen-binding domains contain the same heavy and light chain amino acid sequences and have the same domain arrangement (i.e., conventional or crossed). Furthermore, in these respects, 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-modified” will be made in the constant domains CL and CH1 of the second and third antigen-binding domains, respectively. Alternatively, the amino acid substitutions may be made in the constant domains CL and CH1 of the first antigen-binding domain (which, in a preferred respect, is also a Fab molecule), but not in the constant domains CL and CH1 of the second and third antigen-binding domains.

Like the second antigen-binding domain, the third antigen-binding domain is preferably a conventional Fab molecule. All Fab molecules can share a common light chain. However, aspects in which the second and third antigen-binding domains are cross-Fab molecules (and the first antigen-binding domain is a conventional Fab molecule) are also envisioned. Thus, in some aspects, the second and third antigen-binding domains are each conventional Fab molecules, and the first antigen-binding domain is a cross-Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged/substituted for each other. In other aspects, the second and third antigen-binding domains are each cross-Fab molecules and the first antigen-binding domain is a conventional Fab molecule.

If a third antigen-binding domain is present, in a preferred aspect, the first antigen-binding domain binds to CD3, and the second antigen-binding domain binds to a second antigen, particularly a target cell antigen (such as FolR1).

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

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

The (multispecific, e.g., bispecific) antibody according to the invention can have different conformations, i.e., the first antigen-binding domain, the second antigen-binding domain (and optionally the third antigen-binding domain) can be fused to each other and to the Fc domain in different ways. Components can be fused directly to each other, or preferably via one or more suitable peptide linkers. When the Fab molecule fuses to the N-terminus of a subunit of the Fc domain, the fusion is typically via an immunoglobulin hinge region.

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

In one aspect, the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, with the first antigen-binding domain fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen-binding domain fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain. In a specific aspect, the (multispecific, e.g., bispecific) antibody essentially consists of a first Fab molecule, a second Fab molecule, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. This configuration is schematically depicted in Figures 1G and 1K (in these examples, the first antigen-binding domain is a VH/VL cross-Fab molecule). Optionally, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may additionally fuse with each other.

On the other hand, the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, and each of the first and second antigen-binding domains is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In one specific aspect, a (multispecific, e.g., bispecific) antibody essentially consists of a first Fab molecule and a second Fab molecule, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule and the second Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. Such configurations are schematically shown in Figures 1A and 1D (in these examples, the first antigen-binding domain is a VH/VL cross-linked Fab molecule, and the second antigen-binding domain is a conventional Fab molecule) and Figure 33E (in this example, the light chains of the first and second antigen-binding domains are identical). The first and second Fab molecules can be fused to the Fc domain directly or via peptide linkers. In a preferred embodiment, both the first Fab molecule and the second Fab molecule are fused to the Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is the human _IgG1 hinge region, particularly when the Fc domain is the _IgG1 Fc domain.

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

In one aspect, the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, the second antigen-binding domain being fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the first antigen-binding domain being fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. In a specific aspect, the (multispecific, e.g., bispecific) antibody essentially consists of a first Fab molecule and a second Fab molecule, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such configurations are schematically shown in Figures 1H and 1L (in these examples, the first antigen-binding domain is a VH/VL cross-Fab molecule, and the second antigen-binding domain is a conventional Fab molecule) and Figures 33C and 33D (in these examples, the light chains of the first and second antigen-binding domains are identical). Optionally, the Fab light chains of the first and second Fab molecules can be further fused together.

In a preferred embodiment, the first and third antigen-binding domains are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In one specific embodiment, the (multispecific, e.g., bispecific) antibody essentially comprises a first Fab molecule, a second Fab molecule, and a third Fab molecule, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The first and third Fab molecules may be fused to the Fc domain directly or via peptide linkers. In a preferred embodiment, the first Fab molecule and the third Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. Specifically, the immunoglobulin hinge region is the human _IgG1 hinge region, particularly when the Fc domain is the _IgG1 Fc domain. Optionally, the Fab light chains of the first Fab molecule and the second Fab molecule can be further fused to each other.

In another such aspect, the second and third antigen-binding domains are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. In one specific aspect, the (multispecific, e.g., bispecific) antibody essentially consists of first, second, and third Fab molecules, an Fc domain formed by the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the first subunit of the Fc domain, and wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The second and third Fab molecules may be fused to the Fc domain directly or via peptide linkers. In a preferred aspect, the second and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. Specifically, the immunoglobulin hinge region is the human _IgG1 hinge region, particularly when the Fc domain is the _IgG1 Fc domain. Optionally, the Fab light chains of the first Fab molecule and the second Fab molecule can be further fused to each other.

In the configuration of a (multispecific) antibody, a Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each subunit of the Fc domain via an immunoglobulin hinge region, the two Fab molecules, the hinge region, and the Fc domain essentially forming an immunoglobulin molecule. In a preferred aspect, the immunoglobulin molecule is an IgG class immunoglobulin. In an even more preferred aspect, the immunoglobulin is an IgG ₁ subclass immunoglobulin. In another aspect, the immunoglobulin is an IgG ₄ subclass immunoglobulin. In yet another 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 includes a human constant region, particularly a human Fc region.

In some of the (multispecific) antibodies of the present invention, the Fab light chains of a first Fab molecule and a second Fab molecule are fused together, optionally via peptide linkers. Depending on the configuration of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. The fusion of the Fab light chains of the first and second Fab molecules further reduces mismatches of unmatched Fab heavy and light chains, and also reduces the number of plasmids required to express some of the (multispecific) antibodies of the present invention.

The antigen-binding domain may be fused to the Fc domain directly or via a peptide linker comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, ( _G4S ) _n , (SG4) _n , ( _G4S ) _n , or _G4 ( _SG4 ) _n peptide linkers. “n” is typically an integer from 1 to 10, and typically from 2 to 4. In one aspect, the peptide linker is at least 5 amino acids long; in another aspect, _it is 5 to 100 amino acids long; and in yet another aspect, it is 10 to 50 amino acids long. In one aspect, the peptide linker is (GxS) _n or (GxS) _nGm , where G = glycine, S = serine, and (x = 3, n = 3 _, 4, 5, or 6, and m = 0, 1, 2, or 3) or (x = 4, n = 2, 3, 4, or 5, and m = 0, 1, 2, or 3). In one aspect, x = 4 and n = 2 or 3, and in another aspect, x = 4 and n = 2. In one aspect, the peptide linker is ( _G4S ) ₂ . A particularly suitable peptide linker for fusing the Fab light chains of the first Fab molecule and the second Fab molecule to each other is ( _G4S ) ₂ . An exemplary peptide linker suitable for linking the Fab heavy chains of the first Fab fragment and the second Fab fragment comprises the sequence (D)-( _G4S ) ₂ (SEQ ID NO: 118 and SEQ ID NO: 119). Another suitable such linker comprises the sequence ( _G4S ) ₄ . Additionally, the linker may comprise a portion of an immunoglobulin hinge region. In particular, in the case of fusion of the Fab molecule with the N-terminus of the Fc domain subunit, fusion can occur via the immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

In some aspects, bispecific antigen-binding molecules comprise a common light chain. In one aspect, the present invention provides a bispecific antigen-binding molecule comprising a first antigen-binding portion and a second antigen-binding portion, wherein one antigen-binding portion is a Fab molecule capable of specifically binding to CD3 and the other antigen-binding portion is a Fab molecule capable of specifically binding to FolR1, wherein the first Fab molecule and the second Fab molecule have the same VLCL light chain. In one embodiment, the same light chain (VLCL) comprises the light chain CDR of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. In one embodiment, the same light chain (VLCL) comprises SEQ ID NO:133.

In one embodiment, the present invention provides a T-cell activation bispecific antigen-binding molecule comprising: (i) a first antigen-binding portion, which is a Fab molecule capable of specifically binding to CD3, and comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5 and at least one light chain CDR selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10; and (ii) a second antigen-binding portion, which is a Fab molecule capable of binding to folate receptor 1 (FolR1), and comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:124, SEQ ID NO:125 and SEQ ID NO:126 and at least one light chain CDR selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

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

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

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

In one embodiment, the T-cell activating bispecific antigen-binding molecule further comprises (iii) a third antigen-binding moiety (which is a Fab molecule) capable of specifically binding to FolR1. In one such embodiment, the second and third antigen-binding moieties capable of specifically binding to FolR1 comprise the same heavy chain complementarity-determining region (CDR) and light chain CDR sequences. In one such embodiment, the third antigen-binding moiety is identical to the second antigen-binding moiety.

Therefore, in one embodiment, the present invention provides a T-cell activation bispecific antigen-binding molecule comprising...

(i) A first antigen-binding portion, which is a Fab molecule capable of specifically binding to CD3, and comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39 and at least one light chain CDR selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34;

(ii) A second antigen-binding portion, which is a Fab molecule capable of specifically binding to folic acid receptor 1 (FolR1), and comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 and at least one light chain CDR selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34.

(iii) A third antigen-binding portion, which is a Fab molecule capable of specifically binding to folic acid receptor 1 (FolR1), and comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 and at least one light chain CDR selected from the group consisting of SEQ ID NO:32, SEQ ID NO:533 and SEQ ID NO:34.

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

In one embodiment, the present invention provides a T-cell activating bispecific antigen-binding molecule comprising...

(i) A first antigen-binding portion, which is a Fab molecule capable of specifically binding to CD3, comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the amino acid sequence of SEQ ID NO:36 and the variable light chain comprising the amino acid sequence of SEQ ID NO:31.

(ii) A second antigen-binding portion, which is a Fab molecule capable of specifically binding to folic acid receptor 1 (FolR1), comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the amino acid sequence of SEQ ID NO:15 and the variable light chain comprising the amino acid sequence of SEQ ID NO:31.

(iii) The third antigen-binding portion is a Fab molecule capable of specifically binding to folic acid receptor 1 (FolR1), comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain comprises the amino acid sequence of SEQ ID NO:15 and the variable light chain comprises the amino acid sequence of SEQ ID NO:31.

Therefore, in one embodiment, the present invention relates to a bispecific molecule wherein at least two binding portions have the same light chain and a corresponding remodeled heavy chain, which respectively confer specific binding to the T cell activation antigen CD3 and the target cell antigen FolR1. This so-called "common light chain" principle, that is, combining two binders that share a light chain but still have individual specificity, prevents light chain mismatch. Therefore, there are fewer byproducts during production, which is beneficial for the homogenized preparation of T cell activation bispecific antigen binding molecules.

In some embodiments, the T-cell activated bispecific antigen-binding molecule comprises an Fc domain consisting of a first subunit and a second subunit capable of stable association. Exemplary embodiments of a T-cell activated bispecific antigen-binding molecule comprising the Fc domain are described below.

In one aspect, the present invention provides a (multispecific, e.g., bispecific) antibody comprising...

a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule and includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region includes the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:2, the HCDR 2 of SEQ ID NO:3 and the HCDR 3 of SEQ ID NO:5, and the light chain variable region includes the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:8, the LCDR 2 of SEQ ID NO:9 and the LCDR 3 of SEQ ID NO:10;

b) A second antigen-binding domain that binds to a second antigen, particularly a target cell antigen, and even more particularly FolR1, wherein the second antigen-binding domain is a Fab molecule containing a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8, the LCDR 2 of SEQ ID NO:9, and the LCDR 3 of SEQ ID NO:10;

c) The Fc domain, which is composed of the first subunit and the second subunit;

in

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

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

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

a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule and includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region includes the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:2, the HCDR 2 of SEQ ID NO:3 and the HCDR 3 of SEQ ID NO:5, and the light chain variable region includes the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:8, the LCDR 2 of SEQ ID NO:9 and the LCDR 3 of SEQ ID NO:10;

b) A second antigen-binding domain and a third antigen-binding domain that bind to a second antigen, particularly a target cell antigen, and even more particularly FolR1, wherein each of the second and third antigen-binding domains is a Fab molecule containing a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8, LCDR 2 of SEQ ID NO:9, and LCDR 3 of SEQ ID NO:10; and

c) The Fc domain, which is composed of the first subunit and the second subunit;

in

(i) The first antigen-binding domain according to a) fuses at the C-terminus of the Fab heavy chain with the N-terminus of the second antigen-binding domain according to b), and the second antigen-binding domain according to b) and the third antigen-binding domain according to b) each fuse at the C-terminus of the Fab heavy chain with the N-terminus of one of the subunits of the Fc domain according to c), or

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

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

a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule and includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region includes the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO:2, the HCDR 2 of SEQ ID NO:3 and the HCDR 3 of SEQ ID NO:5, and the light chain variable region includes the light chain complementarity-determining region (LCDR)1 of SEQ ID NO:8, the LCDR 2 of SEQ ID NO:9 and the LCDR 3 of SEQ ID NO:10;

b) A second antigen-binding domain that binds to a second antigen, particularly a target cell antigen, and even more particularly FolR1, wherein the second antigen-binding domain is a Fab molecule containing a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8, the LCDR 2 of SEQ ID NO:9, and the LCDR 3 of SEQ ID NO:10;

c) The Fc domain, which is composed of the first subunit and the second subunit;

in

(i) The first antigen-binding domain according to a) and the second antigen-binding domain according to b) are each fused at the C-terminus of the subunit of the Fc domain according to c) to the N-terminus.

According to any of the above, components of a (multispecific, e.g., bispecific) antibody (e.g., Fab molecules, Fc domains) can be fused directly or via various linkers, particularly peptide linkers containing one or more amino acids, typically about 2 to 20 amino acids, which are described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, ( _G4S ) _n , ( _SG4 ) _n , ( _G4S ) _n , or _G4 ( _SG4 ) _n peptide linkers, where n is typically an integer from 1 to 10, and typically 2 to 4.

In a preferred aspect, the present invention provides a (bispecific) antibody comprising...

a) A first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is Fab and includes a heavy chain variable region (VH), the heavy chain variable region including the heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO:2, HCDR 2 of SEQ ID NO:3 and HCDR 3 of SEQ ID NO:5;

b) A second antigen-binding domain and a third antigen-binding domain that bind to FolR1, wherein each of the second and third antigen-binding domains is a Fab molecule and contains a heavy chain variable region (VH), which includes the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO:124, HCDR 2 of SEQ ID NO:125, and HCDR 3 of SEQ ID NO:126;

c) The Fc domain composed of the first and second subunits; and

The first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain contain light chain variable regions (VLs), which include light chain complementarity-determining regions (LCDRs) 1 of SEQ ID NO:8, LCDR 2 of SEQ ID NO:9, and LCDR 3 of SEQ ID NO:10.

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

In another aspect of these aspects of the invention, in the first subunit of the Fc domain, the serine residue at position 354 is replaced by a cysteine residue (S354C) or the glutamate residue at position 356 is replaced by a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced by a cysteine residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (according to the Kabat EU index number).

In yet another aspect of 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 with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (according to the Kabat EU index number).

In yet another aspect of these aspects of the invention, the Fc domain is the human _IgG1 Fc domain.

In one preferred aspect, the bispecific antibody comprises a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:127, a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:128, and a polypeptide (particularly three polypeptides) containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:129. In another preferred aspect, the (multispecific, e.g., bispecific) antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO:127, a polypeptide containing the amino acid sequence of SEQ ID NO:128, and a polypeptide (particularly three polypeptides) containing the amino acid sequence of SEQ ID NO:129.

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

In one specific aspect, a bispecific antibody comprises a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:130, a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:128, and a polypeptide (particularly three polypeptides) containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:129. In another specific aspect, a (multispecific, e.g., bispecific) antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO:130, a polypeptide containing the amino acid sequence of SEQ ID NO:128, and a polypeptide (particularly three polypeptides) containing the amino acid sequence of SEQ ID NO:129.

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

In one specific aspect, a bispecific antibody comprises a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:131, a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:132, and a polypeptide (particularly three polypeptides) containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:129. In another specific aspect, a (multispecific, e.g., bispecific) antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO:131, a polypeptide containing the amino acid sequence of SEQ ID NO:132, and a polypeptide (particularly three polypeptides) containing the amino acid sequence of SEQ ID NO:129.

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

In one specific aspect, a bispecific antibody comprises a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:133, a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:134, and a polypeptide (particularly three polypeptides) containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:129. In another specific aspect, a (multispecific, e.g., bispecific) antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO:133, a polypeptide containing the amino acid sequence of SEQ ID NO:134, and a polypeptide (particularly three polypeptides) containing the amino acid sequence of SEQ ID NO:129.

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

In one specific aspect, a bispecific antibody comprises a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:135, a polypeptide containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:136, and a polypeptide (particularly two polypeptides) containing at least 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of SEQ ID NO:129. In another specific aspect, a (multispecific, e.g., bispecific) antibody comprises a polypeptide containing the amino acid sequence of SEQ ID NO:135, a polypeptide containing the amino acid sequence of SEQ ID NO:136, and a polypeptide (particularly two polypeptides) containing the amino acid sequence of SEQ ID NO:129.

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

e) Fc domain variants

In a preferred aspect, the (multispecific, e.g., bispecific) antibody of the present invention comprises an Fc domain consisting of a first subunit and a second subunit.

The Fc domain of a (multispecific, e.g., bispecific) antibody consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which contains CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one aspect, the (multispecific, e.g., bispecific) antibody of the present invention comprises no more than one Fc domain.

In one aspect, the Fc domain of the (multispecific, e.g., bispecific) antibody is an IgG Fc domain. In a preferred aspect, the Fc domain is an IgG ₁ Fc domain. In another aspect, the Fc domain is an IgG ₄ Fc domain. In a more specific aspect, the Fc domain is an IgG ₄ Fc domain containing an amino acid substitution (specifically, amino acid substitution S228P) at position S228 (Kabat EU index number). This amino acid substitution reduces in vivo Fab arm exchange of the IgG ₄ antibody (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In yet another 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 sequence of the human IgG ₁ Fc region is given in SEQ ID NO:117.

f) Modification of the Fc domain to promote heterodimerization

The (multispecific, e.g., bispecific) antibody according to the invention comprises a different antigen-binding domain that can fuse with one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically contained in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides result in several possible combinations of the two polypeptides. To improve the yield and purity of the (multispecific, e.g., bispecific) antibody in recombinant production, it is therefore advantageous to introduce modifications into the Fc domain of the (multispecific, e.g., bispecific) antibody that promote the association of the desired polypeptide.

Therefore, in a preferred aspect, the Fc domain of the (multispecific, e.g., bispecific) antibody according to the invention comprises modifications that promote association between the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one aspect, the modification is in the CH3 domain of the Fc domain.

Several methods exist for modifying the CH3 domain of the Fc domain to achieve heterodimerization, and these methods are described in detail, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO2013157954, and WO 2013096291. Typically, in all such approaches, the CH3 domains of the first subunit and the second subunit of the Fc domain are engineered in a complementary manner, such that each CH3 domain (or the heavy chain containing it) is no longer homodimerized with itself, but is instead forced to heterodimerize with other complementaryly engineered CH3 domains (so that the first and second CH3 domains heterodimerize and no homodimers are formed between the two first or two second CH3 domains). These different approaches to achieving improved heavy chain heterodimerization are considered alternatives to heavy-light chain modifications in (multispecific, e.g., bispecific) antibodies (e.g., VH and VL exchange/replacement in one binding arm and the introduction of substitutions of charged amino acids with opposite charges at the CH1/CL interface), which reduce heavy/light chain mismatches and Bence Jones-type byproducts.

In one specific aspect, the modification that promotes association between the first and second subunits of the Fc domain is a so-called "mortar and pestle" modification, which includes a "protrusion" modification in one of the two subunits of the Fc domain and a "pore" modification in the other of the two subunits of the Fc domain.

The pestle-and-mortar structure technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion (“pestle”) at the interface of a first polypeptide and a corresponding cavity (“mortar”) at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote the formation of heterodimers and inhibit the formation of homodimers. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity having the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

Therefore, in a preferred aspect, in the CH3 domain of the first subunit of the Fc domain of a (multispecific, e.g., bispecific) antibody, amino acid residues are replaced by amino acid residues with larger side chain volumes, thereby creating a protrusion within the CH3 domain of the first subunit, which can be positioned in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby creating a cavity within the CH3 domain of the second subunit, where the protrusion within the CH3 domain of the first subunit can be positioned within the cavity.

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

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

Protrusions and cavities can be prepared by altering the nucleic acids encoding polypeptides (e.g., through site-specific mutagenesis or through peptide synthesis).

In one specific aspect, in the first subunit (“mortar” subunit) of the Fc domain (CH3 domain), the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit (“mortar” subunit) of the Fc domain (CH3 domain), the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In another aspect, in the second subunit of the Fc domain, additionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (according to the Kabat EU index number).

In another aspect, in the first subunit of the Fc domain, the serine residue at position 354 is replaced by a cysteine residue (S354C) or the glutamate residue at position 356 is replaced by a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced by a cysteine residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (according to the Kabat EU index number). The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

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

In a preferred aspect, the CD3-binding antigen-binding domain is fused with a first subunit of the Fc domain (containing a "club" modification) via a second antigen-binding domain that binds to a second antigen (i.e., FolR1) and/or a peptide linker. Not wishing to be bound by theory, the fusion of the CD3-binding antigen-binding domain with the club-containing subunit of the Fc domain will (further) minimize the generation of antibodies containing two CD3-binding antigen-binding domains (steric hindrance of two club-containing polypeptides).

Other CH3 modification techniques for implementing heterodimerization are contemplated as alternatives to those described in the present invention, and are described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO2013/157954, and WO 2013/096291.

In one aspect, the heterodimerization method described in EP 1870459 can be used alternatively. This method is based on introducing charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain. A specific aspect of the (multispecific) antibody of the present invention is the amino acid mutation R409D; K370E in one of the two CH3 domains (of the Fc domain); and the amino acid mutation D399K; E357K (according to the Kabat EU index number) in the other CH3 domain of the Fc domain.

In another aspect, the (multispecific, e.g., bispecific) antibody of the present invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally the amino acid mutation R409D; K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutation D399K; and E357K (according to the Kabat EU index number) in the CH3 domain of the second subunit of the Fc domain.

In another aspect, the (multispecific, e.g., bispecific) antibody of the present invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the (multispecific, e.g., bispecific) antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutation R409D; K370E in the CH3 domain of the first subunit of the Fc domain, and amino acid mutation D399K; and E357K in the CH3 domain of the second subunit of the Fc domain (all according to Kabat EU index numbers).

In one aspect, the heterodimerization method described in WO 2013/157953 may be used alternatively. In one aspect, the first CH3 domain contains the amino acid mutation T366K, and the second CH3 domain contains the amino acid mutation L351D (according to the Kabat EU index number). In another aspect, the first CH3 domain contains an additional amino acid mutation L351K. In yet another aspect, the second CH3 domain also contains an amino acid mutation selected from Y349E, Y349D, and L368E (especially L368E) (according to the Kabat EU index number).

In one aspect, the heterodimerization method described in WO 2012/058768 may be used alternatively. In one aspect, the first CH3 domain contains the amino acid mutations L351Y and Y407A, and the second CH3 domain contains the amino acid mutations T366A and K409F. In another aspect, the second CH3 domain contains further amino acid mutations at positions T411, D399, S400, F405, N390, or K392, for example selected from the group consisting of: a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W, b) D399R, D399W, D399Y, or D399F. 9K, 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 (according to Kabat EU index number). In another aspect, the first CH3 domain contains the amino acid mutations L351Y and Y407A, and the second CH3 domain contains the amino acid mutations T366V and K409F. In another aspect, the first CH3 domain contains the amino acid mutation Y407A, and the second CH3 domain contains the amino acid mutations T366A and K409F. In another aspect, the second CH3 domain also contains amino acid mutations K392E, T411E, D399R, and S400R (according to Kabat EU index numbers).

In one respect, the heterodimerization method described in WO 2011/143545 can be used alternatively, for example, by modifying amino acids at positions selected from the group consisting of 368 and 409 (according to the Kabat EU index number).

In one aspect, the heterodimerization method described in WO 2011/090762 can be used alternatively, which also employs the aforementioned mortar and pestle structure technique. In one aspect, the first CH3 domain contains the amino acid mutation T366W, and the second CH3 domain contains the amino acid mutation Y407A. In another aspect, the first CH3 domain contains the amino acid mutation T366Y, and the second CH3 domain contains the amino acid mutation Y407T (according to the Kabat EU index number).

In one respect, (multispecific, e.g., bispecific) antibodies or their Fc domains are _IgG2 subclasses, and alternatively, heterodimerization methods described in WO 2010/129304 may be used.

In one alternative aspect, modifications that promote association between the first and second subunits of the Fc domain include modifications that mediate electrostatic reorientation effects, such as those described in PCT Publication WO 2009/089004. Typically, this method involves substituting one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, making homodimer formation electrostatically unfavorable but heterodimerization electrostatically favorable. In one such aspect, the first CH3 domain comprises an amino acid substitution of 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 of D399, E356, D356, or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), particularly D399K, E356K, D356K, or E357K, more particularly D399K and E356K). In another aspect, the first CH3 domain also comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), particularly K409D or R409D). In another aspect, the first CH3 domain further 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 according to Kabat EU index numbers).

In another aspect, the heterodimerization method described in WO 2007/147901 can be used alternatively. In one aspect, the first CH3 domain contains amino acid mutations K253E, D282K, and K322D, and the second CH3 domain contains amino acid mutations D239K, E240K, and K292D (according to Kabat EU index number).

Alternatively, the heterodimerization method described in WO 2007/110205 can be used.

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

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

The Fc domain endows (multispecific) antibodies with favorable pharmacokinetic properties, including a long serum half-life that facilitates good accumulation in target tissues and a favorable tissue-to-blood partition ratio. However, it can also lead to (multispecific, e.g., bispecific) antibodies undesirably targeting cells expressing Fc receptors instead of preferred antigen-carrying cells. Furthermore, co-activation of the Fc receptor signaling pathway can result in cytokine release, which, combined with T cell activation properties and the long half-life of (multispecific) antibodies, can lead to overactivation of cytokine receptors and severe side effects upon systemic administration. Activation of immune cells other than T cells (carrying Fc receptors) may even reduce the efficacy of (multispecific) antibodies due to potential damage to T cells (e.g., by NK cells).

Therefore, in a preferred aspect, the Fc domain of the (multispecific, e.g., bispecific) antibody according to the invention exhibits reduced binding affinity to the Fc receptor and/or reduced effector function compared to the natural IgG1 _Fc domain. In one such aspect, the Fc domain (or the (multispecific) antibody containing said Fc domain) exhibits less than 50%, particularly less than 20%, more particularly less than 10%, and most particularly less than ₅ % binding affinity compared to the natural IgG1 _Fc domain (or the (multispecific, e.g., bispecific) antibody containing said Fc domain), and/or less than 50%, particularly less than 20%, more particularly less than 10%, and most particularly less than 5% effector function compared to the natural _{IgG1 Fc} domain (or the (multispecific, e.g., bispecific) antibody containing said Fc domain). In one aspect, the Fc domain (or the (multispecific, e.g., bispecific) antibody containing said Fc domain) substantially does not bind to the Fc receptor and/or induce effector function. In a preferred aspect, the Fc receptor is an Fcγ receptor. In another aspect, the Fc receptor is a human Fc receptor. In another 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, and most specifically human FcγRIIIa. In one aspect, the effector function is one or more effector functions selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a preferred aspect, the effector function is ADCC. In one aspect, the Fc domain exhibits a substantially similar binding affinity to the nascent Fc receptor (FcRn) compared to the native _IgG1 Fc domain. When the Fc domain (or a multispecific, e.g., bispecific antibody containing the Fc domain) exhibits a binding affinity for FcRn greater than about 70%, particularly greater than about 80%, and even more particularly greater than about 90% for the natural _IgG1 Fc domain (or a multispecific, e.g., bispecific antibody containing the natural _IgG1 Fc domain), substantially similar binding to FcRn is achieved.

In some respects, the Fc domain is engineered to have reduced binding affinity to the Fc receptor and/or reduced effector function compared to an unengineered Fc domain. In a preferred respect, the Fc domain of a (multispecific, e.g., bispecific) antibody contains one or more amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptor and/or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one respect, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In another respect, 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 the presence of more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, a combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one aspect, antibodies containing engineered Fc domains (multispecific, e.g., bispecific) exhibit a binding affinity of less than 20%, particularly less than 10%, and more particularly less than 5%, to the Fc receptor compared to antibodies containing unengineered Fc domains (multispecific, e.g., bispecific). 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 activated Fc receptor. In a specific aspect, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, the binding affinity with each of these receptors is reduced. In some aspects, the binding affinity to the complementary component, and the specific binding affinity to C1q, is also reduced. In one aspect, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. When the Fc domain (or a multispecific, e.g., bispecific antibody containing the Fc domain) exhibits a binding affinity for FcRn greater than about 70% for the unengineered form of the Fc domain (or a multispecific, e.g., bispecific antibody containing the unengineered form of the Fc domain), substantially similar binding to FcRn is achieved, i.e., the binding affinity of the Fc domain to the receptor is maintained. The Fc domain or the multispecific antibody of the present invention containing the Fc domain can exhibit such an affinity greater than about 80%, and even greater than about 90%. In some aspects, the Fc domain of the multispecific, e.g., bispecific antibody is engineered to have reduced effector function compared to the unengineered Fc domain. The reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signal transduction-induced apoptosis, reduced cross-linking of target-binding antibodies, reduced dendritic cell maturation, or reduced T cell sensitization. In one aspect, the reduced effector function is one or more of the reduced effector functions selected from the group consisting of reduced CDC, reduced ADCC, reduced ADCP, 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 an unengineered Fc domain (or a (multispecific, e.g., bispecific) antibody containing said unengineered Fc domain).

In one aspect, the amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor and/or the effector function is an amino acid substitution. In one aspect, the Fc domain contains an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329 (according to Kabat EU index numbers). In a more specific aspect, the Fc domain contains an amino acid substitution at a position selected from the group consisting of L234, L235, and P329 (according to Kabat EU index numbers). In some aspects, the Fc domain contains amino acid substitutions L234A and L235A (according to Kabat EU index numbers). In one such aspect, the Fc domain is an IgG ₁ Fc domain, particularly the 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, particularly P329G (according to Kabat EU index numbers). In one aspect, the Fc domain contains an amino acid substitution at position P329 and additional amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (according to Kabat EU index numbers). In a more specific aspect, the additional amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a preferred aspect, the Fc domain contains amino acid substitutions at positions P329, L234, and L235 (according to Kabat EU index numbers). In a more preferred aspect, the Fc domain contains amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”). Specifically, in a preferred aspect, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A, and P329G (Kabat EU index number), that is, in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (according to the Kabat EU index number).

In one aspect, the Fc domain is the IgG ₁ Fc domain, particularly the human IgG ₁ Fc domain. The amino acid-substituted “P329G LALA” combination almost completely eliminates Fcγ receptor (and complement) binding of the human IgG ₁ Fc domain, as described in PCT Publication No. WO 2012/130831, the entire contents of which are incorporated herein by reference. WO 2012/130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties, such as Fc receptor binding or effector function.

Compared to _IgG1 antibodies, _IgG4 antibodies exhibit reduced binding affinity to the Fc receptor and reduced effector function. Therefore, in some aspects, the Fc domain of the (multispecific) antibody of the present invention is an _IgG4 Fc domain, particularly a human _IgG4 Fc domain. In one aspect, the _IgG4 Fc domain contains an amino acid substitution at position S228, particularly amino acid substitution S228P (according to Kabat EU index number). To further reduce its binding affinity to the Fc receptor and/or its effector function, in one aspect, the _IgG4 Fc domain contains an amino acid substitution at position L235, particularly amino acid substitution L235E (according to Kabat EU index number). In another aspect, the _IgG4 Fc domain contains an amino acid substitution at position P329, particularly amino acid substitution P329G (according to Kabat EU index number). In a preferred aspect, the IgG ₄ Fc domain comprises amino acid substitutions at positions S228, L235, and P329, particularly amino acid substitutions S228P, L235E, and P329G (according to Kabat EU index number). Such IgG ₄ Fc domain mutants and their Fcγ receptor-binding properties are described in PCT Publication No. WO 2012/130831, the entire contents of which are incorporated herein by reference.

In a preferred aspect, the Fc domain exhibiting reduced binding affinity to the Fc receptor and/or reduced effector function compared to the native _IgG1 Fc domain is a human _IgG1 Fc domain containing amino acid substitutions of L234A, L235A, and optionally P329G, or a human _IgG4 Fc domain containing amino acid substitutions of S228P, L235E, and optionally P329G (according to the Kabat EU index number).

In some respects, N-glycosylation of the Fc domain has been eliminated. In one such respect, the Fc domain contains an amino acid mutation at position N297, specifically replacing the amino acid substitution of asparagine with either alanine (N297A) or aspartic acid (N297D) (according to the Kabat EU index number).

In addition to the Fc domains described above and in PCT Publication WO 2012/130831, Fc domains with reduced Fc receptor binding and/or decreased effector function also include those Fc domains having substitutions for one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056) (according to Kabat's EU Index No.). Such Fc mutants include Fc mutants with substitutions at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

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

Binding to the Fc receptor can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare) and the Fc receptor can be obtained, for example, through recombinant expression. Alternatively, the binding affinity of the Fc domain or (multispecific) antibodies containing the Fc domain to the Fc receptor can be assessed using cell lines known to express a specific Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor).

The effector function of an Fc domain or an Fc domain-containing (multispecific, e.g., bispecific) antibody can be measured by methods known in the art. Examples of in vitro assays for assessing ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., JExp Med 166,1351-1361 (1987). Alternatively, non-radioactive assays can be used (see, for example, the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of molecules of interest can be assessed in vivo, for example, in animal models such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95,652-656 (1998).

In some respects, the binding of the Fc domain to complement components, particularly C1q, is reduced. Therefore, in some respects, the Fc domain is engineered to have reduced effector functions, including reduced CDC. C1q binding assays can be performed to determine whether the Fc domain, or an Fc-containing (multispecific, e.g., bispecific) antibody, is capable of binding C1q and thus possessing CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., JImmunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

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

B. Polynucleotides

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

The polynucleotide encoding the (multispecific, e.g., bispecific) antibody of the present invention can be expressed as a single polynucleotide encoding the complete antibody, or as multiple (e.g., two or more) polynucleotides co-expressed. The polypeptides encoded by the co-expressed polynucleotides can associate via, for example, disulfide bonds or other means to form a functional antibody. For example, the light chain portion of the antibody can be encoded by a polynucleotide separate from the portion of the antibody comprising the heavy chain. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form the antibody. In another example, an antibody comprising a subunit of one of two Fc domain subunits and optionally a portion of one or more Fab molecules can be encoded by a polynucleotide separate from the antibody comprising the other subunit of the two Fc domain subunits and optionally a portion of a Fab molecule. When co-expressed, the Fc domain subunits will associate to form the Fc domain.

In some respects, the isolated polynucleotides encode the complete antibody molecule according to the invention as described herein. In other respects, the isolated polynucleotides encode polypeptides contained in antibodies according to the invention as described herein.

In some respects, the polynucleotide or nucleic acid is DNA. In other respects, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

C. Recombination Methods

The antibodies of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the antibody, such as those described above, are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one aspect, vectors, particularly expression vectors, containing the polynucleotides of the present invention (i.e., single or multiple polynucleotides) are provided. Expression vectors containing the coding sequence of the antibody and appropriate transcription/translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See, for example, the techniques described in the following literature: 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). Expression vectors can be plasmids, part of a virus, or fragments of nucleic acids. An expression vector includes an expression cassette into which a multinucleotide encoding an antibody (i.e., the coding region) is operatively associated with a promoter and/or other transcriptional or translational control elements and cloned. As used herein, a “coding region” is a portion of a nucleic acid consisting of codons that translate into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into amino acids, it can be considered part of the coding region (if present), while 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 multinucleotide construct (e.g., on a single vector) or in separate multinucleotide constructs (e.g., on separate (different) vectors). Furthermore, any vector may contain a single coding region or may contain two or more coding regions; for example, the vectors of the present invention may encode one or more polypeptides that are cleaved into the final protein post-translation or during translation via proteolytic cleavage. Furthermore, the vector, polynucleotide, or nucleic acid of the present invention may encode a heterologous coding region, which may or may not be fused with a polynucleotide or a variant or derivative thereof encoding an antibody of the present invention. The heterologous coding region includes, but is not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association is when the coding region of a gene product (e.g., a polypeptide) is associated in a way that brings the expression of the gene product under the influence or control of the regulatory sequence. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are “operably associated” if the induction of promoter function leads to transcription of mRNA encoding the desired gene product, and if the nature of the connection between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or the ability to interfere with the gene template to be transcribed. Therefore, if the promoter can influence the transcription of the nucleic acid, the promoter region will operably associate with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs the substantial transcription of DNA only in a predetermined cell. In addition to promoters, other transcriptional control elements, such as enhancers, operons, repressors, and transcription termination signals, can operatively associate with polynucleotides to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. A variety of transcriptional control regions are known to those skilled in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g., the immediate early promoter binding intron-A), simian virus 40 (e.g., the early promoter), and retroviruses (e.g., Raoult sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those skilled in the art. These translation control elements include, but are not limited to, ribosome binding sites, translation start and stop codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). Expression cassettes may also include other features such as 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 polynucleotide and nucleic acid coding regions of the present invention can associate with additional coding regions encoding secretory peptides or signal peptides, which in turn direct the secretion of polypeptides encoded by the polynucleotides of the present invention. For example, if antibody secretion is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid of an antibody or fragment thereof of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the protein chain has begun to export across the rough endoplasmic reticulum. Those skilled in the art know that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secretory or “mature” form of the polypeptide. In some aspects, a natural signal peptide (e.g., an immunoglobulin heavy or light chain signal peptide) or a functional derivative of that sequence that retains the ability to direct the secretion of a polypeptide with which it operatively associates can be used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

DNA encoding short protein sequences (which can be used to facilitate subsequent purification (e.g., histidine tagging) or to help label antibodies) can be contained inside or at the end of the antibody (fragment) encoding a polynucleotide.

In another aspect, host cells comprising the polynucleotides (i.e., a single polynucleotide or multiple polynucleotides) of the present invention are provided. In some aspects, host cells comprising the vectors of the present invention are provided. The polynucleotides and vectors may be incorporated, alone or in combination, into any features described herein with respect to the polynucleotides and vectors, respectively. In one such aspect, the host cell comprises one or more vectors (e.g., transformed or transfected with one or more vectors) containing one or more polynucleotides encoding (a portion of) the antibody of the present invention. As used herein, the term “host cell” refers to any kind of cellular system that can be engineered to produce the antibody or fragment thereof of the present invention. Host cells suitable for replicating and supporting antibody expression are well known in the art. Such cells can be appropriately transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculation of large-scale fermenters to obtain sufficient quantities of antibody for clinical application. Suitable host cells include prokaryotic microorganisms, such as *Escherichia coli*, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, peptides can be produced in bacteria, particularly when glycosylation is not required. After expression, peptides can be separated from bacterial cell pastes in soluble fractions and can be further purified. Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding peptides, including fungal and yeast strains whose glycosylation pathways have been “humanized,” resulting in peptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for expressing (glycosylated) peptides also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES ^™ technology for generating antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspensions may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed from SV40; human embryonic kidney lines (293 or 293T cells, as described, for example, in Graham et al., J Gen Virol 36, 59 (1977)); young hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol Reprod 23, 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (as described, for example, in Mather et al., Annals NYAcad Sci 383, 44-68 (1982)); and MRC. 5 cells, as well as FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr ^- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77,4216 (1980)); and myeloma cell lines such as YO, NSO, P3X63, and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255–268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few, as well as cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In one respect, the host cell is a eukaryotic cell, particularly a mammalian cell, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphocytes (e.g., Y0, NSO, Sp20 cells). In another respect, the host cell is not a cell within the human body.

Standard techniques for expressing exogenous genes in these systems are known in the art. Cells expressing polypeptides (e.g., antibodies) containing both heavy and light chains of antigen-binding domains can be engineered to also express another antibody chain, such that the expressed product is an antibody with both heavy and light chains.

In one aspect, a method for producing an antibody according to the invention is provided, wherein the method comprises culturing a host cell containing a polynucleotide encoding an antibody as provided herein under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

The components of the (multispecific, e.g., bispecific) antibody of the present invention can be genetically fused together. The (multispecific, e.g., bispecific) antibody can be engineered such that its components fuse directly or indirectly with each other via adapter sequences. The composition and length of the adapter can be determined according to methods well known in the art, and the efficacy of the adapter can be tested. Examples of adapter sequences between different components of the (multispecific) antibody are provided herein. Additional sequences (e.g., endopeptidase recognition sequences) may be included, if desired, to incorporate cleavage sites to separate the fused components.

The antibodies prepared as described herein can be purified using techniques known in the art, such as high-performance liquid chromatography (HPLC), ion-exchange chromatography (IEC), gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The specific conditions used for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens that bind to the antibody can be used. For example, for affinity chromatography purification of the antibodies of the present invention, a matrix containing protein A or protein G can be used. Essentially as described in the examples, antibodies can be separated using sequential protein A or G affinity chromatography and size exclusion chromatography. The purity of the antibody can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-performance liquid chromatography, etc.

D. Measurement

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

1. Combined with measurement

The binding (affinity) of an antibody to an Fc receptor or target antigen can be determined, for example, using standard instruments such as BIAcore instruments (GE Healthcare) and receptors or target proteins that can be obtained through recombinant expression, by surface plasmon resonance (SPR). Alternatively, cell lines expressing specific receptors or target antigens can be used, for example, by flow cytometry (FACS) to assess the binding of antibodies to different receptors or target antigens. Specific illustrative and exemplary aspects for measuring binding activity with CD3 are described below. The assay shown can be readily adapted to measure binding activity with FolR1 by using the FolR1 antigen instead of the CD3 antigen, and minor adjustments can be easily identified by a skilled technician.

In one respect, the binding activity to CD3 was determined by SPR as follows:

SPR was performed on a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibody (GE Healthcare #28958325) was immobilized on the S-Series sensor chip CM5 (GE Healthcare) using standard amine coupling chemistry at a surface density of 4000 to 6000 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% surfactant P20) was used as the run and dilution buffer. CD3 antibody at a concentration of 2 μg/mL (in 20 mM His, 140 mM NaCl, pH 6.0) was injected at a flow rate of 5 μL/min over approximately 60 s. The CD3 antigen used was a heterodimer of the extracellular domains of CD3δ and CD3ε, fused to a human Fc domain with a club-and-socket structure modification and a C-terminal Avi tag (see SEQ ID NO:28 and SEQ ID NO:29). A concentration of 10 μg/mL of CD3 antigen was injected over 120 s, and dissociation was monitored at a flow rate of 5 μL/min over approximately 120 s. The chip surface was regenerated by injecting 10 mM glycine (pH 2.1) twice consecutively (approximately 60 s each time). Bulk refractive index bias was corrected by subtracting the blank injection and by subtracting the response obtained from the blank control flow cell. For evaluation, the binding response was acquired 5 seconds after injection. To normalize the binding signal, CD3 binding was divided by the anti-Fab response (the signal (RU) obtained when CD3 antibody is captured on immobilized anti-Fab antibody). The binding activity of antibodies to CD3 relative to the binding activity of antibodies after different treatments (also known as relative activity concentration (RAC)) is calculated by referring to the binding activity of antibody samples after specific treatments and the binding activity of corresponding antibody samples after different treatments.

2. Activity Assay

The bioactivity of the (multispecific, e.g., bispecific) antibodies of the present invention can be measured by various assays as described in the examples. Bioactivity may include, for example, induction of T cell proliferation, induction of T cell signaling, induction of expression of activation markers in T cells, induction of T cell cytokine secretion, induction of target cell (e.g., tumor cell) lysis, and induction of tumor regression and/or improved survival.

E. Composition, Formulation and Administration

In another aspect, the present invention provides pharmaceutical compositions comprising any of the (multispecific, e.g., bispecific) antibodies provided herein, for example, in any of the following treatment methods. In one aspect, the pharmaceutical composition comprises an antibody according to the invention, and a pharmaceutical carrier. In another aspect, the pharmaceutical composition comprises a (multispecific, e.g., bispecific) antibody according to the invention, and at least one additional therapeutic agent as described below.

A method for generating the antibody of the invention in a form suitable for in vivo administration is also provided, the method comprising (a) obtaining the antibody according to the invention, and (b) formulating the antibody with at least one pharmaceutical carrier, thereby formulating an antibody preparation for in vivo administration.

The pharmaceutical compositions of the present invention comprise an effective amount of antibody lysed or dispersed in a pharmaceutical carrier. The term "pharmaceutical" means that the molecular entity and composition are generally non-toxic to the recipient at the doses and concentrations employed, i.e., do not produce adverse, allergic, or other adverse reactions when administered to animals (e.g., humans) as appropriate. Preparation of pharmaceutical compositions containing antibodies and optionally additional active ingredients will be known to those skilled in the art according to this disclosure, as illustrated in Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it should be understood that the preparations should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biostandards or other relevant national authorities. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, “pharmaceutical carrier” includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, antioxidants, proteins, pharmaceuticals, pharmaceutical stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, similar substances, and combinations thereof, as should be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). The use of said carriers in pharmaceutical compositions is contemplated, except in cases where any conventional carrier is incompatible with the active ingredient.

The (multispecific, e.g., bispecific) antibodies (and any other therapeutic agents) of the present invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired, for local treatment or intralesional application. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration can be carried out by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term.

Parenteral compositions include those designed for injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intra-arterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the antibodies of the present invention can be formulated in aqueous solutions, particularly in physiologically compatible buffers (e.g., Hanks' solution, Ringer's solution, or saline buffer). The solution may contain a formulatory agent, such as a suspending agent, stabilizer, and/or dispersant. Alternatively, the antibody may be in powder form for use with a suitable medium (e.g., sterile pyrogen-free water) prior to use. Sterile injectable solutions are prepared by incorporating the antibodies of the present invention in the desired amount with various other components listed below into a suitable solvent as needed. For example, sterility can be readily achieved by filtration through a sterile filter membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile medium containing a base dispersion medium and/or other components. In the case of preparing sterile powders for sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or lyophilization, which produces a powder of the active ingredient from a previously sterile filtered liquid medium, plus any additional desired ingredients. If necessary, the liquid medium should be appropriately buffered, and sufficient saline or glucose should be used to make the liquid diluent isotonic before injection. The composition must be stable under the preparation and storage conditions and preserved against contamination by microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a safe level, for example, below 0.5 ng/mg protein. Suitable pharmaceutical carriers include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexachlorocyclohexane quaternary ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, etc. Optionally, the suspension may also contain suitable stabilizers or agents that increase the degree of compound degradation to allow for the preparation of high-concentration solutions. Additionally, suspensions of active compounds can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes.

The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by, for example, cohesive techniques or interfacial polymerization; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include a semi-permeable matrix containing a solid hydrophobic polymer of a polypeptide, said matrix being in the form of a molded article, such as a membrane or microcapsule. In certain aspects, prolonged absorption of the injectable composition can be achieved by using a delayed-absorption agent (e.g., aluminum monostearate, gelatin, or combinations thereof) in said composition.

In addition to the compositions previously described, antibodies can also be formulated into reservoir formulations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular implantation) or by intramuscular injection. Thus, for example, antibodies can be formulated with suitable polymeric or hydrophobic materials (e.g., as emulsions in acceptable oils) or with ion exchange resins, or as slightly soluble derivatives, such as slightly soluble salts.

Pharmaceutical compositions comprising the (multispecific, e.g., bispecific) antibodies of the present invention can be prepared by conventional methods of mixing, lysis, emulsification, encapsulation, embedding, or lyophilization. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate the processing of proteins into pharmaceutically usable preparations. Suitable formulations depend on the chosen route of administration.

Antibodies can be formulated into compositions in the form of free acids or bases, neutral or salts. Pharmaceutical salts are salts that essentially retain the biological activity of the free acid or free base. These pharmaceutical salts include acid addition salts, such as those formed with the free amino groups of protein compositions, or those formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents compared to their corresponding free base forms.

F. Treatment methods and compositions

Any of the (multispecific, e.g., bispecific) antibodies provided herein can be used in therapeutic applications. The antibodies of this invention can be used as immunotherapeutic agents, for example, for the treatment of cancer.

For use in treatments, the (multispecific, e.g., bispecific) antibodies of the present invention will be formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of drug delivery, the method of administration, the timing of administration, and other factors known to the practicing physician.

In one aspect, the (multispecific, e.g., bispecific) antibody of the present invention is provided for use as a medicine. In another aspect, the (multispecific, e.g., bispecific) antibody of the present invention is provided for treating diseases. In some aspects, the (multispecific, e.g., bispecific) antibody of the present invention is provided for use in treatment methods. In one aspect, the present invention provides the (multispecific, e.g., bispecific) antibody of the present invention for treating a disease in an individual in need. In some aspects, the present invention provides a method of using the (multispecific, e.g., bispecific) antibody to treat an individual suffering from a disease, the method comprising administering an effective amount of the antibody to the individual. In some aspects, the disease to be treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In some aspects, if the disease to be treated is cancer, the method further comprises administering an effective amount of at least one other therapeutic agent, such as an anticancer agent, to the individual. In another aspect, the present invention provides the antibody of the present invention for inducing the lysis of target cells, particularly tumor cells. In some aspects, the present invention provides the (multispecific, e.g., bispecific) antibody of the present invention for use in a method of inducing the lysis of target cells, particularly tumor cells, in an individual, the method comprising administering an effective amount of the antibody to the individual to induce the lysis of target cells. According to any of the above, the "individual" is a mammal, preferably a human.

In another aspect, the present invention provides the use of the (multispecific, e.g., bispecific) antibody of the present invention in the preparation or preparation of a medicament. In one aspect, the medicament is used to treat a disease in an individual with this need. In another aspect, the medicament is used in a method of treating a disease, the method comprising administering an effective amount of the medicament to an individual suffering from the disease. In some aspects, the disease to be treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In one aspect, the method further comprises administering a therapeutically effective amount of at least one other therapeutic agent to the individual, for example, an anticancer agent if the disease to be treated is cancer. In another aspect, the medicament is used to induce the lysis of target cells, particularly tumor cells. In yet another aspect, the method of using the medicament to induce the lysis of target cells, particularly tumor cells, in an individual comprises administering an effective amount of the medicament to the individual to induce the lysis of the target cells. The "individual" according to any of the foregoing aspects can be a mammal, preferably a human.

In another aspect, the present invention provides a method for treating a disease. In one aspect, the method includes administering an effective amount of an antibody of the present invention to an individual suffering from the disease. In another aspect, the individual is given a composition comprising an antibody of the present invention in a pharmaceutical form. In some aspects, the disease to be treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In some aspects, if the disease to be treated is cancer, the method further includes administering an effective amount of at least one other therapeutic agent, such as an anticancer agent, to the individual. The "individual" according to any of the foregoing aspects can be a mammal, preferably a human.

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

In some respects, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, leukemia, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other proliferative disorders that can be treated with the antibodies of this invention include, but are not limited to, tumors located in the following sites: abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral nervous systems), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included. In some respects, cancers are 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 respect, particularly where the antibody is a bispecific antibody that binds to FolR1 as a second antigen, the cancer is a cancer that expresses (or overexpresses) FolR1. In another respect, particularly where the antibody is a bispecific antibody that binds to FolR1 as a second antigen, the cancer is ovarian cancer, lung cancer, breast cancer, or kidney cancer. Technicians readily recognize that in many cases, antibodies may not provide a cure, but may only provide partial benefit. In some respects, physiological changes that have certain benefits are also considered to have therapeutic benefits. Therefore, in some respects, the amount of antibody that provides physiological changes is considered an "effective amount." The subjects, patients, or individuals requiring treatment are typically mammals, and more particularly humans.

In some respects, an effective amount of the antibodies of the present invention will be administered to an individual to treat a disease.

For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the antibody, and the judgment of the attending physician. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for the individual subject. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

Antibodies are administered to patients either once or as part of a series of treatments. Depending on the type and severity of the disease, an antibody at a dose of about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg–10 mg/kg) may be an initial candidate dose administered to the patient, for example, by a single or multiple administrations alone or by continuous infusion. Depending on the factors described above, a typical daily dose range may be from about 1 μg/kg to 100 mg/kg or more. For repeated administrations over several days or longer, treatment typically continues until the desired suppression of disease symptoms occurs, depending on the condition. An exemplary dose range for antibodies is from about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, the dosage may also include administration of 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 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg body weight, up to about 1000 mg/kg body weight or more, and any range derived therefrom. In non-limiting examples of ranges that can be derived from the figures listed herein, ranges such as about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 micrograms/kg body weight to about 500 mg/kg body weight, etc., may be administered based on the above values. Therefore, patients can 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 can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about two to about twenty doses, or, for example, about six doses of the antibody). A higher initial loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be useful. Progression to this therapy is easily monitored using routine techniques and assays.

The antibodies of the present invention will generally be used in an amount that effectively achieves the intended purpose. For the treatment or prevention of a condition, the antibodies of the present invention or their pharmaceutical compositions will be administered or applied in an effective amount.

For systemic administration, the effective dose can be initially estimated based on in vitro assays (such as cell culture assays). The dose can then be formulated in animal models to achieve a range of circulating concentrations including the _IC50, as determined in cell cultures. This information can be used to more accurately determine the useful dose for humans.

The initial dose can also be estimated using techniques known in the art, based on in vivo data (e.g., animal models).

The dosage and interval can be individually adjusted to provide sufficient plasma antibody levels to maintain therapeutic efficacy. The usual patient dosage range by injection is approximately 0.1 to 50 mg/kg/day, typically approximately 0.5 to 1 mg/kg/day. Therapeuticly effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by HPLC.

Effective doses of the antibodies of the present invention will generally provide therapeutic benefits without causing significant toxicity. The toxicity and therapeutic efficacy of the antibodies can be determined by standard pharmaceutical methods in cell culture or laboratory animals. Cell culture assays and animal studies can be used to determine the _LD50 (the dose at which 50% of the population is lethal) and _ED50 (the dose at which 50% of the population is therapeutically effective). The dose ratio between toxicity and efficacy is the therapeutic index, which can be expressed as the ratio _LD50 / _ED50 . Antibodies exhibiting a large therapeutic index are preferred. In one aspect, the antibodies according to the present invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of doses suitable for human use. The dose is preferably within a range including cyclic concentrations with little or no toxicity at _ED50 . The dose can vary within this range depending on various factors, such as the dosage form used, the route of administration employed, the patient's condition, etc. The exact formulation, route of administration, and dosage can be chosen by an individual physician based on the patient's condition (see, for example, Fingl et al., 1975, in The Pharmacological Basis of Therapeutics, Chapter 1, page 1, the entire contents of which are incorporated herein by reference).

The attending physician of a patient treated with the antibody of this invention will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The dosage administered in the management of the target condition will vary depending on the severity of the condition being treated, the route of administration, etc. For example, the severity of the condition can be assessed in part by standard prognostic assessment methods. Furthermore, the dosage and possible dosing frequency will also vary based on the individual patient's age, weight, and response.

The (multispecific, e.g., bispecific) antibodies of the present invention can be administered in combination with one or more other agents in treatment. For example, the antibodies of the present invention can be administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat symptoms or diseases of an individual in need of such treatment. Such additional therapeutic agents may contain any active ingredient suitable for the specific disease being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In some aspects, the additional therapeutic agent is an immunomodulator, a cell growth inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis inducers. In a preferred aspect, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, antimetabolite, topoisomerase inhibitor, DNA intercalating agent, alkylating agent, hormone therapy, kinase inhibitor, receptor antagonist, tumor cell apoptosis activator, or antiangiogenic agent.

Other such agents are appropriately available in combinations of amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of antibody used, the type of disease or treatment, and other factors discussed above. Antibodies are typically used at the same dosage and route of administration as described herein, or at approximately 1% to 99% of the dosage described herein, or at any dosage and route of administration empirically/clinically determined to be appropriate.

Such combination therapies include combined administration (where two or more therapeutic agents are included in the same or different compositions) and single administration. In the case of single administration, the administration of the (multispecific, e.g., bispecific) antibody of the present invention can be performed before, simultaneously with, and/or after the administration of additional therapeutic agents and/or adjuvants. The antibody of the present invention can also be used in combination with radiotherapy.

G. Products

In another aspect of the invention, an article is provided containing a substance that can be used to treat, prevent, and/or diagnose the aforementioned conditions. The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container contains a composition that, on its own or in combination with another composition, is effective for treating, preventing, and/or diagnosing the condition, and the container may have a sterile inlet (e.g., the container may be an IV solution bag or vial with a stopper capable of being punctured by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is for treating the selected condition. Furthermore, the article may include (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising additional cytotoxic agents or other therapeutic agents. The article in this aspect of the invention may also include a package insert indicating that the composition is for treating a specific condition. Alternatively or additionally, the article may also include a second (or third) container comprising pharmaceutical buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. The kit may also include other substances required from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

H. Methods and compositions for diagnosis and detection

In some respects, any of the antibodies described herein can be used to detect the presence of their targets (e.g., CD3, FolR1) in biological samples. As used herein, the term "detection" encompasses both quantitative and qualitative detection. In some respects, biological samples include cells or tissues, such as prostate tissue.

In one aspect, an antibody according to the invention is provided for use in diagnostic or detection methods. In another aspect, a method for detecting the presence of CD3 and FolR1 in a biological sample is provided. In some aspects, the method includes contacting the biological sample with the antibody of the invention under conditions that allow the antibody to bind to CD3 and FolR1, and detecting whether a complex is formed between the antibody and CD3 and FolR1. Such methods may be in vitro or in vivo. In one aspect, the antibody of the invention is used to select subjects suitable for treatment with an antibody that binds to CD3 and FolR1, for example, where CD3 and FolR1 are biomarkers for patient selection.

Exemplary conditions that can be diagnosed using the antibodies of this invention include cancer, particularly skin cancer or brain cancer.

In some aspects, antibodies according to the invention are provided, wherein the antibodies are labeled. Labeling includes, but is not limited to, labels or portions that are directly detectable (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detectable (e.g., by enzymatic reactions or molecular interactions) (such as enzymes or ligands). Exemplary labels include, but are not limited to, radioactive isotopes ^32P , ^14C , ^125I , ^3H , and ^131I ; fluorophores, such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl, and umbelliferone; luciferases, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456); insect luciferin, 2,3-dihydrodiazanaphthyldione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosylamylase, and lysozyme; sugar oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as urate oxidase and xanthine oxidase; conjugation to enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase, or microperoxidase); biotin/antibiotin protein, spinning labels, phage labels, stable free radicals, and so on.

III. Sequence

Bispecific CD3/FolR1 antibody sequence:

According to Kabat's CDR definition

IV. Examples

The following are examples of the methods and compositions of the present invention. It should be understood that various other aspects can be practiced given the general description provided above.

Example 1 - Optimized CD3 binder generation

Starting with the previously described (see, for example, WO 2014/131712, which is incorporated herein by reference) CD3 binder (referred to herein as “CD3 _orig ” and containing the VH and VL sequences of SEQ ID NO:6 and SEQ ID NO:11, respectively), the aim is to optimize the properties of the binder by removing two asparagine deamidation sequence motifs at Kabat positions 97 and 100 of the heavy chain CDR3.

To this end, an antibody library suitable for phage display was generated, in which asparagine at positions 97 and 100 of the Kabat was removed and CDRs H1, H2 and H3 were randomly assigned to compensate for the loss of affinity caused by replacing Asn97 and Asn100 through the affinity maturation process.

The library was placed on a filamentous phage by fusing with the minor coat protein p3 (Marks et al. (1991) JMol Biol 222, 581-597) and selectively bound to recombinant CD3ε.

Ten candidate clones were identified in the initial screening, which, as Fab fragments, showed acceptable binding to the recombinant antigen as determined by SPR (produced in E. coli).

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

The selected clone, referred to in this document as “CD3 _opt ”, was further evaluated and contained the VH and VL sequences of SEQ ID NO:7 and SEQ ID NO:11, respectively, and was converted into a bispecific form as described below.

Example 2 - Binding of the optimized CD3 binder to CD3

Combining with recombinant CD3

The binding of the optimized CD3 binder “CD3 _opt ” and the original CD3 binder “CD3 _orig ” to recombinant CD3 was determined using surface plasmon resonance (SPR). Both binders were in the form of human IgG1 containing the P329G L234A L235A (“PGLALA”, according to EU number) mutation in the Fc region (SEQ ID NO:12 and SEQ ID NO:14 (CD3 _orig ) and SEQ ID NO:13 and SEQ ID NO:14 (CD3 _opt )).

To evaluate the effectiveness of deamidation and its impact on antibody stability, the binding of the original and optimized CD3 binding agents to recombinant CD3 was measured after 14 days of storage at 37°C or 40°C. Samples stored at -80°C were used as references. Reference samples, samples stressed at 40°C in 20 mM His, 140 mM NaCl, pH 6.0, and samples stressed at 37°C in PBS, pH 7.4, were used at concentrations ranging from 1.2 to 1.3 mg/ml. After the 14-day stress period, samples in PBS were dialyzed back into 20 mM His, 140 mM NaCl (pH 6.0) for further analysis.

The relative activity concentration (RAC) of the sample was determined using SPR following these steps.

SPR was performed on a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibody (GE Healthcare, #28958325) was immobilized on the S-Series sensor chip CM5 (GE Healthcare) using standard amine coupling chemistry to achieve a surface density of 4000 to 6000 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% surfactant P20) was used as the run and dilution buffer. A 2 μg/mL concentration of CD3 antibody was injected at a flow rate of 5 μL/min over 60 s. A 10 μg/mL concentration of CD3 antigen (see below) was injected over 120 s, and dissociation was monitored at a flow rate of 5 μL/min over 120 s. The chip surface was regenerated by two consecutive injections of 10 mM glycine (pH 2.1) at 60 s each. Large refractive index bias was corrected by subtracting the blank injection and the response obtained from the blank control flow cell. For evaluation, binding responses were acquired 5 seconds after injection. To normalize the binding signal, CD3 binding was divided by the anti-Fab response (the signal (RU) obtained when CD3 antibody is captured on immobilized anti-Fab antibody). Relative activity concentrations were calculated by comparing each temperature-pressurized sample with the corresponding pressure-pressurized sample.

The antigen used is a heterodimer of the extracellular domains of CD3δ and CD3ε, which is fused to a human Fc domain with a club-and-socket structure modification and a C-terminal Avi tag (see SEQ ID NO:28 and SEQ ID NO:29).

The results of this experiment are shown in Figure 2. As can be seen from the figure, compared to the original CD3 binder CD3 _orig , the optimized CD3 binder CD3 _opt showed significantly improved binding to CD3 after temperature stress (exposure at 37°C and pH 7.4 for 2 weeks). This result demonstrates that the removal of the deamidation site was successful and yielded an antibody with excellent stability, the stability of which is related to the in vivo half-life and the formulation of the antibody at neutral pH.

Binding to CD3 on Jurkat cells

The binding of the optimized CD3 binder “CD3 _opt ” and the original CD3 binder “CD3 _orig ” to CD3 on the human reporter T cell line Jurkat NFAT was measured using SPR. Both binders were in the form of human IgG1 containing the P329G L234A L235A (“PGLALA”, according to EU number) mutation in the Fc region (SEQ ID NO:12 and SEQ ID NO:14 (CD3 _orig ) and SEQ ID NO:13 and SEQ ID NO:14 (CD3 _opt )).

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega#CS176501) are a human acute lymphoblastic leukemia reporter cell line containing the NFAT promoter and expressing human CD3. Cells were cultured at densities of 0.1 to 0.5 mI cells/mL in RPMI 1640, 2 g/L glucose, 2 g/L NaHCO3, ₁₀ % FCS, 25 mM HMEPES, 2 mM L-glutamine, 1x NEAA, and 1x sodium pyruvate. Hygromycin B was added to a final concentration of 200 μg/mL at each cell passage.

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

As shown in Figure 3, the optimized CD3 binder "CD3 _opt " and the original CD3 binder "CD3 _orig " both bind well to CD3 on Jurkat cells.

Example 3 – Functional Activity of Optimized CD3 Binder

The functional activity of the optimized CD3 binder "CD3 _opt " was detected in the Jurkat reporter cell assay and compared with that of the original CD3 binder "CD3 _orig ". To detect IgG functional activity, CHO cells expressing anti-PGLALA were co-incubated with Jurkat NFAT reporter cells in the presence of progressively increasing concentrations of either CD3 _opt (human IgG1 PGLALA) or CD3 _{orig (} human IgG1 PGLALA). Following T cell crosslinking, activation of CD3 on Jurkat NFAT reporter cells induced luciferase production, and luminescence was measured as a marker of activation. A negative control containing CD3 _orig (human IgG1 wt) was used, which failed to bind to CHO cells expressing anti-PGLALA and therefore could not undergo crosslinking on Jurkat NFAT cells. A schematic diagram of this assay is provided in Figure 4.

CHO cells expressing anti-PGLALA are CHO-K1 cells engineered to express antibodies (specifically, antibodies binding to human _IgG1 Fc (PGLALA)) on their surface (see WO 2017/072210, which is incorporated herein by reference). These cells were cultured in DMEM/F12 medium containing 5% FCS + 1% GluMax. Jurkat NFAT reporter cells were used as described in Example 2.

Upon simultaneous binding of anti-PGLALA expressed on CD3 huIgG1 PGLALA and CHO cells to CD3 expressed on Jurkat-NFAT reporter cells, the NFAT promoter was activated, leading to the expression of active firefly luciferase. The intensity of the luminescent signal (obtained after addition of the luciferase substrate) was proportional to the intensity of CD3 activation and signal transduction. Jurkat-NFAT reporter cells were grown in suspension and cultured at a density of 0.1–0.5 mI cells/mL in RPMI 1640, 2 g/L glucose, 2 g/L NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1x NEAA, 1x sodium pyruvate, and 200 μg/mL hygromycin. For assays, CHO cells were harvested, and cell viability was measured using ViCell. 30,000 target cells/well were seeded into 100 μL of medium in a flat-bottomed, white-walled 96-well plate (Greiner bio-one #655098), and 50 μL/well of diluted antibody or medium (used as a control) was added to CHO cells. Jurkat-NFAT reporter cells were then harvested and viability was assessed using ViCell. Cells were resuspended at a concentration of 1.2 mI cells/mL in hygromycin B-free cell culture medium and added to CHO cells at a density of 60,000 cells/well (50 μL/well) to obtain a final effector to target (E:T) ratio of 2:1, with a final volume of 200 μL per well. Then, 4 μL of GloSensor (Promega #E1291) was added to each well (2% of the final volume). Cells were incubated in a humidified incubator at 37°C for 24 hours. At the end of incubation, luminescence was detected using a TECAN Spark 10M.

As shown in Figure 5, the optimized CD3 binding agents CD3 _opt and CD3 _orig exhibit similar activity in Jurkat NFAT cells after cross-linking.

Example 4 - Generation of T-cell bispecific antibodies containing optimized CD3 binding agents

TYRP1 TCB

Using the optimized CD3 binding agents identified in Example 1 (“CD3 _opt ”, SEQ ID NO:7(VH) and SEQ ID NO:11(VL)), a T cell bispecific antibody (TCB) targeting CD3 and TYRP1 (“TYRP1 TCB”) was generated.

The TYRP1 binder contained in this TCB is generated by humanization of the TYRP1 binder “TA99” (see GenBank entries AXQ57811 and AXQ57813 for the heavy and light chains, respectively), and contains heavy and light chain variable region sequences as shown in SEQ ID NO:18 and SEQ ID NO:22, respectively.

A schematic diagram of the TCB molecule is provided in Figure 6, and its complete sequence is given in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:27.

Similar molecules with the original CD3 binding sequence (SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26) were also prepared.

The bispecific molecule was generated via transient transfection of HEK293 EBNA cells. Cells were transfected with the appropriate expression vector at a ratio of 1:2:1:1 (“carrier heavy chain (VH-CH1-VL-CH1-CH2-CH3)”:“carrier light chain (VL-CL)”:“carrier heavy chain (VH-CH1-CH2-CH3)”:“carrier light chain (VH-CL)”). Cells were centrifuged and then incubated with preheated CD CHO medium (Thermo Fisher, #10743029). The expression vector was mixed in CD CHO medium, PEI (polyethyleneimine, Polysciences, #23966-1) was added, the solution was vortexed, and incubated at room temperature for 10 min. Then, cells (2 mi/ml) were mixed with the vector/PEI solution, transferred to flasks, and incubated in a shaking incubator at 37°C for 3 h under a 5% _CO2 atmosphere. After incubation, add Excell medium (80% of total volume) containing supplements. One day after transfection, add supplements (feed, 12% of total volume). Seven days later, harvest the cell supernatant by centrifugation and subsequent filtration (0.2 μm filter).

Proteins were purified from filtered cell culture supernatant using standard methods. In short, 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) was used. Elution was achieved at pH 3.0, followed by immediate pH neutralization of the sample. Proteins were concentrated by centrifugation (Millipore ULTRA-15 (#UFC903096)) and the aggregated proteins were separated from monomeric proteins using size exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM histidine and 140 mM sodium chloride (pH 6.0).

The concentration of purified protein was determined by measuring absorbance at 280 nm, using mass extinction coefficients calculated based on amino acid sequences, as described by Pace et al. (1995) (Protein Science 4, 2411-23). Protein purity and molecular weight were analyzed by CE-SDS using a LabChip GXII (Perkin Elmer) in the presence and absence of a reducing agent (Table 1). Aggregate content was determined by HPLC at 25 °C using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffers (25 mM _K₂HPO₄ , 125 mM NaCl, 200 mM L-arginine hydrochloride (pH 6.7) or 200 mM _KH₂PO₄ , 250 _mM KCl (pH 6.2)) ₍ Table 2).

Table 1. CE-SDS analysis of TYRP1 TCB (non-reducing form).

molecular Peak number Size [kDa] purity[%] <![CDATA[TYRP1 TCB CD3_opt]]> 1 221 100 <![CDATA[TYRP1 TCB CD3_orig]]> 1 206 100

Table 2. Summary of the generation and purification of TYRP1 TCB.

Example 5 - Binding of T-cell bispecific antibodies containing optimized CD3 binding agents to CD3 and TYRP1

Combining with recombinant CD3

The binding of TYRP1 TCB to recombinant CD3 was evaluated using SPR, with TCBs containing either an optimized CD3 binding sequence (TYRP1 TCB CD3 _opt ) or the original CD3 binding sequence (TYRP1 TCB CD3 _orig ).

The SPR experiment was performed using a Biacore T200, which used HBS-EP as the run buffer (0.01M MEPES pH 7.4, 0.15M NaCl, 0.05% (v/v) surfactant P20 (GE Healthcare)).

TYRP1 TCB was captured onto the surface of a CM5 sensor chip containing an immobilized antibody that specifically binds to human _IgG1 Fc (PGLALA) (see WO 2017/072210, which is incorporated herein by reference). The capture antibody was conjugated to the sensor chip surface by directly immobilizing approximately 8700 resonance units (RUs) at pH 5.0 using a standard amine conjugation kit (GE Healthcare). TCB molecules were captured for 30 s at a flow rate of 10 μL/min at 5 nM.

Human and cynomolgus monkey antigens (see below) were administered at concentrations ranging from 12.35 to 3000 nM, flowing through the cells at a flow rate of 30 μL/min over 240 s. The dissociation phase was monitored for 240 s and triggered by switching from the sample solution to HBS-EP. After each cycle, 10 mM glycine (pH 2.0) was injected once every 30 s to regenerate the chip surface.

The antigen used is a heterodimer of human or cynomolgus CD3δ and CD3ε extracellular domains fused with a human Fc domain containing a club-and-socket structure modification and a C-terminal Avi tag (see SEQ ID NO:28 and SEQ ID NO:29 (human CD3) and SEQ ID NO:30 and SEQ ID NO:31 (cynomolgus CD3)).

The bulk refractive index bias was corrected by subtracting the response obtained in the reference flow cell (without captured TCB). The affinity constant was obtained by fitting to a 1:1 Langmuir combination using BIAeval software (GE Healthcare) from the kinetic rate constant.

For TYRP1 TCB CD3 _opt , the measured _KD values for binding to CD3 in humans and cynomolgus monkeys were 50 nM and 20 nM, respectively, which are close to the values of TYRP1 TCB CD3 _orig (50 nM and 40 nM, respectively).

This indicates that, under stress-free conditions, both TCBs containing CD3 _opt or CD3 _orig bind quite well to recombinant CD3.

The binding of TYRP1 TCB to recombinant human CD3 was also evaluated after 14 days of temperature stress at 37°C or 40°C, using TCBs containing either an optimized CD3-binding sequence or the original CD3-binding sequence. This experiment was conducted as described in Example 2 above, where TCBs were used instead of IgG molecules.

The results of the experiment are shown in Figure 7.

As shown in Figure 7, compared with TCB containing the original CD3 binder CD3 _orig , TCB containing the optimized CD3 binder CD3 _opt exhibited significantly improved binding to CD3 after stress (exposure at 37°C and pH 7.4 for 2 weeks). This result confirms that the improved properties of the optimized CD3 binder (see Example 2) are maintained at the TCB level.

Combination with recombinant TYRP1

The binding to recombinant TYRP1 was assessed using SPR, which involved the use of TYRP1 Fab fragments prepared by plasmin digestion with the corresponding antibody.

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

Using a standard amine conjugation kit (GE Healthcare), antibodies specifically binding to human _IgG1 Fc (PGLALA) (see WO 2017/072210, which is incorporated herein by reference) were directly conjugated to the CM5 sensor chip at pH 5.0. The antigen (see below) was captured at a flow rate of 10 μL/min for 30 s. A series of samples, three-fold diluted with the TYRP1 Fab fragment, were passed through the flow cell at 30 μL/min for 180 s to record the association phase. The dissociation phase was monitored for 180 s or 1200 s and triggered by switching from the sample solution to HBS-EP. After each cycle, 10 mM glycine (pH 2) was injected once at a flow rate of 30 μL/min over 30 s to regenerate the chip surface.

The antigen used is a monomeric fusion with the extracellular domain (ECD) of human, cynomolgus monkey, or mouse TYRP1 containing a mortar structure (and PG LALA) modified with a C-terminal Avi tag human Fc domain (see SEQ ID NO:32 and SEQ ID NO:35 (human TYRP1), SEQ ID NO:33 and SEQ ID NO:35 (cynomolgus monkey TYRP1) or SEQ ID NO:34 and SEQ ID NO:35 (mouse TYRP1)).

Bulk refractive index bias was corrected by subtracting the response obtained in the reference flow cell (without capturing antigen). The affinity constant (K_{_D}) was derived from the kinetic rate constant using BIAeval software (GE Healthcare) by fitting a 1:1 Langmuir binding.

The measured _KD values for binding to TYRP1 in humans, cynomolgus monkeys, and mice were 130 pM, 180 pM, and 530 pM, respectively, which were similar to the results of the parental TA99 antibody (90 pM, 120 pM, and 310 pM, respectively).

The binding of TYRP1 TCB to recombinant TYRP1 was also evaluated 14 days after temperature stress at 37°C or 40°C, using TCBs containing either an optimized CD3-binding sequence or the original CD3-binding sequence.

The experiment was conducted as described above regarding binding to CD3, using recombinant TYRP1 (SinoBiologicals) as the antigen.

The results of this experiment are shown in Figure 8. The results confirm that the binding of the two TCBs (and the corresponding IgG forms of TYRP1 binders) to human TYRP1 is not affected by stress conditions.

Binding to CD3 on Jurkat cells

As described in Example 2 above, FACS was used to determine the binding of TYRP1 TCB containing the optimized CD3 binder "CD3 _opt " or the original CD3 binder "CD3 _orig " to CD3 on the human reporter T cell line Jurkat NFAT.

As shown in Figure 9, the binding of the TCB containing the optimized CD3 binder "CD3 _opt " to CD3 on Jurkat cells is at least comparable to that of the TCB containing the original CD3 binder "CD3 _orig ".

Example 6 - Functional activity of T-cell bispecific antibodies containing optimized CD3 binding agents

CD3 activation

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

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, leading to the expression of active firefly luciferase. The intensity of the luminescent signal (obtained after addition of the luciferase substrate) is proportional to the intensity of CD3 activation and signal transduction. This assay was performed as described in Example 3, using M150543 cells instead of CHO cells expressing anti-PGLALA.

As shown in the results for IgG (Example 3), the two TCBs containing CD3 _opt or CD3 _orig have similar functional activities on Jurkat NFAT reporter cells and induce CD3 activation in a concentration-dependent manner (Figure 10).

Target cell killing

Next, in the tumor cell killing assay, two TCB molecules were co-incubated with freshly isolated human PBMCs from three different donors and the human melanoma cell line M150543 for analysis. Tumor cell lysis was measured by LDH release after 24 and 48 hours. After 48 hours, activation of CD4 and CD8 T cells was analyzed by upregulation of CD69 and CD25 in two cell subsets.

In short, target cells were harvested with trypsin/EDTA, washed, and seeded in flat-bottomed 96-well plates at a density of 30,000 cells/well. Cell adhesion was allowed overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of fresh blood from healthy human donors. Fresh blood was diluted with sterile PBS and seeded onto Histopaque gradient solution (Sigma#H8889). After centrifugation (450x g, 30 min, room temperature), the plasma above the mesophase containing PBMCs was discarded, and the PBMCs were transferred to new Falcon tubes, which were then filled with 50 mL of PBS. The mixture was centrifuged (400x g, 10 min, room temperature), the supernatant was discarded, and the PBMC pellet was washed twice with sterile PBS (centrifugation step 350x g, 10 min). The resulting PBMC population was automatically counted (ViCell) and stored in RPMI 1640 medium (Biochrom#K0302) containing 10% FCS and 1% L-alanyl-L-glutamine in a cell culture incubator at 37°C and 5% _CO2 until further use (no longer than 24 h). For the killing assay, the specified concentration of antibody was added and repeated three times. PBMCs were added to target cells at a final effector to target (E:T) ratio of 10:1. After incubation at 37°C and 5% _CO2 for 24 h, the killing effect on target cells was assessed by quantifying the LDH released into the cell supernatant using an apoptotic/necrotic cell assay kit (Roche Applied Science#11 644 793 001). Maximum lysis (=100%) of target cells was achieved by incubating the target cells with 1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubated with effector cells, without bispecific constructs.

Flow cytometry was used to assess the activation of CD8 and CD4 T cells following T cell killing of target cells mediated by TCB, using antibodies recognizing T cell activation markers CD25 (a marker of late activation) and CD69 (a marker of early activation). After 48 h of incubation, PBMCs were transferred to round-bottom 96-well plates, centrifuged at 350 x g for 5 min, and washed twice with FACS buffer. Surface staining was performed on CD4 APC (BioLegend#300514), CD8 FITC (BioLegend#344704), CD25BV421 (BioLegend#302630), and CD69 PE (BioLegend#310906) cells according to the supplier's instructions. Cells were washed twice with 150 μL/well of FACS buffer and fixed with 100 μL/well of fixation buffer (BD#554655) at 4 °C for 15 min. After centrifugation, the samples were resuspended in 200 μL/well FACS buffer. The samples were then analyzed using BD FACS Fortessa.

On all three donors, both TCBs containing either the optimized CD3 binder or the original CD3 binder induced T cell activation and tumor cell lysis in a similar manner (Figure 11). The EC50 values of tumor cell lysis for all three antibodies after 48 h are summarized in Table 3.

Table 3. Summary of EC50 values of tumor cell killing by TYRP1 TCB at 48h.

Example 7 - PK Study of T Cell Bispecific Antibody in Mice

The pharmacokinetics (PK) of TYRP1 TCBs containing different CD3 binders (CD3 orig and CD3 opt) were investigated after intravenous bolus administration of 1 mg/kg to human FcRn transgenic mice (line32, _homozygous ) and FcRn knockout mice (Jackson Laboratory strains 003982 and ₀₁₄₅₆₅ ) (n = 3/strain/test compound). Serial micro-blood samples were collected from human FcRn transgenic (tg) mice for up to 672 h (9 samples per mouse from 5 min to 672 h post-dose) and from FcRn knockout (ko) mice for up to 96 h (8 samples per mouse from 5 min to 96 h post-dose). Serum was prepared and cryopreserved until analysis. Mouse serum samples were analyzed using a Roche e411 instrument under non-GLP conditions using a universal ECLIA method targeting the human Ig/Fab CH1/κ domain. Pharmacokinetic assessment was performed using standard non-compartmental analysis.

The results of this study are shown in Table 4. The results indicate that the engineering of the CDR did not induce susceptibility to other sequences that could potentially affect antibody clearance. CD3 _opt was as excellent as CD3 _orig in terms of serum half-life, while also providing the additional benefit of increased CDR stability.

Table 4. Clearance data (mL/day/kg; mean and (CV)) of huFcRn tg mice and FcRn ko mice.

Example 8 – Generation of another T-cell bispecific antibody containing an optimized CD3 binding agent

Using the optimized CD3 binding agent (“CD3 _opt ”, SEQ ID NO:7(VH) and SEQ ID NO:11(VL)) identified in Example 1, a T cell bispecific antibody (TCB) targeting CD3 and EGFRvIII (“EGFRvIII TCB”) was generated.

The EGFRvIII binder contained in this TCB (P063.056) is derived from phage display followed by affinity maturation (see below), and contains the heavy chain variable region sequence and the light chain variable region sequence shown in SEQ ID NO:88 and SEQ ID NO:92, respectively.

A schematic diagram of the TCB molecule is provided in Figure 6, and its complete sequence is given in SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:27.

Similar molecules with the original CD3 binding sequence (SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:26) were also prepared.

Bispecific molecules were generated by transiently transfecting HEK293 EBNA cells and purified and analyzed according to the method described in Example 4 above.

In addition, in a similar manner (for IgG heavy and light chains, HEK EBNA cells were transfected with an expression vector at a 1:1 ratio), human IgG ₁ -derived phage-displayed EGFRvIII antibodies were generated, which were used as described below.

As determined by size exclusion chromatography, all IgG and TCB constructs were purified to a comparable quality, with monomer content exceeding 95%.

Selection of EGFRvIII antibodies

The EGFRvIII antibodies are derived from phage display and are affinity-matured. Using CHO cells stably expressing EGFRvIII and the EGFRvIII-positive human glioblastoma cell line DK-MG, the binding of antibodies exhibiting high affinity and specificity to EGFRvIII (P056.021 (SEQ ID NO:40 and SEQ ID NO:44), P056.052 (SEQ ID NO:48 and SEQ ID NO:52), P047.019 (SEQ ID NO:56 and SEQ ID NO:60), P057.012 (SEQ ID NO:64 and SEQ ID NO:68), P057.011 (SEQ ID NO:72 and SEQ ID NO:76), P056.027 (SEQ ID NO:80 and SEQ ID NO:84)) to EGFRvIII expressed on the cell surface was tested. To confirm specificity and rule out cross-reactivity with wild-type EGFR (EGFRwt), the binding of the selected antibodies to the EGFRwt-positive human tumor cell line MKN-45 was tested (Figure 12). Cetuximab was used as a positive control for binding to EGFRwt, and non-targeting DP47 IgG was used as a negative control. All selected antibodies specifically bound to EGFRvIII and showed no cross-reactivity with EGFRwt, and further characterization was considered.

Next, DK-MG cells were co-incubated with Jurkat NFAT reporter cells expressing anti-PGLALA chimeric antigen receptor (CAR), and the functional activity of these EGFRvIII antibodies (human IgG1 form containing the P329G L234A L235A (“PGLALA”, according to EU number) mutations in the Fc region was assessed by measuring luminescence (CAR J assay, see PCT application number PCT/EP2018/086038, the full text of which is incorporated herein by reference). DP47 IgG1PGLALA was used as a negative control. All tested EGFRvIII antibodies induced significant activation in CAR-expressing Jurkat NFAT reporter cells (Figure 13). Except for P047.019, which showed the weakest binding and activation, all other tested EGFRvIII antibodies were converted to the TCB form (using CD _orig as a CD3 binding agent).

The binding of selected EGFRvIII antibodies converted to TCB form to CHO-EGFRvIII cells was compared with that of the corresponding IgG (Figure 14) to confirm that conversion to TCB form had no effect on EGFRvIII antibody binding ability. Most of the tested EGFRvIII clones retained their binding ability to EGFRvIII after conversion to TCB form; only clone P057.011 showed a slightly reduced binding of EGFRvIII to IgG in TCB form compared to the corresponding IgG (Table 5).

Table 5. Binding of EGFRvIII IgG and TCB to CHO-EGFRvIII (EC50).

EGFRvIII cloning EC50 IgG (nM) EC50 TCB (nM) P056.021 16.5 13.0 P056.027 13.1 15.9 P056.052 18.2 19.5 P057.012 3.0 5.5 P057.011 5.3 12.8

Subsequently, the functional activity of EGFRvIII TCBs was tested in the Jurkat NFAT reporter cell assay targeting EGFRvIII-positive DK-MG cells (Figure 15). All tested EGFRvIII TCBs were active in the Jurkat NFAT reporter cell assay, with P056.021 being the most effective, followed by P056.027, P056.052, and P057.012 with similar activity, while P057.011 showed the lowest activity. Next, EGFRvIII TCBs were tested in tumor cell lysis using PBMCs co-cultured with DK-MG or MKN-45 cells to rule out cross-reactivity between EGFRvIII TCBs and 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 readouts. As previously reported in cell assays, EGFRvIII TCB P056.021 exhibited the highest activity against EGFRvIII-positive cells, but showed no activity against EGFRwt-positive cells. EGFRvIII TCB P057.011 showed nonspecific activity against EGFRwt cells and was therefore excluded. EGFRvIII TCBs P056.027, P056.052, and P057.012 showed comparable activity. Based on these results, EGFRvIII binders P056.021 and P057.012 were selected for another round of affinity maturation.

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

Table 6. Affinity and specificity of selected EGFRvIII binders to EGFRvIII as determined by SPR.

The specific binding of mature EGFRvIII binders (P063.056 (SEQ ID NO:88 and SEQ ID NO:92), P064.078 (SEQ ID NO:96 and SEQ ID NO:100), and P065.036 (SEQ ID NO:104 and SEQ ID NO:108)) to EGFRvIII on U87MG-EGFRvIII and MKN-45 cells was compared with that of parental binders (Figure 19). EGFRvIII binder P063.056, which exhibited the best affinity and specificity for EGFRvIII, was selected and converted to its TCB form, using CD3 _orig or CD3 _opt as the CD3 binder.

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

method

Surface Plasmon Resonance

The affinity of EGFRvIII antibody for EGFRvIII was determined using surface plasmon resonance on a Biacore T200, with HBS-EP used as the run buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% (v/v) surfactant P20; GE Healthcare) at 25 °C. The antibody (specifically binding to human IgG ₁ Fc (PGLALA) immobilized on a CM5 chip (see WO 2017/072210, which is incorporated herein by reference)) captured 25 nM of anti-EGFRvIII PGLALA IgG within 30 s. EGFRvIII-ECD avi his antigen (see below, Example 9) was flowed through the cells at a concentration of 12.4–1000 nM at a flow rate of 30 μL/min over 200 s. The dissociation phase was monitored for 300 s and triggered by switching from the sample solution to HBS-EP. After each cycle, 10 mM glycine (pH 2.0) was injected once every 30 seconds to regenerate the chip surface. Bulk refractive index deviation was corrected by subtracting the response obtained in the reference flow cell. The affinity constant was derived from the kinetic rate constant by fitting to a 1:1 Langmuir binding using BIAeval software (GE Healthcare).

In the specificity assay, 100 nM of EGFRvIII and EGFRwt ECD antigens were captured in 40 s using anti-his (Penta His, Qiagen) immobilized on a CM5 chip. A single injection of 500 nM anti-EGFRvIII antibody was administered over 60 s, followed by regeneration with 10 mM glycine at pH 2.0 over another 60 s. EGFRvIII binding response units greater than 50 were observed. Responses greater than 5 response units (RU) were considered positive for EGFRwt binding, and IgG responses less than 5 RU were classified as specific for EGFRwt.

cell lines

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega#CS176501) are a human acute lymphoblastic leukemia reporter cell line containing the NFAT promoter and expressing human CD3. Cells were cultured at a density of 0.1–0.5 mI cells/mL in RPMI 1640, 2 g/L glucose, ₂ g/L NaHCO3, 10% FCS, 25 mM HEPES, 1% GlutaMAX, 1x NEAA, and 1x sodium pyruvate. Hygromycin B was added to a final concentration of 200 μg/mL at each cell passage.

Jurkat NFAT cells containing the PGLALA CAR are generated internally. The original cell line (Jurkat NFAT; Signosis) is a human acute lymphoblastic leukemia reporter cell whose NFAT promoter leads to luciferase expression via human CD3. They are engineered to express a chimeric antigen receptor capable of recognizing the P293G LALA mutation. After culture, the cells are suspended in RPMI 1640 supplemented with 10% FCS and 1% glutamine and maintained at a concentration between 0.4 and 1.5 miO cells/mL.

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

DK-MG (DSMZ#ACC 277) is a human glioblastoma cell line. EGFRvIII-expressing DK-MG cells were enriched by cell sorting. 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% FCS, and 1% GlutaMAX.

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

Flow cytometry was used to determine the target binding.

Cells used for binding assays were harvested, washed with PBS, and resuspended in FACS buffer. Antibody staining was performed in 96-well circular plates. Cells were harvested, counted, and seeded at 100,000 to 200,000 cells per well. The plates were centrifuged at 400 x g for 4 min, and the supernatant was removed. The detection antibody was diluted with FACS diluent, and 20 μL of the antibody solution was added to the cells over 30 min at 4 °C. To remove unbound antibody, the cells were washed twice with FACS buffer, and then the diluted secondary antibody PE-conjugated AffiniPure F(ab')2 fragment, goat anti-human IgG Fcg fragment specific (Jackson ImmunoResearch, #109-116-170 or #109-116-098) was added. After incubation at 4 °C for 30 min, the unbound secondary antibody was washed away. Prior to measurement, cells were resuspended in 200 μL of FACS buffer and analyzed by flow cytometry using BD Canto II or BDFACS Fortessa.

CAR J NFAT reporter cell assay using EGFRvIII PGLALA IgG

The efficacy of EGFRvIII PGLALA IgG in inducing T cell activation was assessed using the CAR J NFAT reporter cell assay. The principle of this assay is to co-culture Jurkat-NFAT-engineered effector cells with cancer cells expressing tumor antigens. The NFAT promoter is activated only when IgG and CAR are simultaneously bound via the PGLALA mutation and the target antigen EGFRvIII, leading to increased luciferase expression in Jurkat effector cells. Upon addition of an appropriate substrate, active firefly luciferase causes luminescence, which can be measured as a CAR-mediated activation signal. In short, target cells are harvested and their viability is measured. One day prior to the assay, 30,000 target cells/well were seeded into 100 μl of medium in flat-bottomed white-walled 96-well plates (Greiner bio-one, #655098). The next day, the medium was removed, and 25 μl/well of diluted antibody or medium (for control) was added to the target cells. Subsequently, Jurkat-NFAT reporter cells were harvested and their viability was assessed using ViCell. Cells were resuspended in cell culture medium at a concentration of 1.5 mIO cells/mL and added to tumor cells at a density of 75,000 cells/well (50 μL/well) to obtain a final effector to target (E:T) ratio of 2.5:1, with a final volume of 75 μL per well. Then, 4 μL of GloSensor (Promega, #E1291) was added to each well (2% of the final volume). Cells were incubated at 37°C for 24 hours in a humidified incubator. At the end of the incubation period, the plates were brought to room temperature (approximately 15 min). Then, 25 μL of One-Glo luciferase (Promega, #E6120) was added to each well, and the plates were incubated in the dark for 15 min. Luminescence was then detected using a TECAN Spark assay.

Jurkat NFAT reporter cell assay using EGFRvIII TCB

Using EGFRvIII-positive cells and Jurkat-NFAT reporter cells, the ability of EGFRvIII TCBs containing either an improved CD3 binder or the original CD3 binder to induce T cell crosslinking and subsequent T cell activation was evaluated. Upon simultaneous binding of EGFRvIII TCBs to EGFRvIII-positive target cells and the CD3 antigen (expressed on Jurkat-NFAT reporter cells), the NFAT promoter was activated, leading to the expression of active firefly luciferase. The intensity of the luminescent signal (obtained after the addition of the luciferase substrate) was proportional to the intensity of CD3 activation and signal transduction. For assays, target cells were harvested, and cell viability was measured. 30,000 target cells/well were seeded into 100 μL of medium in flat-bottomed white-walled 96-well plates (Greiner bio-one, #655098), and 50 μL/well of diluted antibody or medium (used as a control) was added to the target cells. Subsequently, Jurkat-NFAT reporter cells were harvested, and viability was assessed using ViCell. Cells were resuspended in hygromycin B-free cell culture medium at a concentration of 1.2 mIO cells/mL and added to tumor cells at a density of 60,000 cells/well (50 μL/well) to obtain a final effector to target (E:T) ratio of 2:1, with a final volume of 200 μL per well. Then, 4 μL of GloSensor (Promega, #E1291) was added to each well (2% of the final volume). Cells were incubated in a humidified incubator at 37°C for 24 hours. At the end of incubation, luminescence was detected using a TECAN Spark assay.

T cell-mediated tumor cell killing

Target cells were harvested with trypsin/EDTA, washed, and seeded in flat-bottomed 96-well plates at a density of 30,000 cells/well. Cell adhesion was allowed overnight. Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of fresh blood from healthy human donors. Fresh blood was diluted with sterile PBS and separated on a Histopaque gradient (Sigma, #H8889). After centrifugation (450 x g, 30 min, room temperature), plasma containing PBMCs above the PBMC interval was discarded, and the PBMCs were transferred to new Falcon tubes, which were then filled with 50 ml of PBS. The mixture was centrifuged (400 x g, 10 min, room temperature), the supernatant was discarded, and the PBMC pellet was washed twice with sterile PBS (centrifugation step 350 x g, 10 min). The resulting PBMC population was automatically counted (ViCell) and stored in RPMI 1640 medium containing 10% FCS and 1% GlutaMAX in a cell culture incubator at 37°C and 5% _CO2 until further use (no longer than 24 h). For the killing assay, the specified concentration of antibody was added and repeated three times. PBMCs were added to target cells at a final effector to target (E:T) ratio of 10:1. After incubation at 37°C and 5% _CO2 for 24 hours, target cell killing was assessed by quantifying LDH released from apoptotic/necrotic cells into the cell supernatant (LDH assay kit, Roche Applied Science, #11 644 793 001). Maximum lysis (=100%) of target cells was achieved by incubating target cells with 1% Triton X-100. Minimum lysis (=0%) refers to target cells co-incubated with effector cells without bispecific constructs.

Flow cytometry was used to assess the activation of CD8 and CD4 T cells following T cell killing of target cells mediated by TCB, using antibodies that recognize T cell activation markers CD25 (a marker of late activation) and CD69 (a marker of early activation). After 48 h of incubation, PBMCs were transferred to round-bottom 96-well plates, centrifuged at 350 x g for 5 min, and washed twice with FACS buffer. Surface staining was performed on CD4 APC (BioLegend, #300514), CD8 FITC (BioLegend, #344704), CD25BV421 (BioLegend, #302630), and CD69 PE (BioLegend, #310906) cells according to the supplier's instructions. Cells were washed twice with 150 μL/well of FACS buffer and fixed with 100 μL/well of fixation buffer (BD, #554655) at 4 °C for 15 min. After centrifugation, the samples were resuspended in 200 μL/well FACS buffer. The samples were then analyzed using BD FACS Fortessa.

According to the manufacturer's instructions, cytokine secretion in the supernatant was measured by flow cytometry using a flow cytometry bead array (CBA), but instead of using 50 μL of beads, only 25 μL of supernatant and beads were used. The following CBA kits (BD Biosciences) were used: CBA Human Interferon (IFNγ) Flex Kit, CBA β Flex Kit, and CBA Human TNF Flex Kit. Samples were measured using BD FACS Canto II or BD FACS Fortessa and analyzed using Diva software (BD Biosciences).

Example 9 – T-cell bispecific antibody containing an optimized CD3 binding agent binds to CD3 and EGFRvIII

Combining with recombinant CD3

The binding of EGFRvIII TCB to recombinant CD3 was assessed using SPR, employing TCBs containing either an optimized CD3-binding sequence (EGFRvIII TCB CD3 _opt ) or the original CD3-binding sequence (EGFRvIII TCB CD3 _orig ), as described in Example 5 above for TYRP1 TCB. Approximately 5200 resonance units (RUs) were directly immobilized at pH 5.0 using a standard amine conjugation kit (GE Healthcare) to conjugate the capture antibody to the sensor chip surface, and TCB molecules were captured for 30 s at a flow rate of 10 μL/min at 20 nM.

For TYRP1 TCB CD3 _opt , the measured _KD values for binding to CD3 in humans and cynomolgus monkeys were 30 nM and 20 nM, respectively, which are close to the values of TYRP1 TCB CD3 _orig (40 nM and 30 nM, respectively).

This indicates that, under stress-free conditions, both TCBs containing CD3 _opt or CD3 _orig bind quite well to recombinant CD3.

The binding of EGFRvIII TCB to recombinant human CD3 was also evaluated after 14 days of temperature stress at 37°C or 40°C, using TCBs containing either an optimized CD3-binding sequence or the original CD3-binding sequence. This experiment was conducted as described in Example 2 above, where TCBs were used instead of IgG molecules.

The results of the experiment are shown in Figure 21.

As shown in Figure 21, compared with TCB containing the original CD3 binder CD3 _orig , TCB containing the optimized CD3 binder CD3 _opt exhibited significantly improved binding to CD3 after stress (exposure at 37°C and pH 7.4 for 2 weeks). This result further confirms that the improved properties of the optimized CD3 binder (see Example 2) are maintained at the TCB level.

Binding with recombinant EGFRvIII

The binding of EGFRvIII TCB to recombinant EGFRvIII was assessed using SPR.

The SPR experiment was performed using a Biacore T200, which used HBS-EP as the run buffer (0.01M MEPES pH 7.4, 0.15M NaCl, 0.05% (v/v) surfactant P20 (GE Healthcare)).

Anti-Fc antibody (GE Healthcare) was directly coupled to the CM5 sensor chip surface at pH 5.0 using a standard amine conjugation kit (GE Healthcare). EGFRvIII TCB (5 nM) was captured for 30 s at a flow rate of 10 μL/min. A series of 3-fold diluted EGFRvIII antigen samples were passed through the flow cell at a flow rate of 30 μL/min for 200 s to record the association phase. The dissociation phase was monitored for 300 s and triggered by switching from the sample solution to HBS-EP. After each cycle, 3 M _MgCl2 was injected once at a flow rate of 20 μL/min over 30 s to regenerate the chip surface.

The antigen used contains the extracellular domain of human EGFRvIII fused with the C-terminal Avi tag and His tag (EGFRvIII-ECD avi his; SEQ ID NO:36).

Bulk refractive index bias was corrected by subtracting the response obtained in the reference flow cell (without captured TCB). The affinity constant (K_{_D}) was derived from the kinetic rate constant using BIAeval software (GE Healthcare) by fitting a 1:1 Langmuir binding. The apparent affinity constant K_{_D} was approximated by the rate constant using kinetic analysis, where a 1:1 binding fit was used for this 2:1 interaction.

For EGFRvIII TCBs containing CD3 _opt or CD3 _orig , the _KD value (affinity) for binding with human EGFRvIII was measured to be 6 nM.

The binding of EGFRvIII TCB to recombinant EGFRvIII was also evaluated after 14 days of temperature stress at 37°C or 40°C, using TCBs containing either an optimized CD3-binding sequence or the original CD3-binding sequence. This experiment was conducted as described in Example 5 above, using EGFRvIII-ECD avi his as the antigen (see above).

The results of this experiment are shown in Figure 22. The results confirm that the binding of both TCBs to human EGFRvIII is not affected by stress conditions.

Binding to CD3 on Jurkat cells

As described in Example 2 above, FACS was used to determine the binding of EGFRvIII TCB containing the optimized CD3 binder "CD3 _opt " or the original CD3 binder "CD3 _orig " to CD3 on the human reporter T cell line Jurkat NFAT.

As shown in Figure 23, TCBs containing either the optimized CD3 binder "CD3 _opt " or the original CD3 binder "CD3 _orig " showed good binding to CD3 on Jurkat cells.

Binding of EGFRvIII to U87MG-EGFRvIII cells

The binding of EGFRvIII TCB containing the EGFRvIII binder P063.056 (CD3 _opt or CD3 _orig ) or EGFRvIII clone P056.021 (containing CD3 _orig ) to EGFRvIII on the human glioblastoma cell line U87MG-EGFRvIII was determined using FACS. The EGFRvIII binder P063.056 in IgG form was also used.

As shown in Figure 24, all three TCBs bind to EGFRvIII expressed on U87MG-EGFRvIII cells with high affinity, and the binding is unaffected by conversion to the TCB form.

Example 10 - Functional activity of T-cell bispecific antibodies containing optimized CD3 binding agents

CD3 activation

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

As shown in the results for IgG (Example 3), the two TCBs containing CD3 _opt or CD3 _orig have similar functional activities on Jurkat NFAT reporter cells and induce CD3 activation in a concentration-dependent manner (Figure 20).

Target cell killing

In tumor cell killing assays, in the presence of glioblastoma cell lines U87MG-EGFRvIII and PBMCs (as described above in Example 8), EGFRvIII TCBs containing a selected EGFRvIII binder (P063.056) and either the optimized CD3 binder CD3 _opt or the original CD3 binder CD3 _orig were compared with EGFRvIII TCBs containing the parental EGFRvIII binder P056.021 and the CD3 binder CD3 _orig . Similar to what was observed in Jurkat NFAT reporter cells, TCBs containing CD3 _opt or CD3 _orig exhibited similar functional activity regarding the induction of tumor cell lysis and the activation of CD4 and CD8 T cells, as measured by CD69 upregulation (Figure 25).

Furthermore, the functional activities of EGFRvIII TCBs in a “2+1 form” containing the EGFRvIII binder P063.056 and the optimized CD3 binder CD3 _opt (shown in Figure 6) were compared with those in a “1+1 head-to-tail form” containing the same EGFRvIII and CD3 binder (schematically shown in Figure 1G). Both EGFRvIII TCBs were tested in the Jurkat NFAT reporter cell assay and tumor cell killing assay using the glioblastoma cell line U87MG-EGFRvIII as described above in Example 8. The 2+1 form of EGFRvIII TCBs exhibited superior functional activity in inducing tumor cell killing and T cell activation as measured in the Jurkat NFAT reporter cell assay (Figure 26) and in the killing assay using PBMCs (Figure 27).

Example 11 - Functional characterization of T-cell bispecific antibodies containing optimized CD3 binding agents

T cell proliferation and activation of EGFRvIII TCB

In a T-cell proliferation assay on U87MG-EGFRvIII cells, the functional activities of EGFRvIII TCBs containing the selected EGFRvIII binder (P063.056) and the optimized CD3 binder CD3 _opt or the original CD3 binder CD3 _orig were compared with those containing the parental EGFRvIII binder P056.021 and the CD3 binder CD3 _orig (Figure 28). All three TCBs induced significant proliferation and activation of CD4 T cells and CD8 T cells. The P063.056 EGFRvIII TCB containing CD3 _opt exhibited higher activity than the other two EGFRvIII TCBs.

Utilizing EGFRvIII TCB lysis of tumor cells

Next, in a tumor cell lysis assay co-cultured with PBMCs and DK-MG cells, EGFRvIII TCBs containing the selected EGFR binder (P063.056) and the optimized CD3 binder CD3 _opt were compared with EGFRvIII TCBs containing the parental EGFRvIII binder P056.021 and the CD3 binder CD3 _orig (Figure 29). In this assay, in addition to tumor cell lysis, T cell activation and cytokine release were measured as additional readouts. As previously stated, the P063.056 EGFRvIII TCB containing CD3 _opt showed higher activity than the P056.021 EGFRvIII TCB containing CD3 _orig in terms of tumor cell lysis, T cell activation, and the release of IFNγ and TNFα.

Utilizing TYRP1 TCB activates T cells and lyses tumor cells.

The functional properties of TYRP1TCB-induced cytokine release were tested by co-culturing the primary melanoma cell line M150543 with PBMCs isolated from healthy donors. Tumor cell lysis mediated by T cells via TYRP1TCB was analyzed after 24 and 48 hours of treatment (Fig. 30). After 48 hours of treatment, the release of IFNγ and TNFα into the supernatant, as well as CD4 and CD8 T cell activation, were analyzed. TYRP1TCB was able to induce effective tumor cell lysis after 24 hours. This effect was accompanied by significant activation of CD4 and CD8 T cells as determined by CD25 upregulation and substantial release of IFNγ and TNFα.

method

PBMC separation

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of fresh blood from healthy human donors. Fresh blood was diluted with sterile PBS and separated on a Histopaque gradient (Sigma, #H8889). After centrifugation (450 x g, 30 min, room temperature), plasma containing PBMCs above the PBMC interval was discarded, and the PBMCs were transferred to new Falcon tubes, subsequently filled with 50 ml of PBS. The mixture was centrifuged (400 x g, 10 min, room temperature), the supernatant was discarded, and the PBMC pellet was washed twice with sterile PBS (centrifugation step 350 x g, 10 min). The resulting PBMC population was automatically counted (ViCell) and stored in RPMI 1640 medium containing 10% FCS and 1% _GlutaMAX at 37°C and 5% CO2 until further use (no longer than 24 h) or frozen and stored in liquid nitrogen until further use. Thaw the frozen PBMCs one day before use and incubate them overnight in a medium at 37°C.

T cell proliferation

In short, target cells were harvested, counted, and washed twice with PBS. Cells were resuspended in PBS at a density of 5 mIo cells/mL. Cells were stained for 10 min at 37°C with a final concentration of 5 μM cell proliferation dye eFluor 670 (eBioscience, #65-0840-85). To terminate the staining reaction, 4 volumes of cold complete cell culture medium were added to the cell suspension, and the cells were incubated at 4°C for 5 min, followed by washing three times with culture medium. The labeled target cells were counted and adjusted to 0.1 mIo cells/mL using RPMI 1640, 10% FCS, and 1% GlutaMax. Cells were seeded into 96-well plates at a density of 10,000 target cells/well. Treatment solution was then added at the specified concentration, and finally, 100,000 PBMCs isolated from healthy donors were added to each well. Cells were incubated at 37°C for 5 days, and then PBMCs were harvested and stained with CD3 BUV395 (BioLegend, #563548), CD4 PE (BioLegend, #300508), CD8 APC (BioLegend, #344722), and CD25 PE/Cy7 (BioLegend, #302612). Proliferation of CD4 T cells and CD8 T cells was determined by diluting eFluor 670 dye and measuring the results using flow cytometry (FACS Fortessa, BD Bioscience). Activation of CD4 and CD8 T cells was determined by measuring the upregulation of CD25.

T cell-mediated tumor cell killing

Target cells were harvested with trypsin/EDTA, washed, and seeded in flat-bottomed 96-well plates at a density of 30,000 cells/well. Cell adhesion was allowed overnight. For the killing assay, the specified concentration of antibody was added and repeated three times. PBMCs were added to the target cells at a final effector to target (E:T) ratio of 10:1. After incubation at 37°C and 5% _CO2 for 24 hours, target cell killing was assessed by quantifying LDH released from apoptotic/necrotic cells into the cell supernatant (LDH assay kit, Roche Applied Science, #11 644 793 001). Maximum lysis (=100%) of target cells was achieved by incubating the target cells with 1% Triton X-100. Minimum lysis (=0%) refers to target cells co-incubated with effector cells without bispecific constructs.

T cell activation

Flow cytometry was used to assess the activation of CD8 and CD4 T cells following T cell killing of target cells mediated by TCB, using antibodies recognizing T cell activation markers CD25 (a marker of late activation) and CD69 (a marker of early activation). After 48 h of incubation, PBMCs were transferred to 96-well round-bottom plates, centrifuged at 350 x g for 5 min, and washed twice with FACS buffer. Surface staining was performed on CD4 APC (BioLegend, #300514), CD8 FITC (#344704, BioLegend), CD25BV421 (BioLegend, #302630), and CD69 PE (BioLegend, #310906) cells according to the supplier's instructions. Cells were washed twice with 150 μL/well of FACS buffer and fixed with 100 μL/well of fixation buffer (BD, #554655) at 4 °C for 15 min. After centrifugation, the samples were resuspended in 200 μL/well FACS buffer. The samples were then analyzed using BD FACS Fortessa.

Cytokine secretion

According to the manufacturer's instructions, cytokine secretion in the supernatant was measured by flow cytometry using a flow cytometry bead array (CBA), but instead of using 50 μL of beads, only 25 μL of supernatant and beads were used. The following CBA kits (BD Biosciences) were used: CBA Human Interferon (IFNγ) Flex Kit and CBA Human TNF Flex Kit. Samples were measured using BDFACS Canto II or BD FACS Fortessa and analyzed using Diva software (BD Biosciences).

Example 13 – In vivo efficacy of a T-cell bispecific antibody containing an optimized CD3 binding agent

The antitumor efficacy of TYRP1TCB (containing the optimized CD3 binding agent identified in Example 1) was tested in a mouse model of human tumor cell line xenograft (IGR-1 melanoma xenograft model).

IGR-1 cells (human melanoma) were cultured in DMEM medium containing 10% FCS (Sigma). Cells were cultured at 37°C in a water-saturated atmosphere under 5% _CO2 . The 6th passage was used for transplantation. Cell viability was 96.7%. Using a 1 mL tuberculin syringe (BD Biosciences, Germany), cells in 100 μL of RPMI cell culture medium (Gibco) were subcutaneously injected into the flank of mice at a dose of 2 × ^10⁶ cells per animal.

In accordance with the guidelines (GV-Solas; Felasa; Tiersch G), fully humanized female NSG mice (Roche Glycart AG, Switzerland) were maintained under conditions free of specific pathogens, with a daily cycle of 12 hours of light/12 hours of darkness. The experimental protocol was reviewed and approved by the local government (ZH223/2017). Continuous health monitoring was conducted regularly.

On day 0 of the study, mice were subcutaneously injected with 2 × ^10⁶ IGR-1 cells, randomly assigned to groups, and weighed. Twenty days after tumor cell injection (tumor volume > 200 ^mm³ ), mice were intravenously injected with 10 μg (0.5 mg/kg) TYRP1TCB twice weekly for 5 weeks. All mice were intravenously injected with 200 μl of an appropriate solution. Mice in the vector group were injected with histidine buffer, and mice in the treatment group were injected with the TYRP1TCB construct. To obtain adequate antibody levels per 200 μL, the stock solution was diluted with histidine buffer if necessary. Tumor size was measured three times weekly using calipers and plotted using GrahPadPrism software, with volume units in ^mm³ +/- SEM. Statistical analysis was performed using JMP12 software.

Figure 31 shows that TYRP1 TCB has significant efficacy in inhibiting tumor growth compared with the mediator group (68% TGI, p = 0.0058*).

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

U87 cells (human glioblastoma) were originally obtained from ATCC (Manassas, USA) and stably transfected to express human EGFRvIII protein (Roche Glycart AG, Switzerland). After expansion, the cells were stored in the Roche Glycart internal cell bank. The U87-EGFRvIII cell line was cultured in DMEM medium containing 10% FCS (Sigma) and 0.5 μg/mL puromycin (Invitrogen). The cells were cultured at 37°C in a water-saturated atmosphere at 5% _CO2 . Transplantation was performed using passage 8. Cell viability was 94.7%. Using a 1 mL tuberculin syringe (BD Biosciences, Germany), cells in 100 μL of RPMI cell culture medium (Gibco) were subcutaneously injected into the flank of mice at a rate of 5 × ^10⁵ cells per animal.

In accordance with the guidelines (GV-Solas; Felasa; Tiersch G), fully humanized female NSG mice (Roche Glycart AG, Switzerland) were maintained under conditions free of specific pathogens, with a daily cycle of 12 hours of light/12 hours of darkness. The experimental protocol was reviewed and approved by the local government (ZH223/2017). Continuous health monitoring was conducted regularly.

On day 0 of the study, mice were subcutaneously injected with 5 × ^10⁵ U87-EGFRvIII cells, randomly assigned to groups, and weighed. Two weeks after tumor cell injection (tumor volume > 200 ^mm³ ), mice were intravenously injected with 10 μg (0.5 mg/kg) EGFRvIII TCB twice weekly for 3 weeks. All mice were intravenously injected with 200 μL of the appropriate solution. Mice in the vector group were injected with histidine buffer, and mice in the treatment group were injected with the EGFRvIII TCB construct. To obtain adequate antibody levels per 200 μL, the stock solution was diluted with histidine buffer if necessary. Tumor size was measured three times weekly using calipers and plotted using GrahPad Prism software, with volume units in ^mm³ +/- SEM.

Figure 32 shows that EGFRvIII TCB has significant efficacy in controlling tumor growth, and all mice achieved complete remission.

Example 14 – PK Study of EGFRvIII TCB in Mice

The pharmacokinetics (PK) of EGFRvIII TCB containing an optimized CD3 binder (CD3 _opt ) were investigated after bolus administration of 1 mg/kg to human FcRn transgenic (line32, homozygous) mice and NOD-SCID mice. Serial blood samples were collected from human FcRn transgenic (tg) mice and NOD-SCID mice up to 672 h (9 samples were collected per mouse from 5 min to 672 h post-dose). Serum samples from mice treated with EGFRvIII TCB were analyzed under non-GLP conditions using a specific enzyme-linked immunosorbent assay (ELISA). EGFRvIII TCB was captured with biotinylated EGFRvIII antigen (huEGFRvIII his biotin) on streptavidin-coated microtiter plates (SA-MTP). The binding of EGFRvIII TCB was detected using a digitoxin-labeled anti-human IgG1 Fc (PGLALA) monoclonal antibody (see Example 3), followed by the addition of a second detection antibody against digoxin-POD. A signal was generated by adding a peroxidase substrate (ABTS). The calibration range was 2.35 ng/mL to 150 ng/mL, with 2.5 ng/mL being the limit of quantitation (LLOQ).

The results of this study are shown in Table 7. For both mouse strains tested, the PK characteristics of EGFRvIII TCB were within the expected range. The results indicate that the engineering of the CDR of the CD3 binder did not induce susceptibility to other sequences that could potentially affect antibody clearance.

Table 7. Clearance data (mL/day/kg; mean) of huFcRn tg mice and NOD-SCID mice.

Example 15 – FOLR1 TCB with CD3 _opt as CD3 binder:

Transformation and generation of FOLR1 TCB with CD3 _opt as CD3 binder

To generate CD3 _opt FOLR1 TCB (“classical 2+1 form” = CD3Fab outside the Fc club chain and FOLR1Fab inside, Figure 33A, SEQ ID Nos. 127, 128, 129), the variable domain of the CD3 binder CD3 _opt is inserted into a suitable expression vector. This molecule consists of a human IgG1 backbone with mutations in the Fc region (LL234/235AA and P329G) to eliminate Fc effector function. The T-cell bispecific molecule is generated using a club-and-mortar technique in a proprietary 2+1 heterodimer form (two binding moieties for the target antigen and one binding moiety for CD3).

Transfection and expression of FOLR1 TCB using CD3 _opt as a CD3 binding agent

FOLR1 TCB expression was performed via transient transfection of ExpiCHO-S ^™ cells (ExpiCHO ^™ expression system; Thermo/Gibco#A29133). ExpiCHO-S cells were pre-cultured according to the manufacturer's instructions. On the day of transfection, 500 ml of cells were seeded into sterile disposable shake flasks at a concentration of 6 × 10⁶ live cells/mL. For optimal transfection, a total of 1.0 μg of plasmid DNA and 0.64 μl of ExpiFectamine ^™ CHO reagent were used per milliliter of culture volume.

Incubate the ExpiFectamine ^™ CHO reagent and the DNA separately in cold OptiPRO ^™ medium at room temperature for 5 minutes. Add the diluted ExpiFectamine ^™ CHO reagent to the diluted DNA, mix thoroughly, and incubate again at room temperature ^for 5 minutes. Subsequently, add the composite mixture to the cells and incubate at 37°C on an orbital shaker platform with ≥80% relative humidity and 8% CO2.

Following the supplier's recommendation, a high-titer protocol was used for protein expression: ExpiFectamine ^™ CHO enhancer and a single feed were added on day 1 post-transfection; cells were transferred to 32°C on day 1 post-transfection. The cell supernatant was harvested and purified on day 8 post-transfection.

Purification of FOLR1 TCB

FOLR1 TCB was purified by protein A affinity chromatography, followed by ion exchange and size exclusion chromatography. Briefly, the supernatant was loaded onto a HiTrap MabSelect SuRe column (GE Healthcare), equilibrated with 1x PBS (pH 7.4). After washing with 5 column volumes of equilibration buffer, proTCB was eluted with 100 mM sodium acetate (pH 3.0). The flow rate was set to 5 mL/min. The combined fractions were diluted with water (1:5 v/v) and loaded onto a POROS HS50 column (Thermo Fisher Scientific), equilibrated with 40 mM sodium acetate (pH 5.5). After washing with equilibration buffer, TCB was eluted with a sodium acetate gradient increasing from 40 mM to up to 1 M at 27 column volumes. The flow rate was set to 7 mL/min. The collected fractions were analyzed by size exclusion chromatography (Waters BioSuite) and combined according to monomer concentration. The combined fractions were then concentrated to a final volume of 15 mL using an Amicon Ultra (Millipore) system. The concentrated fraction was loaded onto a HiLoad 26/60 Superdex preparative chromatography column (GE Healthcare) with a column volume of 320 mL. The run buffer was 20 mM histidine hydrochloride and 140 mM NaCl (pH 6.0), and the flow rate was set to 3 mL/min. The fractions were then combined according to monomer concentration.

Table 8: Production/purification results of FOLR1 TCB using Cl22 as a CD3 binding agent

Example 16 – Jurkat NFAT activation mediated by FOLR1-TCB (CD3 _opt )

FOLR1-TCB containing CD3 clone 22 binder was first tested in the Jurkat NFAT reporter cell assay.

A T-cell bispecific antibody (TCB) targeting FOLR1 binds simultaneously to huFOLR1-coated beads and CD3epsilon (Jurkat NFAT) on T cells, thereby inducing T-cell activation. T-cell activation can be measured luminescently because Jurkat NFAT luciferase cells express luciferase upon activation via CD3epsilon (CD3ε). The Jurkat-NFAT reporter cell line (Promega) is a human acute lymphoblastic leukemia reporter cell line with an NFAT promoter that expresses human CD3ε. If the TCB binds to the tumor target and CD3 (crosslinked) binds to CD3ε, luciferase expression can be measured luminescently upon addition of the One-Glo substrate (Promega).

Jurkat NFAT assay medium: RPMI 1640, 2 g/L glucose, 2 g/L NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1x NEAA, 1x sodium pyruvate

Jurkat NFAT medium: RPMI 1640, 2 g/L glucose, 2 g/L NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1x NEAA, 1x sodium pyruvate; freshly added hygromycin B 200 μg/ml.

Dilute 65 μl of streptavidin Dynabeads in 10 ml of PBS. Centrifuge the beads at 400 rcf for 4 min and discard the supernatant. Then add 30 μg of biotinylated FolR1 antigen to 1.5 ml of DPBS, followed by the streptavidin beads. Incubate the bead-antigen mixture at 4 °C for 1 h with slow rotation. After incubation, add 10 ml of DPBS to the bead-antigen conjugate, centrifuge (4 min, 400 rcf) and discard the supernatant. Wash the conjugate again with 5 ml of DPBS, and then resuspend the precipitate in 6 ml of assay medium. Harvest effector cells (Jurkat NFAT), count them, and check their viability. Centrifuge the cells at 350 rcf for 4 min, and then resuspend the cells in 12 ml of assay medium. Add cAMP to the effector cell suspension (2% of the final volume).

Coated beads (10 μl/well) and Jurkat NFAT effector cells (20,000 cells/well, 20 μl/well) were mixed with cAMP and added to 384-well white-walled clear plates (Greiner BioOne). FOLR1-TCB was diluted in Jurkat assay medium and 10 μl was added to each well before preparing the dilution row. Cells were incubated with the beads and TCB/medium at 37°C for 5.5 h in a humidified incubator. After incubation, the cells were removed from the incubator and allowed to acclimatize for approximately 10 minutes. Nucleotide readings were then taken using a Tecan Spark assay with a detection time of 0.5 s/well.

FOLR1-TCB uses huFOLR1-coated beads for crosslinking to induce dose-dependent Jurkat NFAT activation (Figure 34).

Example 17 – T-cell killing mediated by FOLR1 pro-TCB (CD3 clone 22)

To evaluate the efficacy of FOLR1-TCB containing CD3 _opt , FOLR1-positive Ovcar-3 cells were used to assess FOLR1-TCB-mediated cytotoxicity and T cell activation in target cells. Human PBMCs were used as effector cells, and T cell activation markers were stained after 48 h of incubation with the molecules and cells. Human peripheral blood mononuclear cells (PBMCs) were isolated from erythrocyte sedimentation rate (ESR) amber layer (obtained from healthy human donors). The ESR amber layer was diluted 1:1 with sterile PBS and separated on a Histopaque gradient (Sigma, #H8889). After centrifugation (450 x g, 30 min, uninterrupted, room temperature), the PBMC-containing intermediate phase was transferred to new Falcon tubes, followed by filling with 50 ml of PBS. The mixture was centrifuged (400 x g, 10 min, room temperature), the supernatant was discarded, and the PBMC precipitate was resuspended in 2 ml of ACK buffer for erythrocyte lysis. After incubating at 37°C for approximately 2 to 3 minutes, fill the tubes to 50 ml with sterile PBS and centrifuge at 350 x g for 10 minutes. Repeat this washing step once before resuspending the PBMCs in advanced RPMI 1640 medium containing 2% FCS, 1X GlutaMax, and 10% DMSO. Slowly freeze the PBMCs at -80°C in a cryogenic container (BioCision) and then transfer them to liquid nitrogen. One day before the assay, harvest adherent target cells with trypsin/EDTA, count them, check their viability, and resuspend them in assay medium (RPMI 1640, 2% FCS, 1X GlutaMax). Approximately 24 hours before the assay, thaw the PBMCs in RPMI 1640 medium (10% FCS, 1X GlutaMax). Centrifuge the PBMCs at 350 g for 7 minutes and resuspend them in fresh medium (RPMI 1640, 10% FCS, 1X GlutaMax). PBMCs should be stored for no more than 24 hours before use in the assay. The assay medium is RPMI + 2% FCS + 1% GlutaMax.

Target cells were seeded in 96-well flat-bottomed plates at a density of 20,000 cells/well (in 50 μl/well of assay medium). Cells were incubated overnight in a humidified incubator at 37°C. Molecules were diluted in assay medium and added at the specified concentration, repeated three times. PBMCs were harvested and centrifuged at 350 g for 7 min, then resuspended in assay medium. Before incubating the plates at 37°C for 48 h, 0.2 mi hu PBMCs (E:T 10:1, based on the number of seeded target cells) were added at 100 μl/well. After incubation at 37°C and 5% CO2 for 48 h, the killing effect of target cells was assessed by quantifying LDH released into the cell supernatant using an apoptotic/necrotic cell assay kit (Roche Applied Science, #11 644 793 001). Maximal lysis (100%) of target cells was achieved by incubating them with 1% Triton X-100 for 1 hour before LDH readings. Minimum lysis (0%) was defined as co-incubation of target cells with effector cells free of any TCBs. LDH release was measured by measuring absorbance (A492nm–A650nm).

After incubation at 37°C and 5% CO2 for 48 hours, T cell activation was assessed by quantifying CD69 on CD4-positive and CD8-positive T cells.

Add DPBS to the wells containing PBMCs, then centrifuge the plate at 400x g for 4 min. Aspirate the supernatant and wash the cells again with PBS, centrifuge, remove the supernatant, and resuspend the cell pellet by carefully vortexing the plate. Dilute 5 μl of LIVE/DEAD ^™ Fixable Aqua dead cell stain with DPBS at a ratio of 1:1000. Add 50 μl of diluted dye to the wells containing PBMCs. Prepare an empty well by adding 1 drop of compensating beads (Invitrogen Arc™, active beads) and 1 μl of undiluted LIVE/DEAD dye. Incubate the plate at 4°C for 30 min. To remove excess stain, wash the cells twice, first with 150 μl of PBS and then with 150 μl of FACS buffer. Centrifuge the plate at 400x g for 4 min. Remove the supernatant and resuspend the cells by carefully vortexing. Add one drop of negative control beads (Invitrogen Arc™, negative control beads) to a compensation control well containing LIVE-stained beads. Add 25 μl of diluted CD4/CD8/CD25/CD69 antibody mixture (0.8 μl/well for each color), a single antibody (for compensation control; 0.8 μl AB + 24 μl FACS buffer), or FACS buffer (for unstained control) to the wells to resuspend the cells, and incubate the plate at 4°C for 60 min. To remove unbound antibodies, wash the cells twice with 150 μl of FACS buffer per well. After centrifugation, remove the supernatant and resuspend the cells by careful vortexing. Fix the cells overnight in 150 μl of FACS + 1% PFA buffer for analysis the following week. The next day, resuspend the cells in 150 μl of FACS buffer. Perform fluorescence measurements using FACS LSR Fortessa.

Ovcar-3 cells incubated with huPBMCs and FOLR1-TCB (CD3 _opt ) exhibited dose-dependent target cell killing (Fig. 35A). Dashed lines show huPBMCs incubated with target cells but not with TCB. For CD4 and CD8 positive T cells, target cell cytotoxicity correlated with T cell activation as measured by CD69 (Fig. 35B).

***

Although the invention has been described in considerable detail above by way of example and embodiments for the purpose of clarity, such descriptions and embodiments should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

sequence list

<110> Hofmeister AG

<120> Antibodies that bind to CD3 and FolR1

<130> P36031

<160> 137

<170> PatentIn Version 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig HCDR1

<400> 1

Thr Tyr Ala Met Asn

1 5

<210> 2

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3opt HCDR1

<400> 2

Ser Tyr Ala Met Asn

1 5

<210> 3

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig / CD3opt HCDR2

<400> 3

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

1 5 10 15

Val Lys Gly

<210> 4

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig HCDR3

<400> 4

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

1 5 10

<210> 5

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3opt HCDR3

<400> 5

His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr Gly Tyr

1 5 10

<210> 6

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig VH

<400> 6

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 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

115 120 125

<210> 7

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3opt VH

<400> 7

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 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

115 120 125

<210> 8

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig / CD3opt LCDR1

<400> 8

Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig / CD3opt LCDR2

<400> 9

Gly Thr Asn Lys Arg Ala Pro

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig / CD3opt LCDR3

<400> 10

Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

<210> 11

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig / CD3opt VL

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

<210> 12

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig IgG HC

<400> 12

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 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 Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Leu Ser Pro

450

<210> 13

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3opt IgG HC

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Leu Ser Pro

450

<210> 14

<211> 216

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3orig / CD3opt IgG LC

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 15

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> TYRP1 HCDR1

<400> 15

Asp Tyr Phe Leu His

1 5

<210> 16

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> TYRP1 HCDR2

<400> 16

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

1 5 10 15

Gly

<210> 17

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> TYRP1 HCDR3

<400> 17

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

1 5 10

<210> 18

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> TYRP1 VH

<400> 18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 11

<212> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> TYRP1 LCDR2

<400> 20

Asp Ala Lys Thr Leu Ala Asp

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> TYRP1 LCDR3

<400> 21

Gln His Phe Trp Ser Leu Pro Phe Thr

1 5

<210> 22

<211> 107

<212> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> TYRP1 VH-CH1(EE) - CD3orig/CD3opt VL-CH1 - Fc (PESTLE, 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 Ser Leu Ser Ser Val Val Thr Val

180 185 190

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

405 410 415

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 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> PRT

<213> Artificial Sequence

<220>

<223> TYRP1 VH-CH1(EE)-Fc (mortar, 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 Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 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> PRT

<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> PRT

<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 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> PRT

<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 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> PRT

<213> Artificial Sequence

<220>

<223> Human CD3 ε Handle - Fc (Pepper) - 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 Ser 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> PRT

<213> Artificial Sequence

<220>

<223> Human CD3 δ handle - Fc (mortis) - 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> PRT

<213> Artificial Sequence

<220>

<223> Crab-eating macaque CD3 ε handle - Fc (pestle) - 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 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> PRT

<213> Artificial Sequence

<220>

<223> Crab-eating macaque CD3 δ shank - Fc (pore) - 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> PRT

<213> Artificial Sequence

<220>

<223> Person TYRP1 ECD - Fc (Pepper) - 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 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> PRT

<213> Artificial Sequence

<220>

<223> Crab-eating macaque TYRP1 ECD - Fc (pestle) - 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 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> PRT

<213> Artificial Sequence

<220>

<223> Mouse TYRP1 ECD - Fc (pallet) - 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> PRT

<213> Artificial Sequence

<220>

<223> Fc (mortar)

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII ECD - 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

385 390

<210> 37

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P056.021 HCDR1

<400> 37

Ser Tyr Trp Ile Ala

1 5

<210> 38

<211> 17

<212> PRT

<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> PRT

<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> PRT

<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 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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P056.021 LCDR2

<400> 42

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 43

<211> 10

<212> PRT

<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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P056.052 HCDR1

<400> 45

Asn Tyr Trp Ile Gly

1 5

<210> 46

<211> 17

<212> PRT

<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> PRT

<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> PRT

<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 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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P056.052 LCDR2

<400> 50

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 51

<211> 10

<212> PRT

<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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P047.019 HCDR1

<400> 53

Ser Ile Trp Ile His

1 5

<210> 54

<211> 17

<212> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P047.019 HCDR3

<400> 55

Thr Gly Pro Gly Leu Ala Phe Asp Tyr

1 5

<210> 56

<211> 118

<212> PRT

<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 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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P047.019 LCDR2

<400> 58

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 59

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P047.019 LCDR3

<400> 59

Gln Gln Ser Tyr Ser Thr Pro Ile Thr

1 5

<210> 60

<211> 113

<212> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P057.012 HCDR1

<400> 61

Asn Tyr Trp Ile Ala

1 5

<210> 62

<211> 17

<212> PRT

<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> PRT

<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> PRT

<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 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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P057.012 LCDR2

<400> 66

Trp Ala Ser Lys Arg Glu Ser

1 5

<210> 67

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P057.012 LCDR3

<400> 67

Gln Gln Ser Tyr Ser Ala Pro Ile Thr

1 5

<210> 68

<211> 113

<212> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P057.011 HCDR1

<400> 69

Arg Arg Trp Ile Ala

1 5

<210> 70

<211> 17

<212> PRT

<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> PRT

<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> PRT

<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 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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P057.011 LCDR2

<400> 74

Trp Ala Ser Lys Arg Glu Ser

1 5

<210> 75

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P057.011 LCDR3

<400> 75

Gln Gln Ser Tyr Ser Ala Pro Ile Thr

1 5

<210> 76

<211> 113

<212> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P056.027 HCDR1

<400> 77

Asn Asn Trp Ile Ala

1 5

<210> 78

<211> 17

<212> PRT

<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> PRT

<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> PRT

<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 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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P056.027 LCDR2

<400> 82

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 83

<211> 10

<212> PRT

<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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P063.056 HCDR1

<400> 85

Ser Tyr Trp Ile Ala

1 5

<210> 86

<211> 17

<212> PRT

<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> PRT

<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> PRT

<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 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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P063.056 LCDR2

<400> 90

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 91

<211> 10

<212> PRT

<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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P064.078 HCDR1

<400> 93

Ser Tyr Trp Ile Ala

1 5

<210> 94

<211> 17

<212> PRT

<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> PRT

<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> PRT

<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 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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P064.078 LCDR2

<400> 98

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 99

<211> 10

<212> PRT

<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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P065.036 HCDR1

<400> 101

Ser Tyr Trp Ile Ala

1 5

<210> 102

<211> 17

<212> PRT

<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> PRT

<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> PRT

<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 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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII P065.036 LCDR2

<400> 106

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 107

<211> 10

<212> PRT

<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> PRT

<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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII VH-CH1(EE) - CD3orig/CD3opt VL-CH1 - Fc (PESTLE, 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 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 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 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 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> PRT

<213> Artificial Sequence

<220>

<223> EGFRvIII VH-CH1(EE) -Fc (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 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 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> PRT

<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> PRT

<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 Ser 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> PRT

<213> Crab-eating macaques

<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> PRT

<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 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> PRT

<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 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 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> PRT

<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 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> PRT

<213> Artificial Sequence

<220>

<223> hIgG1 Fc region

<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> PRT

<213> Artificial Sequence

<220>

<223> Connector GGGGSGGGGS

<400> 118

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 119

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector DGGGGSGGGGS

<400> 119

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 120

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Human κ CL domain

<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> PRT

<213> Artificial Sequence

<220>

<223> Human λ CL structural domain

<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> PRT

<213> Artificial Sequence

<220>

<223> Human IgG1 heavy chain constant region (CH1-CH2-CH3)

<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

<210> 123

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> 16D5 VH

<400> 123

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Pro 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 Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 124

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> 16D5 CDRH1

<400> 124

Asn Ala Trp Met Ser

1 5

<210> 125

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> 16D5 CDRH2

<400> 125

Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 126

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 16D5 CDRH3

<400> 126

Pro Trp Glu Trp Ser Trp Tyr Asp Tyr

1 5

<210> 127

<211> 687

<212> PRT

<213> Artificial Sequence

<220>

<223> FORL1 TCB 16D5-CD3opt 2+1 Classic Form K Chain

<400> 127

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 Tyr Ala Asp

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu

225 230 235 240

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

245 250 255

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala Trp

260 265 270

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

275 280 285

Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

290 295 300

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

305 310 315 320

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

325 330 335

Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln Gly

340 345 350

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

355 360 365

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

370 375 380

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

625 630 635 640

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

645 650 655

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

660 665 670

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

675 680 685

<210> 128

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> FORL1 TCB 16D5-CD3opt 2+1 Classic Form H Chain

<400> 128

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Pro 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 Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 129

<211> 215

<212> PRT

<213> Artificial Sequence

<220>

<223> Common Light Chain

<400> 129

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

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

115 120 125

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

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 130

<211> 687

<212> PRT

<213> Artificial Sequence

<220>

<223> FORL1 TCB 16D5-CD3opt 2+1 Inverted Form K Chain

<400> 130

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Pro 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 Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

245 250 255

Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr Ala Met Asn Trp Val Arg

260 265 270

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys

275 280 285

Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

305 310 315 320

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

325 330 335

Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr Gly Tyr Trp Gly Gln Gly

340 345 350

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

355 360 365

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

370 375 380

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

625 630 635 640

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

645 650 655

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

660 665 670

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

675 680 685

<210> 131

<211> 687

<212> PRT

<213> Artificial Sequence

<220>

<223> FORL1 TCB 16D5-CD3opt 1+1 Head-to-tail inverted form K chain

<400> 131

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Pro 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 Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

245 250 255

Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr Ala Met Asn Trp Val Arg

260 265 270

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys

275 280 285

Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

290 295 300

Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

305 310 315 320

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

325 330 335

Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr Gly Tyr Trp Gly Gln Gly

340 345 350

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

355 360 365

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

370 375 380

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

625 630 635 640

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

645 650 655

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

660 665 670

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

675 680 685

<210> 132

<211> 225

<212> PRT

<213> Artificial Sequence

<220>

<223> FORL1 TCB 16D5-CD3opt 1+1 Head-to-tail inverted form H chain

<400> 132

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 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 133

<211> 687

<212> PRT

<213> Artificial Sequence

<220>

<223> FORL1 TCB 16D5-CD3opt 1+1 Head-to-Tail Classic Form K Chain

<400> 133

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 Tyr Ala Asp

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu

225 230 235 240

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

245 250 255

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala Trp

260 265 270

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

275 280 285

Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

290 295 300

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

305 310 315 320

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

325 330 335

Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln Gly

340 345 350

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

355 360 365

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

370 375 380

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

625 630 635 640

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

645 650 655

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

660 665 670

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

675 680 685

<210> 134

<211> 225

<212> PRT

<213> Artificial Sequence

<220>

<223> FORL1 TCB 16D5-CD3opt 1+1 Classic H-chain (Head to Tail)

<400> 134

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 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 135

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> FOLR1 TCB 16D5-CD3opt 1+1 IgG-like form K chain

<400> 135

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Pro 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 Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 136

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> FOLR1 TCB 16D5-CD3opt 1+1 IgG-like form H chain

<400> 136

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Pro 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 Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 137

<211> 257

<212> PRT

<213> Homo sapiens

<400> 137

Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val

1 5 10 15

Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu

20 25 30

Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly

35 40 45

Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala

50 55 60

Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr

65 70 75 80

Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys

85 90 95

Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn

100 105 110

Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg

115 120 125

Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu

130 135 140

Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp

145 150 155 160

Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln

165 170 175

Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile

180 185 190

Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg

195 200 205

Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu

210 215 220

Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala

225 230 235 240

Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu

245 250 255

Ser

Claims (31)

1. A bispecific antibody that binds to CD3 and folate receptor 1 (FolR1), wherein the bispecific antibody comprises (i) a first antigen-binding domain capable of specifically binding to CD3, the first antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises 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 the light chain variable region comprises the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO:8, LCDR 2 of SEQ ID NO:9, and LCDR 3 of SEQ ID NO:10; and (ii) A second antigen-binding domain that can specifically bind to FolR1. 2. The bispecific antibody according to claim 1, wherein the VH of the first antigen-binding domain comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:7, and/or the VL comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:11. 3. A bispecific antibody that binds to CD3 and FolR1, wherein the bispecific antibody comprises (i) a first antigen-binding domain capable of specifically binding to CD3, the first antigen-binding domain comprising the VH sequence of SEQ ID NO:7 and the VL sequence of SEQ ID NO:11; and (ii) A second antigen-binding domain that can specifically bind to FolR1. 4. The bispecific antibody according to any one of claims 1 to 3, wherein the first antigen-binding domain is a Fab molecule. 5. The bispecific antibody according to any one of claims 1 to 4, comprising an Fc domain, said Fc domain being composed of a first subunit and a second subunit. 6. The bispecific antibody according to any one of claims 1 to 5, comprising a third antigen-binding domain capable of specifically binding to FolR1. 7. The bispecific antibody according to claim 6, wherein the second antigen-binding domain and/or the third antigen-binding domain, when present, are Fab molecules. 8. The bispecific antibody according to any one of claims 1 to 7, wherein the first antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are interchanged with each other or the constant domains CL and CH1 are interchanged with each other, particularly the variable domains VL and VH. 9. The bispecific antibody according to any one of claims 6 to 8, wherein the second antigen-binding domain and the third antigen-binding domain, when present, are conventional Fab molecules. 10. The bispecific antibody according to any one of claims 6 to 9, wherein the second antigen-binding domain and the third antigen-binding domain, when present, are Fab molecules, wherein in the constant domain CL, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (according to Kabat numbering); and in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU indexing). 11. The bispecific antibody according to any one of claims 6 to 10, wherein the first antigen-binding domain and the second antigen-binding domain are fused to each other, optionally via a peptide linker. 12. The bispecific antibody according to any one of claims 6 to 11, wherein the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, and (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. 13. The bispecific antibody according to any one of claims 6 to 12, wherein the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain, when present, are each Fab molecules, and the bispecific antibody comprises an Fc domain, the Fc domain being composed of a first subunit and a second subunit; and wherein (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; and the third antigen-binding domain, when 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 bispecific antibody according to any one of claims 5 to 13, wherein the Fc domain is an IgG Fc domain, particularly an IgG ₁ Fc domain. 15. The bispecific antibody according to any one of claims 5 to 14, wherein the Fc domain is a human Fc domain. 16. The bispecific antibody according to any one of claims 5 to 15, wherein the Fc comprises a modification that promotes association between the first subunit and the second subunit of the Fc domain. 17. The bispecific antibody according to any one of claims 5 to 16, wherein the Fc domain comprises one or more amino acid substitutions, the one or more amino acid substitutions reducing binding to the Fc receptor and/or reducing effector function. 18. The bispecific antibody according to any one of claims 6 to 17, wherein the second antigen-binding domain and the third antigen-binding domain, when present, comprise VH and VL, wherein VH comprises HCDR 1 of SEQ ID NO: 124, HCDR 2 of SEQ ID NO: 125, and HCDR 3 of SEQ ID NO: 126, and VL comprises LCDR 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9, and LCDR 3 of SEQ ID NO: 10. 19. The bispecific antibody according to claim 19 or 20, wherein the second antigen-binding domain and the third antigen-binding domain, when present, comprise VH and/or VL, wherein the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 123, and the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11. 20. An isolated polynucleotide encoding a bispecific antibody according to any one of claims 1 to 19. 21. A host cell comprising the isolated polynucleotide according to claim 20. 22. A method for producing a bispecific antibody that binds to CD3 and FolR1, comprising the steps of: (a) culturing a host cell according to claim 21 under conditions suitable for expressing the bispecific antibody, and optionally (b) recovering the bispecific antibody. 23. A bispecific antibody that binds to CD3 and FolR1, produced by the method according to claim 22. 24. A pharmaceutical composition comprising: a bispecific antibody according to any one of claims 1 to 19 or 23, and a pharmaceutical carrier. 25. The bispecific antibody according to any one of claims 1 to 19 or 23, or the pharmaceutical composition according to claim 24, used as a drug. 26. The bispecific antibody according to any one of claims 1 to 19 or 23, or the pharmaceutical composition according to claim 24, for use in the treatment of cancer. 27. Use of the bispecific antibody according to any one of claims 1 to 19 or 23, or the pharmaceutical composition according to claim 24, in the preparation of a medicament. 28. Use of the bispecific antibody according to any one of claims 1 to 19 or 23, or the pharmaceutical composition according to claim 24, in the preparation of a medicament for treating cancer. 29. A method of treating a disease in an individual, comprising administering to the individual an effective amount of a bispecific antibody according to any one of claims 1 to 19 or 23 or a pharmaceutical composition according to claim 24. 30. The method of claim 29, wherein the disease is cancer. 31. The present invention as described above.