Antibodies that bind to CD3 and uses therefor

Abstract

The invention provides antibodies that bind to CD3, particularly pH responsive anti-CD3 antibodies and methods of using the same.

Description

[0001] ANTIBODIES THAT BIND TO CD3 AND USES THEREFOR

[0002] TECHNICAL FIELD

[0003] The present invention relates to antibodies that bind to cluster of differentiation 3 (CD3), particularly pH responsive antibodies that bind to CD3, pH responsive T cell bispecifics (TCBs) containing the pH responsive antibodies that bind to CD3 and methods of using them, e.g. for preferential binding to CD3 at acidic pH as compared to neutral pH and / or preferential activation of T cells at acidic pH as compared to neutral pH. In addition, the present invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies, and to methods of using them in the treatment of disease.

[0004] BACKGROUND

[0005] CD3 (cluster of differentiation 3) is a protein complex composed of four subunits, the CD3γ chain, the CD35 chain, and two CD3ε chains. CD3 associates with the T-cell receptor and the ζ, chain to generate an activation signal in T lymphocytes.

[0006] CD3 has been extensively explored as a drug target. Monoclonal antibodies targeting CD3 have been used as immunosuppressant therapies in autoimmune diseases such as type I diabetes, or in the treatment of transplant rejection. The CD3 antibody muromonab-CD3 (OKT3) was the first monoclonal antibody ever approved for clinical use in humans, in 1985.

[0007] A more recent application of CD3 antibodies is in the form of bispecific antibodies, binding CD3 on the one hand and a target cell antigen e.g. a tumor cell antigen on the other hand. The simultaneous binding of such an antibody to both of its targets will force a temporary interaction between target cell and T cell, causing activation of any cytotoxic T cell and subsequent lysis of the target cell. An example of a CD3 antibody used in TCBs is disclosed in WO2015 / 095392A1.

[0008] The approaches taken so far with regard to CD3 bispecific antibodies have many disadvantages, such as lack of tumor specificity resulting in on target - off tumor toxicity, limited T cell infiltration in the tumor microenvironment and impact on T cell effector function levels (doi: 10.3390 / cancersl3020287).

[0009] To maximize tumor specificity is a primary goal in cancer therapy. One approach to obtain this is by making use of the acidic tumor cell surface and acidic tumor microenvironment. (Warburg, O., Posener, K., & Negelein, E. (1924) Uber den Stoffwechsel der Carcinomzelle. Naturwissenschaften, 12(50), 1131-1137). (Krahling, H. et al. The glycocalyx maintains a cell surface pH nanoenvironment crucial for integrin-mediated migration of human melanoma cells. 2009; doi.org / 10.1007 / s00424-009-0694-7).

[0010] Several approaches have been tried to harness the acidic tumor microenvironment for therapeutic applications.

[0011] Sulea and coworkers applied a method of dual-pH histidine-scanning mutagenesis for pH selectivity optimization (Sulea et al. doi.org / 10.1080 / 19420862.2019.1682866). Binding selectivity toward acidic pH was improved by as much as 25 fold relative to a parental anti- Her2 antibody, albeit with an overall lowered affinity. Indeed, pH engineering of antibodies generally results in affinity loss. To retain initial affinity or obtain higher affinity binders at the desired acidic pH conditions, while decreasing binding at neutral pH levels is therefore a tremendous engineering effort and challenge.

[0012] US2022135680A1 describes OKT3 antibodies binding to human CD3 at acidic pH. WO2020247932A1 and WO2022266660A1 describe 40G5 derived engineered pH-dependent anti-CD3 antibodies. Obtaining pH-responsive antibodies is difficult. After three rounds of phage selections and screening of about six hundred clones only two clones showed pH responsive binding. To further increase affinity and / or the pH window of pH responsive clones is also difficult.

[0013] Given the tremendous therapeutic potential of pH responsive antibodies, particularly bispecific antibodies for the activation of T cells, and given the difficulty of generating and optimizing pH responsive CD3 antibodies, there is a need to provide alternative and also optimized pH responsive CD3 and pH responsive TCB antibodies.

[0014] In addition, pH responsive antibodies are also a valuable tool for imaging purposes and the measuring of acidification. SUMMARY

[0015] The invention provides antibodies that bind to CD3, including multispecific (e.g. bispecific) antibodies, and methods of using the same. The antibodies according to the invention bind to CD3 in a pH dependent manner. The (multispecific) antibodies provided can thus increase tumor specificity.

[0016] In one aspect, the invention provides an antibody that binds to CD3, wherein the antibody binds to CD3 at neutral pH with a first KD and at an acidic pH with a second KD, wherein the first KD is greater than the second KD and the first KD and the second KD are measured by surface plasmon resonance (SPR) at 25 °C.

[0017] In a certain aspect, the acidic pH ranges from 6.0 to 6.6 and the neutral pH ranges from 7.0 to 7.4, particularly wherein the acidic pH is 6.5 or 6.1 and the neutral pH is 7.0 or 7.4, more particularly wherein the acidic pH is 6.5 and the neutral pH is 7.4.

[0018] In a certain aspect, the first KD is greater than the second KD by a factor of at least 2.0, at least 2.2, at least 2.8, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 4.6, at least 5.0, at least 10.0, at least 10.9, at least 14.3, at least 16.2, at least 18.0, at least 19.6, at least 20.0, at least

[0019] 21.0, at least 23.0, at least 24.0, at least 25.0, at least 28.2, at least 29.7, at least 32.4, at least

[0020] 33.3, at least 38.6, at least 43.5, at least 43.9, at least 52.8, at least 61.9, at least 70.0, at least

[0021] 80.0, at least 90.2, at least 100.0, at least 113.2, at least 120.0, at least 130.0, at least 140.0, at least 150.0, at least 160.0, at least 170.0, at least 181.6, least 190.0, at least 200.0, at least 210.0, at least 220.0, at least 230.0, at least 236.0 or more.

[0022] In a certain aspect, the second KD is equal to 4 nM or less at pH 6.5 and the first KD is equal to 9 nM or more at pH 7.4.

[0023] In a certain aspect, the first KD is greater than the second KD by a factor ranging from 2.0 to 236.0 or from 4.5 to 236.0.

[0024] In a certain aspect, the KD reflects affinity, wherein affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of the antibody and its binding partner, here the CD3 antigen. The affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair, here antibody and CD3 antigen.

[0025] In a certain aspect, the antibody is a monoclonal antibody. In a certain aspect, the antibody is a human antibody.

[0026] In a certain aspect, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of NX13X14IH (SEQ ID NO:225), (b) CDR-H2 comprising the amino acid sequence of WIX7PGX8GX9TX10YNX11KFX12G (SEQ ID NO:226), and (c) CDR-H3 comprising the amino acid sequence of DX1X2X3X4YX5X6DY (SEQ ID NO:227), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of KSSQSLLNX22RTRKNYLA (SEQ ID NO:222), (e) CDR-L2 comprising the amino acid sequence of WASX19RX20X21 (SEQ ID NO:223), and (f) CDR-L3 comprising the amino acid sequence of XisXieSFXnXisRT (SEQ ID NO:224), wherein Xi is T, S or Q, X2is Y, V or R, X3is S or V, X4is N, Q or H, X5is F or Y, X6is F, Y or H, X7is Y, L, V, H, E, K or R, X8is D, Y, T, R or E, X9is N, D, Q or E, X10 is R or K, Xu is E, Q or H, X12 is K, Q or E, X13 is Y or E, X14 is Y or H, X15 is S, N or H, Xi6 is T or N, X17 is E or Q, Xis is S or H, X19 is T or D, X20 is E or Q, X21 is I or H, X22 is L, H or D.

[0027] In one aspect, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR- H1 comprising the amino acid sequence of NX13YIH SEQ (ID NO:228), (b) CDR-H2 comprising the amino acid sequence of WIX23PGDGX24TKYNEKFX25G (SEQ ID NO:229), and (c) CDR-H3 comprising the amino acid sequence of DX26X27HX28YYX29DY (SEQ ID NO:230), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO:231), (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of TX30SFILRT (SEQ ID NO:233) wherein X13 is Y or E, X23 is Y or E, X24 is N or D, X25 is E or K, X26 is Q, T or H, X27 is Y or V, X28 is H or Q, X29 is F or Y, X30 is Q or E, optionally wherein X23 is E when X24 is D and / or X23 is Y when X24 is N.

[0028] In one aspect, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR- H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 266, 272, 169 and 3, (b) CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 170 and 174, and (c) CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 273, 267, 159, 171 and 175, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7 or 271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 12. In a certain aspect, the antibody comprises

[0029] A) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 169, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 170, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9;

[0030] B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12;

[0031] C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:159, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; or

[0032] D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12

[0033] E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 266, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:267, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0034] F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 272, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NON, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:273, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0035] In one aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises

[0036] A) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 169, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 170, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9;

[0037] B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NON, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NON, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12;

[0038] C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NON, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NON, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:159, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; or

[0039] D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12

[0040] E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 266, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:276, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0041] F) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 272, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:273, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0042] In a certain aspect, the antibody further comprises a light chain variable domain framework FR1 sequence of SEQ ID NO:253, FR2 sequence of SEQ ID NO:254 or SEQ ID NO: 261, FR3 sequence of SEQ ID NO:255:, FR4 sequence of SEQ ID NO:256, and / or a heavy chain variable domain framework FR1 sequence of SEQ ID NO:257, FR2 sequence of SEQ ID NO:258 or sequence of SEQ ID NO: 261, FR3 sequence of SEQ ID NO:259, and FR4 sequence of SEQ ID NO:260. In a certain aspect, the antibody comprises a VH comprising an amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASNX13YIHWVRQAPGQGLEWIGWIX23PGDGX24 TKYNEKFX25GRATLTADTSTSTAYLELSSLRSEDTAVYYCARDX26X27HX28YYX29DY WGQGTLVTVSS (SEQ ID NO: 262), and / or a VL comprising an amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRX32NYLAWYQQKPGQPPKLLIX31 WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTX30SFILRTWGQGTLVTVS S (SEQ ID NO: 263), wherein X13 is Y or E, X23 is Y or E, X24 is N or D, X25 is E or K, X26 is Q, T or H, X27 is Y or V, X28 is H or Q, X29 is F or Y, X30 is Q or E and X31 is H or Y, X32 is K or H, optionally wherein X23 is E when X24 is D and / or X23 is Y when X24 is N and / or X31 is Y when X30 is Q and / or X31 is H when X30 is E.

[0043] In a certain aspect, the antibody comprises

[0044] A) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 172, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10;

[0045] B) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 176, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13;

[0046] C) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 160, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 206;

[0047] D) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 208, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 206 E) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 268, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 269; or

[0048] F) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 270, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 265.

[0049] In a certain aspect, the antibody comprises

[0050] A) a VH comprising an amino acid sequence of SEQ ID NO: 172, and / or a VL comprising an amino acid sequence of SEQ ID NO: 10;

[0051] B) a VH comprising an amino acid sequence of SEQ ID NO: 176, and / or a VL comprising an amino acid sequence of SEQ ID NO: 13;

[0052] C) a VH comprising an amino acid sequence of SEQ ID NO: 160, and / or a VL comprising an amino acid sequence of SEQ ID NO: 206;

[0053] D) a VH comprising an amino acid sequence of SEQ ID NO: 208, and / or a VL comprising an amino acid sequence of SEQ ID NO: 206;

[0054] E) a VH comprising an amino acid sequence of SEQ ID NO: 268, and / or a VL comprising an amino acid sequence of SEQ ID NO: 269 ; or

[0055] F) a VH comprising an amino acid sequence of SEQ ID NO: 270, and / or a VL comprising an amino acid sequence of SEQ ID NO: 265.

[0056] In one aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises

[0057] A) a VH comprising an amino acid sequence of SEQ ID NO: 172, and / or a VL comprising an amino acid sequence of SEQ ID NO: 10; B) a VH comprising an amino acid sequence of SEQ ID NO: 176, and / or a VL comprising an amino acid sequence of SEQ ID NO: 13;

[0058] C) a VH comprising an amino acid sequence of SEQ ID NO: 160, and / or a VL comprising an amino acid sequence of SEQ ID NO: 206;

[0059] D) a VH comprising an amino acid sequence of SEQ ID NO: 208, and / or a VL comprising an amino acid sequence of SEQ ID NO: 206;

[0060] E) a VH comprising an amino acid sequence of SEQ ID NO: 268, and / or a VL comprising an amino acid sequence of SEQ ID NO: 269 ; or

[0061] F) a VH comprising an amino acid sequence of SEQ ID NO: 270, and / or a VL comprising an amino acid sequence of SEQ ID NO: 265.

[0062] In a certain aspect, the antibody is an antibody fragment that binds CD3.

[0063] In a certain aspect, the antibody fragment is a Fab molecule.

[0064] In a certain aspect, the antibody is a full length IgG1antibody.

[0065] In a certain aspect, the antibody comprises an Fc domain composed of a first and a second subunit.

[0066] In a certain aspect, the antibody is multispecific.

[0067] In a certain aspect, the antibody is a bispecific antibody comprising at least one domain that binds to CD3 and at least one domain that binds to a target cell antigen.

[0068] In a certain aspect, the domain that binds to CD3 is a Fab molecule wherein the variable domains VL and VH or the constant domains CL and CHI, particularly the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced by each other.

[0069] In a certain aspect, the domain that binds to CD3 is a Fab molecule wherein in the constant domain CL the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat EU index) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

[0070] In a certain aspect, the Fab molecule is a conventional Fab molecule.

[0071] In a certain aspect, the target cell antigen is a cancer cell antigen. In a certain aspect, the Fc domain is an IgG Fc domain, particularly an IgG1Fc domain, more particularly a human IgG1Fc domain.

[0072] In a certain aspect, the Fc domain is a human Fc domain.

[0073] In a certain aspect, the Fc comprises a modification promoting the association of the first and the second subunit of the Fc domain.

[0074] In a certain aspect, the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function.

[0075] In a certain aspect, the antibody comprises an Fc region derived from a human IgG1Fc region and comprising the substitutions L234A, L235A and P329G (LALA-PG).

[0076] In on aspect, the AUC fold change pH 6.5 to pH 7.4 is at least 2.9, at least 3.0, at least 4.8, at least 4.9, at least 5.0 or at least 9.0 as measured by binding of the antibody as monovalent IgGi to human CD3 on Jurkat NF AT T cell line quantified as the percentage of CD3 positive cells at 4°C with a BD Symphony A3 cell analyzer.

[0077] In one aspect, the AUC fold change pH 6.5 to pH 7.4 is at least 3.7, at least 4.0, at least 4.6, at least 4.9, at least 5.4, at least 6.0, at least 8.9 or at least 9.3 as measured by binding of the antibody as monovalent IgGi to human CD3 on Jurkat NF AT T cell line quantified as the Mean Fluorescent Intensity (MFI) at 4°C with a BD Symphony A3 cell analyzer.

[0078] In one aspect, the invention provides an isolated nucleic acid encoding the antibody of the invention.

[0079] In one aspect, the invention provides a host cell comprising the nucleic acid according to the invention.

[0080] In one aspect, the invention provides a method of producing an antibody that binds to CD3 comprising culturing the host cell comprising the nucleic acid according to the invention under conditions suitable for the expression of the antibody.

[0081] In a certain aspect, the invention provides further a method of producing an antibody that binds to CD3 comprising culturing the host cell comprising the nucleic acid according the invention under conditions suitable for the expression of the antibody comprising recovering the antibody from the host cell.

[0082] In one aspect, the invention provides an antibody produced by the method according to the invention.

[0083] In one aspect, the invention provides a pharmaceutical composition comprising the antibody according to the invention and a pharmaceutically acceptable carrier.

[0084] In a certain aspect, the invention provides the antibody of the invention or the pharmaceutical composition according to the invention for use as a medicament. In a certain aspect, the invention provides the antibody according to the invention or the pharmaceutical composition according to the invention for use in treating cancer.

[0085] In one aspect, the invention provides the use of an antibody according to the invention or the pharmaceutical composition according to the invention in the manufacture of a medicament for treatment of cancer.

[0086] In one aspect, the invention provides the use of the antibody according to the invention or the pharmaceutical composition according to the invention in the manufacture of a medicament for pH dependent T-cell redirection and pH dependent T-cell mediated cancer cell killing.

[0087] In one aspect, the invention provides a method of treating an individual having cancer, comprising administering to the individual an effective amount of the antibody according to the invention or the pharmaceutical composition according to the invention.

[0088] In one aspect, the invention provides a method of pH dependent T-cell redirection and T-cell mediated cancer cell killing in an individual, comprising administering to the individual an effective amount of the antibody according to the invention or the pharmaceutical composition according to the invention to redirect T cells and mediate T-cells to kill cancer cells in a pH dependent manner.

[0089] BRIEF DESCRIPTION OF THE FIGURES

[0090] Figure 1 A-F Binding of anti-CD3 IgGl to human CD3, expressed on Jurkat NFAT cells and CD3 negative control cell line HEK-293T cells. (Fig. 1 A) non pH responsive parental clone P1AD5407 (40G5c bivalent, FV000595), (Fig. 1 B-F) engineered pH responsive clones P1AE0916 (FV002390), P1AF7430 (FV004700), P1AF7431 (FV004702), P1AF7433 (FV004708) and P1AF7438 (FV007415). Results show binding of the antibodies to cells at pH 6.5 and 7.4. All clones shown led to dose dependent and specific binding to human CD3 and no binding to CD3 negative cell line. Non pH responsive control IgG (FIG. 1 A) led to similar binding at both pH conditions whereas pH responsive clones P1AE0916 (FV002390), P1AF7430 (FV004700), P1AF7431 (FV004702), P1AF7433 (FV004708) and P1AF7438 (FV007415) led to higher binding at acidic pH 6.5 and low or no binding at neutral pH 7.4 (Fig. 1 B-F). Depicted are average values of technical duplicates and error bars indicate SD (standard deviation). Figure 2 A-D Reporter cell activation assay with Jurkat NF AT cells determined by luminescence. The latter is induced upon simultaneous binding of the anti-CD3 IgGls to CD3 expressed on Jurkat cell and PGLALA mutation bearing Fc region to CH0-K1 expressing anti-PGLALA Fab used as cross-linking cells. (Fig. 2 A) non pH responsive parental clone P1AD5407 (40G5c bivalent, FV000595) , (Fig. 2 B-D) engineered pH responsive clones P1AE0916 (FV002390), P1AF7433 (FV004708) and P1AF7438 (FV007415). Results show relative luminescence emitted RLU (500ms / sample) by reporter cells 6 hours after treatment with anti-CD3 antibodies in assay media set at pH 6.5 and 7.4. As shown, named constructs show dose dependent increase in signal and pH responsive CD3 antibodies P1AE0916 (FV002390), P1AF7433 (FV004708) and P1AF7438 (FV007415) (FIG 2 B-D) led to higher activation at acidic pH 6.5 as compared to neutral pH 7.4. Depicted are average values of technical duplicates and error bars indicate SD.

[0091] Figure 3 A-C. Primary human T cell activation with plate coated anti-CD3 antibodies (Fig. 3A) non pH responsive parental clone P1AD5407 (40G5c bivalent, FV000595), (Fig. 3B and 3C) engineered pH responsive clones P1AE0916 (FV002390) and P1AF7438 (FV007415) as bivalent IgGls. Results show expression of T cell activation marker CD69 on CD4 T cells derived from two PBMC donors, 48h after treatment with plate coated anti-CD3 antibodies in assay media set at pH 6.5 and 7.4. As shown all named constructs led to dose dependent increase in percentage of CD69+ CD4+ T cells. pH responsive clones P1AE0916 (FV002390) and P1AF7438 (FV007415) (FIG 3 B and C) led to higher activation at acidic pH 6.5 as compared to neutral pH 7.4, non pH responsive control P1AD5407 (40G5c bivalent, FV000595) (FIG 3 A) showed similar activation at both pH. Depicted are average values of technical replicates (n=3) and error bars represent SD. Results are baseline corrected to signal from cells with respective buffers containing no antibody.

[0092] Figure 4 A-F. Binding of anti-CD3 IgGl to human CD3, expressed on Jurkat NF AT cells (Fig. 4A) non pH responsive parental clone P1AD5407 (40G5c bivalent, FV000595), ( Fig. 4B-F) engineered pH responsive clones P1AE0916 (FV002390), P1AF3715 (FV004747), P1AF7438 (FV007415), P1AG3716 (FV014505) and P1AG3719 (FV014508). Results show binding of the antibodies to cells co-incubated in buffer with pH 6.5 and 7.4. All tested clones led to dose dependent increase in binding, pH responsive clones P1AE0916 (FV002390), P1AF3715 (FV004747), P1AF7438 (FV007415), P1AG3716 (FV014505) and P1AG3719 (FV014508) (FIG 4 B-F) led to higher binding at acidic pH 6.5 and lower binding at neutral pH 7.4, non pH responsive clone P1AD5407 (40G5c bivalent, FV000595) (FIG 4 A), led to similar binding at both pH conditions. Depicted are average values of technical duplicates and error bars represent SD. Results are baseline corrected to signal from cells containing no antibody and further divided by 1000.

[0093] Figure 5 A-I. Reporter cell activation induced with Jurkat NFAT cells determined by luminescence. The latter is induced upon simultaneous binding of the anti-CD3 IgGls to CD3 expressed on Jurkat cell and PGLALA mutation bearing Fc region to CHO-K1 expressing anti- PGALA Fab used as cross-linking cells. (Fig. 5 A) non pH responsive parental clone Pl AD5407 (40G5c bivalent, FV000595), (Fig. 5 B-I) engineered pH responsive clones P1AG2189 (FV013439), P1AG2194 (FV013444), P1AE0916 (FV002390), P1AG2192 (FV013442), P1AG3716 (FV014505), P1AF7438 (FV007415), P1AG2193 (FV013443) and P1AG3720 (FV014509). Results show relative luminescence emitted RLU (500ms / sample) by reporter cells 6 hours after treatment with anti-CD3 antibodies in assay media set at pH 6.5 and 7.4. As shown, tested constructs show dose dependent increase in signal and pH responsive CD3 antibodies P1AG2189 (FV013439), P1AG2194 (FV013444), P1AE0916 (FV002390), P1AG2192 (FV013442), P1AG3716 (FV014505), P1AF7438 (FV007415), P1AG2193 (FV013443) and P1AG3720 (FV014509) (FIG 5 B-I) led to higher activation at acidic pH 6.5 as compared to neutral pH 7.4. Depicted are average values of technical duplicates and error bars indicate SD. Results are baseline corrected to signal from cells with respective buffers containing no antibody.

[0094] Figure 6 A-F. Reporter cell activation induced with Jurkat NFAT cells determined by luminescence. The latter is induced upon simultaneous binding of the anti-CD3 monovalent IgGls to CD3 expressed on Jurkat cell and PGLALA mutation bearing Fc region to CHO-K1 expressing anti-PGALA Fab used as cross-linking cells. Results show relative luminescence emitted RLU (500ms / sample) by reporter cells 6 h after treatment with anti-CD3 antibodies in assay media set at pH 6.5 and 7.4. As shown, all pH responsive constructs show dose dependent increase in signal and pH responsive CD3 antibodies P1AG1816 (FV013233), P1AG1817 (FV013234), P1AG1835 (FV013252), P1AG1840 (FV013257) and P1AG1841 (FV013258) (FIG 6 A-F) led to higher activation at acidic pH 6.5 as compared to neutral pH 7.4. Depicted are average values of technical duplicates and error bars indicate SD. Results are baseline corrected to signal from cells with respective buffers containing no antibody. Figure 7 A-L. Primary human T cell activation with plate coated anti-CD3 antibodies as bivalent or monovalent IgGls. (FIG 7 A and FIG 7 G) depict non pH responsive parental clone as bivalent IgG P1AD5407 (40G5c) and monovalent IgG P1AG1814 (40G5c monovalent) respectively, (FIG7 B-F and FIG 7 H-L) depict engineered pH responsive clones as bivalent IgGs P1AE0916 (FV002390), P1AF7438 (FV007415), P1AG2194 (FV013444), P1AG3716 (FV014505) and P1AG3720 (FV014509) and monovalent IgGs P1AG1816 (FV013233), P1AG1817 (FV013234), P1AG1839 (FV013256), P1AG1840 (FV013257) and P1AG1841 (FV013258) respectively. Results show expression of T cell activation marker CD69 on PBMC derived CD4 T cells, 62 h after treatment with plate coated anti-CD3 antibodies in assay media set at pH 6.5 and 7.4. As shown all tested constructs led to dose dependent increase in percentage of CD69+ CD4+ T cells. pH responsive clones P1AE0916 (FV002390), P1AF7438 (FV007415), P1AG2194 (FV013444), P1AG3716 (FV014505), P1AG3720 (FV014509), P1AG1816 (FV013233), P1AG1817 (FV013234), P1AG1839 (FV013256), P1AG1840 (FV013257) and P1AG1841 (FV013258) (FIG 7 B-F and H-L) led to higher activation at acidic pH 6.5 as compared to neutral pH 7.4. Depicted are average values of triplicates and error bars represent SD. Results are baseline corrected to signal from cells with respective buffers containing no antibody.

[0095] Figure 8 A-F. Binding of anti-CD3 IgGl to human CD3, expressed on Jurkat NF AT cells and CD3 negative control cell line MKN-45. (FIG 8 A -F) engineered pH responsive clones P1AJ7610 (FV030298), P1AJ7611 (FV030299), P1AJ7612 (FV030300), P1AJ7613 (FV030301), P1AJ7614 (FV030302) and P1AJ7615 (FV030303). Results show binding of the antibodies to cells co-incubated in buffer with pH 6.5 and 7.4. Clones P1AJ7610 (FV030298), P1AJ7611 (FV030299), P1AJ7612 (FV030300), P1AJ7613 (FV030301), P1AJ7614 (FV030302) led to dose dependent increase in binding, with higher binding at acidic pH 6.5 and lower binding at neutral pH 7.4. Depicted are average values of technical duplicates and error bars represent SD (standard deviation). Results are baseline corrected to signal from cells with buffer containing no antibody and further divided by 1000.

[0096] Figure 9 A-L. Reporter cell activation with Jurkat NF AT cells determined by luminescence. The latter is induced upon simultaneous binding of anti-CD3 IgGls to CD3 expressed on Jurkat cell and PGLALA mutation bearing Fc region to CHO-K1 expressing anti-PGLALA Fab used as cross-linking cells. (FIG 9 A) non pH responsive parental clone P1AD5407 (40G5c bivalent, FV000595), (FIG 9 B-L) engineered pH responsive clones Pl AJ7604 (FV030293), Pl AJ7606 (FV030294), P1AJ7607 (FV030295), P1AJ7608 (FV030296), P1AJ7609 (FV030297), P1AJ7610 (FV030298), P1AJ7611 (FV030299), P1AJ7612 (FV030300), P1AJ7613 (FV030301), P1AJ7614 (FV030302), P1AJ7615 (FV030303). Results show relative luminescence emitted RLU (500 ms / sample) by reporter cells 6 hours after treatment with anti- CD3 antibodies in assay media set at pH 6.5 and 7.4 As shown, tested constructs show dose dependent increase in signal and pH responsive CD3 antibodies P1AJ7604 (FV030293), P1AJ7606 (FV030294), P1AJ7607 (FV030295), P1AJ7608 (FV030296), P1AJ7609 (FV030297), P1AJ7610 (FV030298), P1AJ7611 (FV030299), P1AJ7612 (FV030300), P1AJ7613 (FV030301), P1AJ7614 (FV030302), P1AJ7615 (FV030303) (FIG 9 B-L) led to higher activation at acidic pH 6.5 as compared to neutral pH 7.4. Depicted are average values of technical duplicates and error bars represent SD. Results are baseline corrected to signal from cells containing no antibody and further divided by 1000.

[0097] Figure 10 A-I. Binding of CD3 IgGl to human CD3, expressed on Jurkat NFAT cells (FIG 10 A) non pH responsive parental clone Pl AD4232 (40G5c) (FV000595), (FIG 10 B-I) engineered pH responsive clones P1AJ7612 (FV030300), P1AK5678 (FV034288), P1AK5679 (FV034289), P1AK5718 (FV034327), P1AK5723 (FV034332), P1AK5725 (FV034334), P1AK5726 (FV034335), P1AK5728 (FV034337). Results show binding of the antibodies to cells co-incubated in buffer with pH 6.5 and 7.4. All clones led to dose dependent increase in binding, pH responsive clones P1AJ7612 (FV030300), P1AK5678 (FV034288), P1AK5679 (FV034289), P1AK5718 (FV034327), P1AK5723 (FV034332), P1AK5725 (FV034334), P1AK5726 (FV034335), P1AK5728 (FV034337) (FIG 10 B-I) led to higher binding at acidic pH 6.5 and lower binding at neutral pH 7.4, non pH responsive clone Pl AD4232 (40G5c) (FIG 10 A), led to similar binding at both pH conditions. Depicted are average values of technical duplicates and error bars represent SD. Results are baseline corrected to signal from cells containing no antibody and further divided by 1000.

[0098] Figure 11 A and B. Monovalent and bivalent IgGl formats used for experimental assessment of pH-dependent CD3 binders. (A) Monovalent format consisted in a one-armed (OA) Fc fusion where the VH, CHI, and hinge region were fused to the knob Fc chain, while the hole Fc was occasionally C-terminally fused to a 6x-His tag. Both PGLALA and non PGLALA Fc domains were used. Similarly, the classical bivalent IgGl format (B) was used with PGLALA mutations in the FC domain. Figure 12 Figure 12 A-D. Binding of CD3 IgG to human CD3, expressed on Jurkat NF AT cells (A) non pH responsive parental clone as a TCB (B-D) engineered pH responsive clones. Results show binding of the antibodies to cells co-incubated in buffer with pH 6.5 and 7.4. All tested clones led to dose dependent increase in binding, pH responsive clones B-D led to higher binding at acidic pH 6.5 and lower binding at neutral pH 7.4, non pH responsive clone A, led to similar binding at both pH conditions. Depicted are average values of technical duplicates and error bars represent SD. Results are baseline corrected to signal from cells containing no antibody and further divided by 1000.

[0099] Figure 13 A-M. Binding of monovalent CD3 binders to CD3 expressing Jurkat cells NF AT cells depicted as percentage of CD3 positive cells at pH 6.5 and pH 7.4. (A) non pH responsive parental clone P1AK5490 (B-E) engineered pH responsive clones of the present invention P1AK5488, P1AK5489, P1AK5719 and P1AK5723 (F-M) comparator clones P1AM7114 (SEQ ID NOs: 282 and 283), P1AM7115 (SEQ ID NOs:284 and 285), P1AM7118 (SEQ ID NOs: 286 and 287), P1AM7117 (SEQ ID Nos: 298 and 299), P1AM7119 (SEQ ID NOs: 288 and 289), P1AM7120 (SEQ ID NOs: 290 and 291), P1AM7121 (SEQ ID NOs: 292 and 293), P1AM7122 (SEQ ID NOs: 294 and 295) and Pl AM7123 (SEQ ID NOs: 296 and 297). Results show binding of the antibodies to cells co-incubated in buffer with pH 6.5 and 7.4. Depicted are average values of technical duplicates and error bars represent SD. Results are baseline corrected to signal from cells containing no antibody.

[0100] Figure 14 A-M. Binding of monovalent CD3 binders to CD3 expressing Jurkat cells NF AT cells depicted as mean fluorescent intensity at pH 6.5 and pH 7.4. (A) non pH responsive parental clone P1AK5490 (B-E) engineered pH responsive clones of the present invention P1AK5488, P1AK5489, P1AK5719 and P1AK5723 (F-M) comparator clones P1AM7114, P1AM7115, P1AM7118, P1AM7117, P1AM7118, P1AM7119, P1AM7120, P1AM7121, P1AM7122 and P1AM7123. Results show binding of the antibodies to cells co-incubated in buffer with pH 6.5 and 7.4. Depicted are average values of technical duplicates and error bars represent SD. Results are baseline corrected to signal from cells containing no antibody.

[0101] Figure 15 A-O. Binding of monovalent CD3 binders to CD3 expressing Jurkat NF AT cells depicted as percentage of CD3 positive cells at pH 6.5 and pH 7.4. (A) non pH responsive parental clone P1AK5490 (B-E) engineered pH responsive clones of the present invention P1AK5488, P1AK5489, P1AK5719 and P1AK5723 (F-0) Comparator clones P1AM7114, P1AM7115, P1AM7118, P1AM7117, P1AM7118, P1AM7119, P1AM7120, P1AM7121, P1AM7122 and P1AM7123. Results show binding of the antibodies to cells co-incubated in buffer with pH 6.5 and 7.4. Depicted are average values of technical duplicates and error bars represent SD. Results are baseline corrected to signal from cells containing no antibody.

[0102] DETAILED DESCRIPTION

[0103] I DEFINITIONS

[0104] Terms are used herein as generally used in the art, unless otherwise defined in the following.

[0105] Use of the terms “first” or “second” is not intended to confer a specific order or orientation of the moiety unless explicitly so stated.

[0106] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “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 a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described in the following. If not stated otherwise, the KDS herein refer to affinity.

[0107] An “affinity matured” antibody refers to an antibody with one or more alterations in one or more complementary determining regions (CDRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

[0108] “Avidity” refers to the strength of the sum total of noncovalent interactions between multiple binding sites of a molecule (e.g., an antibody) and its binding partner(s) (e.g., an antigen). The avidity of a molecule X for its partner(s) can generally be represented by the dissociation constant (KD). Avidity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring avidity are described herein. “Reduced binding”, for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term also includes reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity (lower absolute value for KD measurement) for the respective interaction.

[0109] “CD3” refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CD3 as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, e.g., splice variants or allelic variants. In one aspect, CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3s). The amino acid sequence of human CD3s is shown in SEQ ID NO: 211 (without signal peptide). See also UniProt (www.uniprot.org) accession no. P07766 (version 209), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. In another aspect, CD3 is cynomolgus (Macaca fascicularis) CD3, particularly cynomolgus CD3e. The amino acid sequence of cynomolgus CD3s is shown in SEQ ID NO: 212 (without signal peptide). See also NCBI GenBank no. BAB71849.1. In certain aspects the antibody of the invention binds to an epitope of CD3 that is conserved among the CD3 antigens from different species, particularly human and cynomolgus CD3. In preferred aspects, the antibody binds to human CD3.

[0110] The terms “anti-CD3 antibody” and “an antibody that binds to CD3” refer to an antibody that is capable of binding CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD3. The terms “anti-CD3 antibody” and “an antibody that binds to CD3” include pH responsive antibodies. The term “pH responsive antibody” as used in the current application refers to an antibody with preferential binding at acidic pH as compared to neutral pH. “Acidic pH” refers to a pH lower than 7.0 particularly to a pH ranging from 6.0 to 6.6 and more particularly the acidic pH refers to pH 6.1 and / or pH 6.5. “Neutral pH” refers to a pH ranging from 7.0 to 7.4, particularly to pH 7.4. An antibody is said to “preferentially bind” to CD3 at acidic pH as compared to neutral pH or have “preferential binding” at acidic pH as compared to neutral pH, when the antibody binds to CD3 at neutral pH 7.4 with a first KD and at acidic pH 6.5 with a second KD, wherein the first KD is greater than the second KD as measured by SPR at 25°C. In certain aspects the first KD at pH 7.4 is greater than the second KD at pH 6.5 by a factor of at least 2.0 or 4.5 as measured by SPR at 25°C. In a further aspect, the first KD at pH 7.4 is greater than the second KD at pH 6.5 by a factor of at least 2.0, at least 2.2, at least 2.8, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 4.6, at least 5.0, at least 10.0, at least 10.9, at least 14.3, at least 16.2, at least 18.0, at least

[0111] 19.6, at least 20.0, at least 21.0, at least 23.0, at least 24.0, at least 25.0, at least 28.2, at least

[0112] 29.7, at least 32.4, at least 33.3, at least 38.6, at least 43.5, at least 43.9, at least 52.8, at least 61.9, at least 70.0, at least 80.0, at least 90.2, at least 100.0, at least 113.2, at least 120.0, at least 130.0, at least 140.0, at least 150.0, at least 160.0, at least 170.0, at least 181.6, least 190.0, at least 200.0, at least 210.0, at least 220.0, at least 230.0, at least 236.0 or more, as measured by SPR at 25°C, particularly wherein the second KD is equal to 47nM or less at pH 6.5 and the first KD is equal to 0.2 nM or more at pH 7.4 as measured by SPR at 25°C. In one aspect, the extent of binding of an antibody that binds to CD3 to an unrelated, non-CD3 protein is less than about 10% of the binding of the antibody to CD3 as measured, e.g., by surface plasmon resonance (SPR) at 25°C. An antibody is said to “specifically bind” to CD3 when the antibody has a KD of IpM or less at pH 6.5 as measured by SPR at 25°C. In certain aspects, an antibody that binds to CD3 binds to an epitope of CD3 that is conserved among CD3 from different species.

[0113] The term “pH window” herein encompasses the fold difference in KD between pH 6.5 and 7.4.

[0114] The term “antibody” encompasses various antibody structures exhibiting the desired antigen-binding activity, including but not limited to: monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments.

[0115] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0116] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0117] The term “immunoglobulin molecule” herein refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1(IgA1) and α2(IgA2). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.

[0118] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgGs, IgG4, IgA1, and IgA2. In certain aspects, the antibody is of the IgG1isotype. In certain aspects, the antibody is of the IgG1isotype with the P329G, L234A and L235A mutation to reduce Fc- region effector function. In other aspects, the antibody is of the IgG2isotype. In certain aspects, the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve stability of IgG4 antibody. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, 7, and p, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

[0119] A “Fab molecule” refers to a protein consisting of the VH and CHI domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.

[0120] By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (VL-CH1, inN- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in N- to C-terminal direction). For clarity, in a crossover Fab molecule wherein the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CHI is referred to herein as the “heavy chain” of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule wherein the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the “heavy chain” of the (crossover) Fab molecule.

[0121] In contrast thereto, by a “conventional” Fab molecule is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant domains (VH-CH1, in N- to C-terminal direction), and a light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction).

[0122] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-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 an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case in particular where the final two C- terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, Kabat EU numbering). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including an Fc region are denoted herein without C-terminal lysine if not indicated otherwise. The corresponding sequence including a C-terminal lysine residue is also encompassed, however. Accordingly, in one aspect, a heavy chain including an Fc region as specified herein comprises an additional C-terminal lysine residue (K447, Kabat EU numbering). Also encompassed is the corresponding sequence without the C-terminal glycine residue. Accordingly, in one aspect, a heavy chain including an Fc region as specified herein lacks the C-terminal glycine residue (G446, Kabat EU numbering). In such a heavy chain, the C-terminal amino acid residue may be proline (P445, Kabat EU numbering) or proline amide (P445-NH2, Kabat EU numbering). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system, also called the EU index, as described in Kabat 1991.

[0123] By “fused” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0124] The term “multispecific” means that the antibody is able to specifically bind to at least two distinct antigenic determinants. A multispecific antibody can be, for example, a bispecific antibody. Typically, a bispecific antibody comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain aspects the multispecific (e.g. bispecific) antibody is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

[0125] The term “valent” as used herein denotes the presence of a specified number of antigen binding sites in an antigen binding molecule. As such, the term “monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antigen binding molecule.

[0126] An “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site.

[0127] As used herein, the term "antigenic determinant" or "antigen" refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of noncontiguous amino acids) on a polypeptide macromolecule to which a domain that binds to the antigen binds, forming a complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and / or in the extracellular matrix (ECM). In a preferred aspect, the antigen is a human protein.

[0128] A “target cell antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma (in that case a “tumor cell antigen”). Preferably, the target cell antigen is not CD3, and / or is expressed on a different cell than CD3. The terms “constant region derived from human origin” or “human constant region” as used in the current application denotes a constant heavy chain region of a human antibody of the subclass IgG1, IgG2, IgGs, or IgG4 and / or a constant light chain kappa or lambda region. Such constant regions are well known in the state of the art and e.g. described by Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also e.g. Johnson, G., and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785- 2788). Unless otherwise specified herein, numbering of amino acid residues in the constant region is according to the EU numbering system, also called the EU index of Kabat, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0129] “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0130] An “effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0131] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-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 an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including an Fc region are denoted herein without C-terminal glycine-lysine dipeptide if not indicated otherwise. In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to EU index). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called 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.

[0132] “Framework” or “FR” refers to variable domain residues other than complementary determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR-H2(CDR-L2)- FR3- CDR-H3(CDR-L3)-FR4.

[0133] The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0134] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0135] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human-repertoire inspired antibody-encoding synthetic libraries. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0136] The term "domain that binds to" refers to the part of an antibody that comprises the area which binds to and is complementary to part or all of an antigen. A domain that binds to an antigen may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). In preferred aspects, a domain that binds to an antigen e.g. CD3 comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0137] A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one aspect, for the VL, the subgroup is subgroup kappa II ( hIGKV2-28-01 ) as in Kabat et al., supra. In one aspect, for the VH, the subgroup is subgroup I and III ( hIGHVl-46-01, hIGHVl-46-02, hIGHV3-23-05) as in Kabat et al., supra.

[0138] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0139] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, 6thed., W.H. Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As used herein in connection with variable region sequences, "Kabat numbering" refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0140] Glutamine or glutamate residues at the N-terminus of antibody heavy or light chains may be converted to pyro-glutamate spontaneously (see e.g. Liu et al., Journal of Pharmaceutical Sciences 97, 2426-2447 (2008), Rehder et al., Journal of Chromatography A 1102, 164-175 (2006), Chelius et al., Anal Chem 78, 2370-2376 (2006)). Hence, variable regions or variable domains disclosed herein which comprise either a glutamine (Q) or a glutamate (E) amino acid residue at the N-terminus of an the antibody heavy or light chain, may comprise an N- terminal pyro-glutamate (pyroE) residue instead of the N-terminal Q or E residue. Likewise, antibody heavy chains or light chains disclosed herein which comprise either a glutamine (Q) or a glutamate (E) amino acid residue at the N-terminus, may comprise an N terminal pyroglutamate (pyroE) residue instead of the N-terminal Q or E residue. Accordingly, for each antibody heavy chain, light chain, or variable domain or region sequence disclosed herein that contains an N-terminal Q or E residue, the corresponding sequence with an N-terminal pyroE residue is also encompassed.

[0141] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to as “numbering according to Kabat” or “Kabat numbering” herein. Specifically the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotype and the Kabat EU index numbering system (see pages 661- 723) is used for the heavy chain constant domains (CHI, hinge, CH2 and CH3), which is herein further clarified by referring to “numbering according to Kabat EU index” or “Kabat EU index numbering” in this case.

[0142] The term “complementarity determining region” or “CDR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity. Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). CDRs are defined by a variety of methods / sy stems by those skilled in the art. These systems and / or definitions have been developed and refined over a number of years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and generally is the most commonly used. The Chothia definition is based on the location of the structural loop regions. The IMGT system is based on sequence variability and location within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analyses of the available antibody crystal structures. Software programs (e g-, abYsis: http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi) are available and known to those of skill in the art for analysis of antibody sequences and determination of CDRs.

[0143] Exemplary CDRs herein include (numbering of amino acid residues according to the reference cited, i.e. Chothia numbering for the Chothia and Contact definition, Kabat numbering for the Kabat definition and IMGT numbering for the IMGT definition):

[0144] (a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3), according to Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) (“Chothia definition”);

[0145] (b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35B (Hl), 50-65 (H2), and 95-102 (H3), according to Kabat 1991 (“Kabat definition”);

[0146] (c) antigen contacts occurring at amino acid residues 30-36 (LI), 46-55 (L2), 89-96 (L3), 30-35 (Hl), 47-58 (H2), and 93-101 (H3), according to MacCallum et al. J. Mol. Biol. 262: 732-745 (1996) (“Contact definition”); and

[0147] (d) CDRs occurring at amino acid residues residues 27-38 (LI), 56-65 (L2), 105-117 (L3), 27-38 (Hl), 56-65 (H2), and 105-117 (H3), according to Lefranc et al. Dev. Comp. Immunol. 27: 55-77 (2003) (“IMGT definition”).

[0148] Unless otherwise indicated, the CDRs are determined herein according to Kabat 1991. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, MacCallum, Lefranc, or any other scientifically accepted definiti on / sy stem .

[0149] In one aspect, CDR residues comprise those identified in the Sequences Table below or elsewhere in the specification.

[0150] An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0151] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

[0152] An “isolated” antibody is one which has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0153] The term “nucleic acid molecule” or “polynucleotide” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the 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 expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 Bl).

[0154] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0155] “Isolated nucleic acid encoding an antibody that binds to CD3” refers to one or more nucleic acid molecules encoding anti-CD3 antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0156] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, 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 making monoclonal antibodies being described herein.

[0157] A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0158] “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.

[0159] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0160] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN- 2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU5 10087 and is described in WO 2001 / 007611.

[0161] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227- 258; and Pearson et. al. (1997) Genomics 46:24-36 and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www. ebi.ac.uk / Tools / sss / fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare the sequences, using the ggsearch (global protein: protein) program and default options (BLOSUM50; open: -10; ext: - 2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

[0162] The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0163] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0164] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

[0165] “T cell activation” as used herein refers to one or more cellular responses of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays to measure T cell activation are known in the art and described herein.

[0166] A “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein preferably includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be non-identical in the sense that further components fused to each of the subunits (e.g. domains that bind to an antigen e.g. CD3) are not the same. In some aspects, the modification promoting the association of the first and the second subunit of the Fc domain comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a preferred aspect, the modification promoting the association of the first and the second subunit of the Fc domain comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.

[0167] The term “effector functions” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.

[0168] An “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRIIIa (CD 16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).

[0169] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives therefore comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006 / 082515 or PCT publication no. WO 2012 / 130831).

[0170] As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.

[0171] The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino- and / or carb oxy -terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non-naturally occurring amino acids or by 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 and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly.

[0172] The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

[0173] II. COMPOSITIONS AND METHODS

[0174] In one aspect, the invention is based, in part, on antibodies that bind CD3 preferentially at acidic pH as compared to neutral pH. In certain aspects, antibodies that bind to CD3, wherein the antibodies bind to CD3 at neutral pH 7.4 with a first KD and at an acidic pH 6.5 with a second KD, wherein the first KD is greater than the second KD and the first KD and the second KD are measured by surface plasmon resonance (SPR) at 25 °C are provided. The antibodies show preferential binding at acidic pH as compared to neutral pH. Preferential binding at acidic pH as compared to neutral pH is a favorable property for therapeutic applications such as in cancer, for example with respect to safety and efficacy. Antibodies of the invention are useful, e.g., for the diagnosis or treatment of diseases such as cancer.

[0175] A. Exemplary pH responsive Antibodies that bind to CD3

[0176] In one aspect, the invention provides antibodies that bind to CD3, wherein the antibodies bind to CD3 at neutral pH 7.4 with a first KD and at an acidic pH 6.5 with a second KD, wherein the first KD is greater than the second KD and the first KD and the second KD are measured by surface plasmon resonance (SPR) at 25 °C. In one aspect, provided are isolated antibodies that bind to CD3, wherein the antibodies bind to CD3 at neutral pH 7.4 with a first KD and at an acidic pH 6.5 with a second KD, wherein the first KD is greater than the second KD and the first KD and the second KD are measured by surface plasmon resonance (SPR) at 25 °C. In one aspect, the invention provides antibodies that specifically bind to CD3, wherein the antibodies bind to CD3 at neutral pH 7.4 with a first KD and at an acidic pH 6.5 with a second KD, wherein the first KD is greater than the second KD and the first KD and the second KD are measured by surface plasmon resonance (SPR) at 25 °C. In one aspect, the first KD at neutral pH 7.4 is greater than the second KD at acidic pH 6.5 by a factor of at least 2.0, at least 2.2, at least 2.8, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 4.6, at least 5.0, at least 10.0, at least 10.9, at least 14.3, at least 16.2, at least 18.0, at least 19.6, at least 20.0, at least 21.0, at least 23.0, at least 24.0, at least 25.0, at least 28.2, at least 29.7, at least 32.4, at least 33.3, at least 38.6, at least 43.5, at least 43.9, at least 52.8, at least 61.9, at least 70.0, at least 80.0, at least 90.2, at least 100.0, at least 113.2, at least 120.0, at least 130.0, at least 140.0, at least 150.0, at least 160.0, at least 170.0, at least 181.6, least 190.0, at least 200.0, at least 210.0, at least 220.0, at least 230.0, at least 236.0 or more. Most particularly wherein the first KD at neutral pH 7.4 is greater than the second KD at acidic pH 6.5 by a factor of at least 4.5. In certain aspects, an antibody that binds to CD3 preferentially binds to CD3 at acidic pH as compared to neutral pH. In some aspects, an antibody that binds to CD3 has a dissociation constant (KD) of < 47 nM (e.g. 47xl0'9M or less) at acidic pH 6.5 as measured by SPR at 25°C, particularly, the antibody that binds to CD3 additionally has a dissociation constant (KD) of > 0.2 nM (e.g. 0.2 x 10'9M or more) at neutral pH 7.4 as measured by SPR at 25°C. More particularly, the antibody that binds to CD3 has a dissociation constant (KD) of < 4 nM (e.g. 4xl0'9M or less) at acidic pH 6.5 as measured by SPR at 25°C, and a dissociation constant (KD) of > 9 nM (e.g. 9 x 10'9M or more) at neutral pH 7.4 as measured by SPR at 25°C. In certain aspects, the first KD is greater than the second KD by a factor ranging from 2.0 to 236.0 or from 4.5 to 236.0. In a certain aspect the KD reflects monovalent affinity.

[0177] In one aspect, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of NX13X14IH (SEQ ID NO:225), (b) CDR-H2 comprising the amino acid sequence of WIX7PGX8GX9TX10YNX11KFX12G (SEQ ID NO:226), and (c) CDR-H3 comprising the amino acid sequence of DX1X2X3X4YX5X6DY (SEQ ID NO:227), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of KSSQSLLNX22RTRKNYLA (SEQ ID NO:222), (e) CDR-L2 comprising the amino acid sequence of WASX19RX20X21 (SEQ ID NO:223), and (f) CDR-L3 comprising the amino acid sequence of XisXieSFXnXisRT (SEQ ID NO:224), wherein Xi is T, S or Q, X2is Y, V or R, X3is S or V, X4is N, Q or H, X5is F or Y, X6is F, Y or H, X7is Y, L, V, H, E, K or R, X8is D, Y, T, R or E, X9is N, D, Q or E, X10 is R or K, Xu is E, Q or H, X12 is K, Q or E, X13 is Y or E, X14 is Y or H, X15 is S, N or H, Xi6 is T or N, X17 is E or Q, Xis is S or H, X19 is T or D, X20 is E or Q, X21 is I or H, X22 is L, H or D.

[0178] In one aspect, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of NX13YIH (SEQ ID NO:228), (b) CDR-H2 comprising the amino acid sequence of WIX23PGDGX24TKYNEKFX25G (SEQ ID NO:229), and (c) CDR-H3 comprising the amino acid sequence of DX26X27HX28YYX29DY (SEQ ID NO:230), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO:231), (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of TX30SFILRT (SEQ ID NO:233) wherein X13 is Y or E, X23 is Y or E, X24 is N or D, X25 is E or K, X26 is Q, T or H, X27 is Y or V, X28 is H or Q, X29 is F or Y, X30 is Q or E, optionally wherein X23 is E when X24 is D and / or X23 is Y when X24 is N.

[0179] In one aspect, the antibody comprises a VH comprising an amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASNX13YIHWVRQAPGQGLEWIGWIX23PGDGX24 TKYNEKFX25GRATLTADTSTSTAYLELSSLRSEDTAVYYCARDX26X27HX28YYX29DY WGQGTLVTVSS (SEQ ID NO: 262), and / or a VL comprising an amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRX32NYLAWYQQKPGQPPKLLIX31 WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTX30SFILRTWGQGTLVTVS S (SEQ ID NO: 263), wherein X13 is Y or E, X23 is Y or E, X24 is N or D, X25 is E or K, X26 is Q, T or H, X27 is Y or V, X28 is H or Q, X29 is F or Y, X30 is Q or E and X31 is H or Y, X32 is K or H, optionally wherein X23 is E when X24 is D and / or X23 is Y when X24 is N and / or X31 is Y when X30 is Q and / or X31 is H when X30 is E.

[0180] In one aspect, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 266, 272, 169 and 3, (b) CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 170 and 174, and (c) CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 273, 267, 159, 171 and 175, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7 or 271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 12.

[0181] In one aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NOs: 172 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 10.

[0182] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 172 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 172. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 172 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 172. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 172 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 172.

[0183] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 10 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 10. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 10 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 10. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 10 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 10.

[0184] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 169; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 170; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:171; (d) CDR- L1 comprising the amino acid sequence of SEQ ID NO:7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 172. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0185] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 172. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 172. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO: 172, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 169, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 170, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 172. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 10. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 10, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:7, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NOV.

[0186] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO: 172 and SEQ ID NO: 10, respectively, including post-translational modifications of those sequences.

[0187] In further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CORED amino acid sequences of the VH domain of SEQ ID NOs: 176 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 13.

[0188] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 176 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 176. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 176 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 176. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 176 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 176.

[0189] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 13 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 13. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 13 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 13. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 13 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 13.

[0190] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NOD; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NOD; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 176. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0191] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 176. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 176. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO: 176, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 175. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 13. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 13, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:7, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 12.

[0192] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO: 176 and SEQ ID NO: 13, respectively, including post-translational modifications of those sequences.

[0193] In a further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH domain of SEQ ID NOs: 160 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 206.

[0194] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 160 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 160. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 160 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 160. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 160 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 160.

[0195] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206.

[0196] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 159; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 160, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:206; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 160. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:206.

[0197] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 160. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 160. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 160. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO: 160, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 159. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:206. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:206. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:206. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 206, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO:7, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID N0:12.

[0198] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO: 160 and SEQ ID NO:206, respectively, including post-translational modifications of those sequences.

[0199] In a further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH domain of SEQ ID NOs: 208 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 206.

[0200] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:208 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:208. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:208 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:208. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:208 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:208.

[0201] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206.

[0202] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:208, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:206; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:208. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:206. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:208. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:208. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:208. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO:208, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 171. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:206. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:206. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:206. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 206, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO:7, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID N0:12.

[0203] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:208 and SEQ ID NO:206, respectively, including post-translational modifications of those sequences.

[0204] In a further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH domain of SEQ ID NOs: 268 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 269.

[0205] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:268 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:268. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:268 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:268. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:268 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:268.

[0206] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:269 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:269. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:269 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:269. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:269 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:269. In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:266; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:276; (d) CDR- L1 comprising the amino acid sequence of SEQ ID NO:271; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:268, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:269; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:268. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:269.

[0207] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:268. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:268. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:268. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO:268, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:266, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO:267. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:269. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:269. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:269. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 269, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO:271, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 12.

[0208] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:270 and SEQ ID NO:265, respectively, including post-translational modifications of those sequences.

[0209] In a further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH domain of SEQ ID NOs: 270 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 265.

[0210] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:270 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:270. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:270 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:270. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:270 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:270.

[0211] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:265 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:265. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:265 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:265. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:265 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:265.

[0212] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:272; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:273; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:270, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:265; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:270. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:265.

[0213] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:270. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:270. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:270. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO:270, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:272, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO:273. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:265. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:265. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:265. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO:265, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO:271, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 12.

[0214] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:270 and SEQ ID NO:265, respectively, including post-translational modifications of those sequences. In a further aspect of the invention, an antibody that binds to CD3 according to any of the above aspects is a monoclonal antibody, including a humanized antibody. In one aspect, an antibody that binds to CD3 is an antibody fragment, e.g., a Fv, Fab, Fab’, scFv, diabody, or F(ab’)2 fragment.

[0215] In another aspect, the antibody is a full length antibody, IgG1antibody or other antibody class or isotype as defined herein.

[0216] In a further aspect, the antibody as described herein is of IgG1isotype / subclass and comprises a constant heavy chain domain of SEQ ID NO:234 or the constant parts of the heavy chain amino acid sequence. In one aspect, additionally the C-terminal glycine (Gly446) is present. In one aspect, additionally the C-terminal glycine (Gly446) and the C-terminal lysine (Lys447) is present.

[0217] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO: 235 and the full heavy chain sequence (full H) of SEQ ID NO:239.

[0218] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO: 236 and the full heavy chain sequence (full H) of SEQ ID NO:240.

[0219] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO: 237 and the full heavy chain sequence (full H) of SEQ ID NO:241.

[0220] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO: 238 and the full heavy chain sequence (full H) of SEQ ID NO:242.

[0221] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO:275 and the full heavy chain sequence (full H) of SEQ ID NO:274.

[0222] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO:277 and the full heavy chain sequence (full H) of SEQ ID NO:276.

[0223] In one aspect, the antibody that binds to CD3 comprises a full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 235 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:239. In one aspect, the antibody that binds to CD3 comprises a full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 235 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 239.

[0224] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 236 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:240. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 236 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 240.

[0225] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 237 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:241. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 237 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0226] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 238 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:242. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 238 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 242.

[0227] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:275 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:274. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:275 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:274.

[0228] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:277 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:276. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:277 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:276.

[0229] Table 1 shows the molecule IDs with their respective CD3 IDs and format.

[0230] Table 2 shows an overview of sequences of antibodies of the invention. It also includes full H and full L sequences of full length IgGs.

[0231] In a further aspect, an antibody that binds to CD3 according to any of the above aspects may incorporate any of the features, singly or in combination, as described in Sections 1-6 below:

[0232] 1. Antibody Fragments In certain aspects, an antibody provided herein is an antibody fragment.

[0233] In one aspect, the antibody fragment is a Fab, Fab’, Fab’-SH, or F(ab’)2 fragment, in particular a Fab fragment. Papain digestion of intact antibodies produces two identical antigen -binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI). The term “Fab fragment” thus refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CHI domain. “Fab’ fragments” differ from Fab fragments by the addition of residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab’-SH are Fab’ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites (two Fab fragments) and a part of the Fc region. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0234] In another aspect, the antibody fragment is a diabody, a triabody or a tetrabody. “Diabodies” are antibody fragments with two antigen-binding sites that may 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). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0235] In a further aspect, the antibody fragment is a single chain Fab fragment. A “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, b) VL-CL-linker-VH-CHl, c) VH-CL-linker-VL-CHl or d) VL- CH1 -linker- VH-CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CHI domain. In addition, these single chain Fab fragments might be further stabilized by generation of interchain disulfide bonds via 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).

[0236] In another aspect, the antibody fragment is single-chain variable fragment (scFv). A “singlechain variable fragment” or “scFv” is a fusion protein of the variable domains of the heavy (VH) and light chains (VL) of an antibody, connected by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. For a review of scFv fragments, see, e.g., Pliickthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. In another aspect, the antibody fragment is a single-domain antibody. “Single-domain antibodies” are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain aspects, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

[0237] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as recombinant production by recombinant host cells (e.g., E. coli), as described herein.

[0238] 2. Humanized Antibodies

[0239] In one aspect, the anti-CD3 antibody provided herein is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In one aspect, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0240] 3. Library-Derived Antibodies

[0241] In certain aspects, an antibody provided herein is derived from a library. Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are reviewed, e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Frenzel et al. in mAbs 8: 1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8: 1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016) as well as in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001) and in Marks and Bradbury in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003). In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. in Annual Review of Immunology 12: 433-455 (1994). Phage typically display antibody fragments, either as singlechain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al. in EMBO Journal 12: 725-734 (1993). Furthermore, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter in Journal of Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: US Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as US Patent Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0242] Further examples of methods known in the art for screening combinatorial libraries for antibodies with a desired activity or activities include ribosome and mRNA display, as well as methods for antibody display and selection on bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are reviewed, e.g., in Scholler et al. in Methods in Molecular Biology 503: 135-56 (2012) and in Cherf et al. in Methods in Molecular biology 1319: 155-175 (2015) as well as in Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, e.g., in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and in Hanes et al. in PNAS 94:4937-4942 (1997).

[0243] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0244] 4. Multispecific And Bispecific Antibodies

[0245] In one aspect, the anti-CD3 antibody provided herein is a multispecific, e.g. a bispecific, antibody.

[0246] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob -in-hole” engineering (see, e.g., Carter et al., J Immunol Meth 248, 7-15 (2001)). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011 / 034605); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98 / 50431); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).

[0247] Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH / VL domains (see e.g., WO 2009 / 080252 and WO 2015 / 150447), the CH1 / CL domains (see e.g., WO 2009 / 080253) or the complete Fab arms (see e.g., WO 2009 / 080251, WO 2016 / 016299). Also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20. In one aspect, the multispecific antibody comprises a cross-Fab fragment. The term “cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CHI), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL).

[0248] Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab (i.e. heavy and light chain) pairing. Exemplary Fab pairing amino acid mutations are Q39E (Kabat numbering) and S183K (Kabat EU numbering) in the heavy chain and Q38K (Kabat numbering) and V133E (Kabat EU numbering) in the light chain, or Q39K (Kabat numbering) and S183E (Kabat EU numbering) in the heavy chain and Q38E (Kabat numbering) and V133K (Kabat EU numbering) in the light chain (see e.g. WO 2016 / 172485). Further Fab pairing mutations include 124K, 124R or 124H (Kabat numbering) and 123K, 123R or 123H (Kabat numbering) in the light chain and 147E or 147D (Kabat EU numbering) and 213E or 213D (Kabat EU numbering) in the heavy chain (see e.g. WO 2015 / 150447). To promote the correct association of heavy chains in asymmetric multispecific (e.g. bispecific) antibodies, their heavy chains may be engineered to comprise e.g. sterically (“knob- in-hole”) or electrostatically complementary amino acid mutations, salt bridges, and / or disulfide bonds. The knob-in-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., ProtEng 9, 617-621 (1996), Atwell et al., J. Mol. Biol. 270, 26 (1997), Merchant et al., Nat Biotechnol 16, 677-681 (1998), and Carter, J Immunol Meth 248, 7-15 (2001). In one aspect, a multispecific (e.g. bispecific) antibody comprising a human IgGl Fc domain may comprise the“knob” mutation T366W on the first heavy chain, and “hole” mutations Y407V and optionally T366S and L368A (all Kabat EU numbering) on the second heavy chain (T366W / T366S:L368A:Y407V). Additionally, the antibody may comprise a S354C substitution on the first heavy chain and a Y349C substitution (both Kabat EU numbering) on the second heavy chain, forming a disulfide bond ((T366W:S354C / Y349C:T366S:L368A:Y407V).

[0249] Further examples of amino acid mutations (e.g. substitutions) that may be comprised in multispecific (e.g. bispecific) antibodies include the substitution S228P (Kabat EU numbering) in antibodies comprising an IgG4 Fc region, e.g. for preventing Fab arm exchange (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)).

[0250] Various further molecular formats for multispecific antibodies are known in the art and are included herein (see e.g., Spiess et al., Mol Immunol 67: 95-106 (2015)).

[0251] A particular type of multispecific antibodies, also included herein, are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to an activating, invariant component of the T cell receptor (TCR) complex, such as CD3, for retargeting of T cells to kill target cells. Hence, in one aspect, the antibody provided herein is a multispecific antibody, particularly a bispecific antibody, wherein one of the binding specificities is for a target cell antigen and the other is for the component of the TCR complex, i.e. CD3.

[0252] Certain aspects of the antibody of the present invention are described in the following.

[0253] In one aspect, the invention provides an antibody that binds to CD3 and a target cell antigen, comprising a domain that binds to CD3 as described herein and a second domain that binds to a target cell antigen.

[0254] According to certain aspects of the invention, the domains comprised in the antibody are Fab molecules (i.e. domains that bind to an antigen e.g. CD3 composed of a heavy and a light chain, each comprising a variable and a constant domain). In one aspect, the domains that bind to CD3 are a Fab molecule. In one aspect, said Fab molecule is human. In another aspect, said Fab molecule is humanized. In yet another aspect, said Fab molecule comprises human heavy and light chain constant domains.

[0255] Preferably, at least one of the domains that bind to the CD3 or target cell antigen e.g. tumor cell antigen is a crossover Fab molecule. Such modification reduces mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the multi specific antibody according to the invention in recombinant production. In a preferred crossover Fab molecule useful for the multispecific antibody according to the invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are exchanged. Even with this domain exchange, however, the preparation of the multispecific antibody may comprise certain side products due to a so-called Bence Jones-type interaction between mispaired heavy and light chains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To further reduce mispairing of heavy and light chains from different Fab molecules and thus increase the purity and yield of the desired multispecific antibody, charged amino acids with opposite charges may be introduced at specific amino acid positions in the CHI and CL domains of either the Fab molecule binding to CD3, or the Fab molecule(s) binding to the target cell antigen e.g. tumor cell antigen, as further described herein. Charge modifications are made either in the conventional Fab molecule(s) comprised in the multispecific antibody, or in the VH / VL crossover Fab molecule(s) comprised in the multispecific antibody (but not in both). In preferred aspects, the charge modifications are made in the conventional Fab molecule(s) comprised in the multispecific antibody (which in preferred aspects bind(s) to the target cell antigen such as a tumor cell antigen).

[0256] In a certain aspect of the invention, the multispecific antibody is capable of simultaneous binding to CD3 and the target cell antigen e.g. tumor cell antigen. In one aspect, the multispecific antibody is capable of crosslinking a T cell and a target cell by simultaneously binding to CD3 and a target cell antigen such as a tumor cell antigen. In an even more preferred aspect, such simultaneous binding results in lysis of the target cell, particularly a tumor cell antigen-expressing target cell such as a tumor cell. In one aspect, such simultaneous binding results in activation of the T cell. In other aspects, such simultaneous binding results in a cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from the group of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In one aspect, binding of the multispecific antibody to CD3 without simultaneous binding to the target cell antigen e.g. tumor cell antigen does not result in T cell activation.

[0257] In one aspect, the multispecific antibody is capable of re-directing cytotoxic activity of a T cell to a target cell. In a preferred aspect, said re-direction is independent of MHC-mediated peptide antigen presentation by the target cell and and / or specificity of the T cell.

[0258] Preferably, a T cell according to any of the aspects of the invention is a cytotoxic T cell. In some aspects the T cell is a CD4+or a CD8+T cell, particularly a CD8+T cell. a) CD3 binding domain

[0259] In one aspect, a (multispecific) antibody according to the invention comprises at least one domain that binds to CD3. In preferred aspects, CD3 is human CD3 (SEQ ID NO: 211) or cynomolgus CD3 (SEQ ID NO: 212) most particularly human CD3. In one aspect the domain that binds to CD3 is cross-reactive for (i.e. specifically binds to) human and cynomolgus CD3. In some aspects, CD3 is the epsilon subunit of CD3 (CD3 epsilon).

[0260] In one aspect, the domain that binds to CD3 is an antibody fragment selected from the group of an Fv molecule, a scFv molecule, a Fab molecule, and a F(ab’)2 molecule. In a preferred aspect, the domain that binds to CD3 is a Fab molecule.

[0261] In preferred aspects, the domain that binds to CD3 is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other. In such aspects, the domain(s) that binds to the target cell antigen e.g. tumor cell antigen is preferably a conventional Fab molecule. In aspects where there is more than one domain that binds to antigens e.g. CD3 and / or target cell antigen such as tumor cell antigen, particularly Fab molecule, that binds to the target cell antigen e.g. tumor cell antigen comprised in the (multispecific) antibody, the domain that binds to CD3 preferably is a crossover Fab molecule and the domain that binds to the target cell antigen e.g. tumor cell antigen are conventional Fab molecules.

[0262] In alternative aspects, the domain that binds to CD3 is a conventional Fab molecule. In such aspects, the domain(s) that binds to the target cell antigen (e.g. tumor cell antigen) is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other. In aspects where there is more than one domain that binds to antigen(s) e.g. target cell antigen such as tumor cell antigen and / or CD3, particularly Fab molecule, that binds to CD3 comprised in the (multispecific) antibody, the domain that binds to the target cell antigen e.g. tumor cell antigen preferably is a crossover Fab molecule and the domains that bind to CD3 are conventional Fab molecules.

[0263] In certain aspects, the domain that binds to CD3 is a Fab molecule wherein the variable domains VL and VH or the constant domains CL and CHI, particularly the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced by each other (i.e. according to such aspect, the domain that binds to CD3 is a crossover Fab molecule wherein the variable or constant domains of the Fab light chain and the Fab heavy chain are exchanged). In one such aspect, the antigen domain(s) that binds to the target cell antigen e.g. tumor cell antigen is a conventional Fab molecule.

[0264] In one aspect, the CD3 binding domain comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of NX13X14IH (SEQ ID NO:225), (b) CDR-H2 comprising the amino acid sequence of WIX7PGX8GX9TX10YNX11KFX12G (SEQ ID NO:226), and (c) CDR-H3 comprising the amino acid sequence of DX1X2X3X4YX5X6DY (SEQ ID NO:227), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of KSSQSLLNX22RTRKNYLA (SEQ ID NO:222), (e) CDR-L2 comprising the amino acid sequence of WASX19RX20X21 (SEQ ID NO:223), and (f) CDR-L3 comprising the amino acid sequence of XisXieSFXnXisRT (SEQ ID NO:224), wherein Xi is T, S or Q, X2is Y, V or R, X3is S or V, X4is N, Q or H, X5is F or Y, X6is F, Y or H, X7is Y, L, V, H, E, K or R, X8is D, Y, T, R or E, X9is N, D, Q or E, X10 is R or K, Xu is E, Q or H, X12 is K, Q or E, X13 is Y or E, X14 is Y or H, X15 is S, N or H, Xi6 is T or N, X17 is E or Q, Xis is S or H, X19 is T or D, X20 is E or Q, X21 is I or H, X22 is L, H or D.

[0265] In one aspect, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of NX13YIH (SEQ ID NO:228), (b) CDR-H2 comprising the amino acid sequence of WIX23PGDGX24TKYNEKFX25G (SEQ ID NO:229), and (c) CDR-H3 comprising the amino acid sequence of DX26X27HX28YYX29DY (SEQ ID NO:230), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO:231), (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of TX30SFILRT (SEQ ID NO:233) wherein X13 is Y or E, X23 is Y or E, X24 is N or D, X25 is E or K, X26 is Q, T or H, X27 is Y or V, X28 is H or Q, X29 is F or Y, X30 is Q or E, optionally wherein X23 is E when X24 is D and / or X23 is Y when X24 is N.

[0266] In one aspect, the antibody comprises a VH comprising an amino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASNX13YIHWVRQAPGQGLEWIGWIX23PGDGX24 TKYNEKFX25GRATLTADTSTSTAYLELSSLRSEDTAVYYCARDX26X27HX28YYX29DY WGQGTLVTVSS (SEQ ID NO: 262), and / or a VL comprising an amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRX32NYLAWYQQKPGQPPKLLIX31 WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTX30SFILRTWGQGTLVTVS S (SEQ ID NO: 263), wherein X13 is Y or E, X23 is Y or E, X24 is N or D, X25 is E or K, X26 is Q, T or H, X27 is Y or V, X28 is H or Q, X29 is F or Y, X30 is Q or E and X31 is H or Y, X32 is K or H, optionally wherein X23 is E when X24 is D and / or X23 is Y when X24 is N and / or X31 is Y when X30 is Q and / or X31 is H when X30 is E.

[0267] In one aspect, the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 266, 272, 169 and 3, (b) CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 170 and 174, and (c) CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 273, 267, 159, 171 and 175, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7 or 271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 12.

[0268] In one aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of the VH domain of SEQ ID NOs: 172 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 10.

[0269] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 172 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 172. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 172 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 172. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 172 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 172.

[0270] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 10 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 10. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 10 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 10. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 10 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 10.

[0271] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 169; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 170; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:171; (d) CDR- L1 comprising the amino acid sequence of SEQ ID NO:7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 172. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0272] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 172. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 172. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO: 172, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 169, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 170, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 172. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 10. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 10, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:7, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NOV.

[0273] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO: 172 and SEQ ID NO: 10, respectively, including post-translational modifications of those sequences. In further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH domain of SEQ ID NOs: 176 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 13.

[0274] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 176 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 176. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 176 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 176. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 176 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 176.

[0275] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 13 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 13. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 13 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 13. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 13 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 13.

[0276] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 176. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0277] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 176. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 176. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO: 176, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 175. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:13. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 13, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:7, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 12.

[0278] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO: 176 and SEQ ID NO: 13, respectively, including post-translational modifications of those sequences.

[0279] In a further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH domain of SEQ ID NOs: 160 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 206.

[0280] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 160 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 160. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 160 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 160. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 160 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 160.

[0281] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206.

[0282] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 159; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 160, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:206; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 160. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:206.

[0283] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 160. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 160. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 160. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO: 160, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NON, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 159. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:206. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:206. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:206. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 206, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO:7, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID N0:12.

[0284] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO: 160 and SEQ ID NO:206, respectively, including post-translational modifications of those sequences.

[0285] In a further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH domain of SEQ ID NOs: 208 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 206.

[0286] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:208 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:208. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:208 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:208. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:208 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:208.

[0287] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:206 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:206.

[0288] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:208, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:206; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:208. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:206.

[0289] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:208. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:208. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:208. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO:208, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 171. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:206. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:206. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:206. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 206, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO:7, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID N0:12. In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:208 and SEQ ID NO:206, respectively, including post-translational modifications of those sequences.

[0290] In a further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH domain of SEQ ID NOs: 268 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 269.

[0291] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:268 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:268. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:268 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:268. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:268 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:268.

[0292] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:269 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:269. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:269 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:269. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:269 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:269.

[0293] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:266; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:276; (d) CDR- LI comprising the amino acid sequence of SEQ ID NO:271; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:268, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:269; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:268. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:269.

[0294] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:268. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:268. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:268. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO:268, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:266, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO:267. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:269. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:269. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:269. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO: 269, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO:271, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 12.

[0295] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:270 and SEQ ID NO:265, respectively, including post-translational modifications of those sequences.

[0296] In a further aspect, an antibody that binds to CD3 comprises the CDR-H1, CDR-H2 and CDR- H3 amino acid sequences of the VH domain of SEQ ID NOs: 270 and the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of the VL domain of SEQ ID NO: 265.

[0297] In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:270 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:270. In one aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:270 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:270. In another aspect, the antibody that binds to CD3 comprises the three heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:270 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:270.

[0298] In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:265 and a framework of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:265. In one aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:265 and a framework of at least 95% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:265. In another aspect, the antibody that binds to CD3 comprises the three light chain CDR amino acid sequences of the VL domain of SEQ ID NO:265 and a framework of at least 98% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:265.

[0299] In one aspect, the antibody that binds to CD3 comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:272; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:273; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12, and a VH domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:270, and a VL domain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:265; wherein the antibody specifically binds to CD3. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:270. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:265.

[0300] In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:270. In one aspect, an antibody that binds to CD3 comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:270. In certain aspects, a VH sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:270. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VH sequence in SEQ ID NO:270, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO:272, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO:273. In another aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:265. In one aspect, an antibody that binds to CD3 comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:265. In certain aspects, a VL sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody that binds to CD3 comprising that sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO:265. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the antibody that binds to CD3 comprises the VL sequence in SEQ ID NO:265, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR- Ll, comprising the amino acid sequence of SEQ ID NO:271, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:8, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO: 12.

[0301] In another aspect, an antibody that binds to CD3 comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:270 and SEQ ID NO:265, respectively, including post-translational modifications of those sequences.

[0302] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO: 235 and the full heavy chain sequence (full H) of SEQ ID NO:239.

[0303] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO: 236 and the full heavy chain sequence (full H) of SEQ ID NO:240. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO: 237 and the full heavy chain sequence (full H) of SEQ ID NO:241.

[0304] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO: 238 and the full heavy chain sequence (full H) of SEQ ID NO:242.

[0305] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO:275 and the full heavy chain sequence (full H) of SEQ ID NO274:.

[0306] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) of SEQ ID NO:277 and the full heavy chain sequence (full H) of SEQ ID NO:276.

[0307] In one aspect, the antibody that binds to CD3 comprises a full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 235 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:239. In one aspect, the antibody that binds to CD3 comprises a full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 235 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 239.

[0308] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 236 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:240. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 236 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 240.

[0309] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 237 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:241. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 237 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0310] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 238 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:242. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 238 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 242.

[0311] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:275 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:274. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:275 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:274.

[0312] In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:277 and the full heavy chain sequence (full H) having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:276. In one aspect, the antibody that binds to CD3 comprises the full light chain sequence (full L) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:277 and the full heavy chain sequence (full H) having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:276. b) Target cell antigen binding domain

[0313] A multispecific antibody according to the invention comprises at least one domain , particularly a Fab molecule, that binds to a target cell antigen e.g. a tumor cell antigen. The domain that binds to the target cell antigen is able to direct the multispecific antibody to a target site, for example to a specific type of cell that expresses the target cell antigen.

[0314] In one aspect, the domain that binds to the target cell antigen is an antibody fragment selected from the group of an Fv molecule, a scFv molecule, a Fab molecule, and a F(ab’)2 molecule. In a preferred aspect, the domain that binds to the target cell antigen is a Fab molecule.

[0315] In certain aspects, the multispecific antibody comprises two domains, particularly Fab molecules, that bind to the target cell antigen. In a preferred aspect, all of these domains are identical, i.e. they have the same molecular format (e.g. conventional or crossover Fab molecule) and comprise the same amino acid sequences including the same amino acid substitutions in the CHI and CL domain as described herein (if any). In one aspect, the multispecific antibody comprises not more than two domains, particularly Fab molecules, that bind to the target cell antigen .

[0316] In preferred aspects, the domain(s) that bind to the target cell antigen is / are a conventional Fab molecule. In such aspects, the domain(s) that binds to CD3 is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other.

[0317] In alternative aspects, the domain(s) that bind to the target cell antigen is / are a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CHI and CL of the Fab heavy and light chains are exchanged / replaced by each other. In such aspects, the domain(s) that binds to CD3 is a conventional Fab molecule.

[0318] In one aspect, the domain(s) that bind to the target cell antigen comprises a human constant region. In one aspect, the domain(s) that binds to the target cell antigen is a Fab molecule comprising a human constant region, particularly a human CHI and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 213 and 214 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 215 (human IgG1heavy chain constant domains CH1-CH2-CH3).

[0319] In one aspect, the domain(s) that bind to the target cell antigen comprises any of the features of the antibodies that bind to the target cell antigen described above. c) Charge modifications

[0320] The (multi specific) antibody of the invention may comprise amino acid substitutions in Fab molecules comprised therein which are particularly efficient in reducing mispairing of light chains with non-matching heavy chains (Bence-Jones-type side products), which can occur in the production of Fab-based (multi specific) antibodies with a VH / VL exchange in one (or more, in case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT publication no. WO 2015 / 150447, particularly the examples therein, incorporated herein by reference in its entirety). The ratio of a desired (multispecific) antibody compared to undesired side products, in particular Bence Jones-type side products occurring in (multi specific) antibodies with a VH / VL domain exchange in one of their binding arms, can be improved by the introduction of charged amino acids with opposite charges at specific amino acid positions in the CHI and CL domains (sometimes referred to herein as “charge modifications”).

[0321] Accordingly, in some aspects wherein the domain that binds to CD3 and the domain(s) that bind to the target cell antigen of the (multispecific) antibody are both Fab molecules, and in one of the domains (particularly the domain that binds to CD3) the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, i) in the constant domain CL of the domain(s) that bind to the target cell antigen the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CHI of the domain(s) that bind to the target cell antigen the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index); or ii) in the constant domain CL of the domain that binds to CD3 the amino acid at position 124 is substituted by a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CHI of the domain that binds to CD3 the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to Kabat EU index).

[0322] The (multi specific) antibody does not comprise both modifications mentioned under i) and ii). The constant domains CL and CHI of the domain(s) having the VH / VL exchange are not replaced by each other (i.e. remain unexchanged).

[0323] In a more specific aspect, i) in the constant domain CL of the domain(s) that bind to the target cell antigen the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the domain(s) that bind to the target cell antigen the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index); or ii) in the constant domain CL of the domain that binds to CD3 the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the domain that binds to CD3 the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

[0324] In one such aspect, in the constant domain CL of the domain(s) that bind to the target cell antigen the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the domain(s) that bind to the target cell antigen the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

[0325] In a further aspect, in the constant domain CL of the domain(s) that bind to the target cell antigen the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the domain(s) that bind to the target cell antigen the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index). In one aspect, in the constant domain CL of the domain(s) that bind to the target cell antigen the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the domain(s) that bind to the target cell antigen the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

[0326] In another aspect, in the constant domain CL of the domain(s) that bind to the target cell antigen the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and in the constant domain CHI of the domain(s) that bind to the target cell antigen the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0327] In yet another aspect, in the constant domain CL of the domain(s) that bind to the target cell antigen the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and in the constant domain CHI of the domain(s) that bind to the target cell antigen the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0328] In preferred aspects, if amino acid substitutions according to the above aspects are made in the constant domain CL and the constant domain CHI of the domain(s) that bind to the target cell antigen, the constant domain CL of the domain(s) that bind to the target cell antigen is of kappa isotype.

[0329] Alternatively, the amino acid substitutions according to the above aspects may be made in the constant domain CL and the constant domain CHI of the domain that binds to CD3 instead of in the constant domain CL and the constant domain CHI of the domain(s) that bind to the target cell antigen. In preferred such aspects, the constant domain CL of the domain that binds to CD3 is of kappa isotype. Accordingly, in one aspect, in the constant domain CL of the domain that binds to CD3 the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the domain that binds to CD3 the amino acid at position 147 or the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

[0330] In a further aspect, in the constant domain CL of the domain that binds to CD3 the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the domain that binds to CD3 the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

[0331] In still another aspect, in the constant domain CL of the domain that binds to CD3 the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI of the domain that binds to CD3 the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

[0332] In one aspect, in the constant domain CL of the domain that binds to CD3 the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and in the constant domain CHI of the domain that binds to CD3the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0333] In another aspect, in the constant domain CL of the domain that binds to CD3 the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and in the constant domain CHI of the domain that binds to CD3 the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index). d) Multispecific antibody formats

[0334] The multispecific antibody according to the present invention can have a variety of configurations.

[0335] In some aspects, the domain(s) that bind to CD3 and / or the target cell antigen comprised in the multispecific antibody are Fab molecules.

[0336] In some aspects, the (multispecific) antibody of the invention comprises an Fc domain, particularly a human IgG1Fc domain, composed of a first and a second subunit. The first and the second subunit of the Fc domain are capable of stable association.

[0337] The domain(s) may be fused to the Fc domain or to each other directly or through a peptide linker, comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable, non-immunogenic peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n, G4(SG4)nor (G4S)nG5peptide linkers, “n” is generally an integer from 1 to 10, typically from 2 to 4. In one aspect said peptide linker has a length of at least 5 amino acids, in one aspect a length of 5 to 100, in a further aspect of 10 to 50 amino acids. In one aspect said peptide linker is (GxS)nor (GxS)nGmwith G=glycine, S=serine, and (x=3, n= 3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=l, 2, 3, 4 or 5 and m= 0, 1, 2, 3, 4 or 5), in one aspect x=4 and n=2 or 3, in a further aspect x=4 and n=2, in yet a further aspect x=4, n=l and m=5. In one aspect said peptide linker is (648)2. In another aspect, said peptide linker is G4SG5. A particularly suitable peptide linker for fusing the Fab light chains of the first and the second Fab molecule to each other is (648)2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and the second Fab fragments comprises the sequence (D)-(G4S)2(SEQ ID NOs: 216 and 217). Another suitable such linker comprises the sequence (D)-G4SG5(SEQ ID NOs: 218 and 219). Additionally, linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where a Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0338] In certain aspects, the (multispecific) antibody does not comprise an Fc domain. In preferred such aspects, the (first) domain that binds to the target cell antigen and, if present, second domain that binds to the target cell antigen are each a conventional Fab molecule, and the domain that binds CD3 is a crossover Fab molecule as described herein, i.e. a Fab molecule wherein the variable domains VH and VL or the constant domains CL and CHI of the Fab heavy and light chains are exchanged / replaced by each other. In other such aspects, the first and, if present, second domain are each a crossover Fab molecule and the domain that binds to CD3 is a conventional Fab molecule.

[0339] 5. Fc domain variants

[0340] In preferred aspects, the (multispecific) antibody of the invention comprises an Fc domain composed of a first and a second subunit.

[0341] The Fc domain of the (multispecific) antibody consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one aspect, the (multispecific) antibody of the invention comprises not more than one Fc domain.

[0342] In one aspect, the Fc domain of the (multispecific) antibody is an IgG Fc domain. In a preferred aspect, the Fc domain is an IgG1Fc domain, more particularly a human IgG1Fc domain. In another aspect the Fc domain is an IgG4 Fc domain. In a more specific aspect, the Fc domain is an IgG4Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further preferred aspect, the Fc domain is a human Fc domain. In an even more preferred aspect, the Fc domain is a human IgG1Fc domain. An exemplary sequence of a human IgG1Fc region is given in SEQ ID NO: 243 (Fc knob chain) or SEQ ID NO: 244 (Fc hole chain). a) Fc domain modifications promoting heterodimerization

[0343] (Multi specific) antibodies according to the invention comprise different domains that bind to the target cell antigen and CD3, which may be fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two nonidentical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of (multispecific) antibodies in recombinant production, it will thus be advantageous to introduce in the Fc domain of the (multispecific) antibody a modification promoting the association of the desired polypeptides.

[0344] Accordingly, in preferred aspects, the Fc domain of the (multispecific) antibody according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect said modification is in the CH3 domain of the Fc domain.

[0345] There exist several approaches for modifications in the CH3 domain of the Fc domain in order to enforce heterodimerization, which are well described e.g. in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed). These different approaches for improved heavy chain heterodimerization are contemplated as different alternatives in combination with the heavy -light chain modifications (e.g. VH and VL exchange / replacement in one binding arm and the introduction of substitutions of charged amino acids with opposite charges in the CH1 / CL interface) in the (multispecific) antibody which reduce heavy / light chain mispairing and Bence Jones-type side products.

[0346] In a specific aspect said modification promoting the association of the first and the second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.

[0347] The knob-into-hole technology is described e.g. 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, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). Accordingly, in a preferred aspect, in the CH3 domain of the first subunit of the Fc domain of the (multispecific) antibody an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

[0348] Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0349] Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0350] The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.

[0351] In a specific aspect, in (the CH3 domain of) the first subunit of the Fc domain (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in (the CH3 domain of) the second subunit of the Fc domain (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one aspect, in the second subunit of the Fc domain additionally 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) (numberings according to Kabat EU index).

[0352] In yet a further aspect, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). In a preferred aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).

[0353] In a preferred aspect the domain that binds to CD3 is fused (optionally via the first domain that binds to the target cell antigen, which binds to the target cell antigen, and / or a peptide linker) to the first subunit of the Fc domain (comprising the “knob” modification). Without wishing to be bound by theory, fusion of the domain that binds CD3 to the knob -containing subunit of the Fc domain will (further) minimize the generation of antibodies comprising two domains that bind to CD3 (steric clash of two knob -containing polypeptides).

[0354] Other techniques of CH3 -modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g. in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0355] In one aspect, the heterodimerization approach described in EP 1870459, is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. A particular aspect for the (multispecific) antibody of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).

[0356] In another aspect, the (multispecific) antibody of the invention comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index).

[0357] In another aspect, the (multispecific) antibody of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said (multispecific) antibody 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 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to Kabat EU index).

[0358] In one aspect, the heterodimerization approach described in WO 2013 / 157953 is used alternatively. In one aspect, a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (numberings according to Kabat EU index). In a further aspect, the first CH3 domain comprises further amino acid mutation L351K. In a further aspect, the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (particularly L368E) (numberings according to Kabat EU index).

[0359] In one aspect, the heterodimerization approach described in WO 2012 / 058768 is used alternatively. In one aspect a first CH3 domain comprises amino acid mutations L351 Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further aspect the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numberings according to Kabat EU index). In a further aspect a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F. In a further aspect, a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further aspect, the second CH3 domain further comprises amino acid mutations K392E, T41 IE, D399R and S400R (numberings according to Kabat EU index).

[0360] In one aspect, the heterodimerization approach described in WO 2011 / 143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Kabat EU index).

[0361] In one aspect, the heterodimerization approach described in WO 2011 / 090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one aspect a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A. In one aspect, a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Kabat EU index).

[0362] In one aspect, the (multispecific) antibody or its Fc domain is of IgG2subclass and the heterodimerization approach described in WO 2010 / 129304 is used alternatively.

[0363] In an alternative aspect, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009 / 089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In one such aspect, a first CH3 domain comprises 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 a second CH3 domain comprises 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, and more particularly D399K and E356K). In a further aspect, the first CH3 domain further comprises 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 a further aspect the first CH3 domain further or alternatively comprises amino acid substitution of K439 and / or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Kabat EU index).

[0364] In yet a further aspect, the heterodimerization approach described in WO 2007 / 147901 is used alternatively. In one aspect, a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index).

[0365] In still another aspect, the heterodimerization approach described in WO 2007 / 110205 can be used alternatively.

[0366] In one aspect, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to Kabat EU index). b) Fc domain modifications reducing Fc receptor binding and / or effector function

[0367] The Fc domain confers to the (multispecific) antibody favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the (multispecific) antibody to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the T cell activating properties and the long half-life of the (multispecific) antibody, results in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptorbearing) immune cells other than T cells may even reduce efficacy of the (multi specific) antibody due to the potential destruction of T cells e.g. by NK cells.

[0368] Accordingly, in preferred aspects, the Fc domain of the (multispecific) antibody according to the invention exhibits reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgG1Fc domain. In one such aspect the Fc domain (or the (multispecific) antibody comprising said Fc domain) exhibits less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the binding affinity to an Fc receptor, as compared to a native IgG1Fc domain (or a (multi specific) antibody comprising a native IgG1Fc domain), and / or less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the effector function, as compared to a native IgG1Fc domain domain (or a (multispecific) antibody comprising a native IgG1Fc domain). In one aspect, the Fc domain domain (or the (multispecific) antibody comprising said Fc domain) does not substantially bind to an Fc receptor and / or induce effector function. In a preferred aspect the Fc receptor is an Fey receptor. In one aspect the Fc receptor is a human Fc receptor. In one aspect the Fc receptor is an activating Fc receptor. In a specific aspect the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one aspect the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a preferred aspect, the effector function is ADCC. In one aspect, the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgG1Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the (multispecific) antibody comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgG1Fc domain (or the (multispecific) antibody comprising a native IgG1Fc domain) to FcRn.

[0369] In certain aspects the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a non-engineered Fc domain. In preferred aspects, the Fc domain of the (multispecific) antibody comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In aspects where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one aspect the (multispecific) antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to a (multispecific) antibody comprising a non-engineered Fc domain. In a preferred aspect, the Fc receptor is an Fey receptor. In some aspects, the Fc receptor is a human Fc receptor. In some aspects, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. Preferably, binding to each of these receptors is reduced. In some aspects, binding affinity to a complement component, specifically binding affinity to Clq, is also reduced. In one aspect, binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or the (multispecific) antibody comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or the (multispecific) antibody comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or (multispecific) antibodies of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain aspects, the Fc domain of the (multispecific) antibody is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex -mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one aspect, the reduced effector function is one or more selected from the group 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 a non-engineered Fc domain (or a (multispecific) antibody comprising a non-engineered Fc domain).

[0370] In one aspect, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or effector function is an amino acid substitution. In one aspect, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific aspect, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some aspects, the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In one such aspect, the Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one aspect, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific aspect, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In preferred aspects, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more preferred aspects, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in preferred aspects, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second subunit 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 by a glycine residue (P329G) (numbering according to Kabat EU index).

[0371] In one such aspect, the Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. The “P329GLALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgG1Fc domain, as described in PCT publication no. WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.

[0372] IgG4antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgG1antibodies. Hence, in some aspects, the Fc domain of the (multispecific) antibodies of the invention is an IgG4Fc domain, particularly a human IgG4Fc domain. In one aspect, the IgG4Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index). To further reduce its binding affinity to an Fc receptor and / or its effector function, in one aspect, the IgG4Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index). In another aspect, the IgG4Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index). In a preferred aspect, the IgG4Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index). Such IgG4Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012 / 130831, incorporated herein by reference in its entirety.

[0373] In a preferred aspect, the Fc domain exhibiting reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgG1Fc domain, is a human IgG1Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).

[0374] In certain aspects, N-glycosylation of the Fc domain has been eliminated. In one such aspect, the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index). In addition to the Fc domains described hereinabove and in PCT publication no. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (numberings according to Kabat EU index). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

[0375] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.

[0376] Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. Alternatively, binding affinity of Fc domains or (multispecific) antibodies comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.

[0377] Effector function of an Fc domain, or a (multi specific) antibody comprising an Fc domain, can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499- 1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays may be employed (see, for example, ACTI™ non- radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0378] In some aspects, binding of the Fc domain to a complement component, specifically to Clq, is reduced. Accordingly, in some aspects wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. Clq binding assays may be carried out to determine whether the Fc domain, or the (multi specific) antibody comprising the Fc domain, is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0379] FcRn binding and in vivo clearance / half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’L Immunol. 18(12): 1759-1769 (2006); WO 2013 / 120929).

[0380] 6. Antibody Variants

[0381] In one aspect, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. a. Substitution, Insertion, and Deletion Variants

[0382] In one aspect, antibody variants having one or more amino acid substitutions are provided. Substitutions are possible in the CDRs, FRs, or constant regions. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0383] Amino acids may be grouped according to common side-chain properties:

[0384] (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0385] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0386] (3) acidic: Asp, Glu;

[0387] (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro;

[0388] (6) aromatic: Trp, Tyr, Phe.

[0389] Conservative substitutions will entail exchanging members of one of these classes for another member in the class.

[0390] One type of substitutional variant involves substituting one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display -based affinity maturation techniques. Briefly, one or more. CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0391] Alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR “hotspots”, i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)). In one aspect of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4- 6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0392] In one aspect, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0393] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionine residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide which increases the serum half-life of the antibody. b. Glycosylation variants

[0394] In one aspect, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0395] Where the antibody comprises an Fc region, the oligosaccharide attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Jennewein et al. Trends in Immunology 38:P358-372 (2017). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In one aspect, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

[0396] In one aspect, antibody variants are provided having a non-fucosylated oligosaccharide, i.e. an oligosaccharide structure that lacks fucose attached (directly or indirectly) to an Fc region. Such non-fucosylated oligosaccharide (also referred to as “afucosylated” oligosaccharide) particularly is an N-linked oligosaccharide which lacks a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region as compared to a native or parent antibody. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have enhanced FcyRIIIa receptor binding and / or improved effector function, in particular enhanced ADCC function. See, e.g., US 2003 / 0157108; US 2004 / 0093621. In one aspect, the antibody having a non-fucosylated oligosaccharide is produced by a FUT8K0 CHO cell line. See, e.g., Jennewein et al. Trends in Immunology 38:P358-372 (2017).

[0397] In a further aspect, antibody variants are provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described, e.g., 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.

[0398] Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764. c. Fc region variants

[0399] In one aspect, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region sequence) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0400] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which increase binding to human FcyR (e.g. FcyRIIIa) and / or effector function (e.g. ADCC), e.g., substitutions at positions 239, 298, 330, 332, 333 and / or 334 of the Fc region (Kabat EU numbering of residues). In one aspect, the substitutions are S298A, E333A and K334A in an Fc region derived from a human IgGl Fc region (see e.g. Shields et al. (2001) J. Biol. Chem. 276, 6591-6604). In one aspect, the substitutions are S239D, I332E and optionally A330L in an Fc region derived from a human IgGl Fc region (see e.g Lazar et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 4005-4010).

[0401] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which reduce binding to human FcyR (e.g. FcyRIIIa) and / or effector function (e.g. ADCC), e.g., substitutions at positions 228, 233, 234, 235, 265, 267, 297, 329 and / or 331 of the Fc region (Kabat EU numbering of residues).

[0402] In one aspect, the substitutions are L234A and L235A (LALA) in an Fc region derived from a human IgGl Fc region. In one aspect, the Fc region further comprises a D265A and / or P329G substitution. In one aspect, the substitutions are L234A, L235A and P329G (LALA-PG) in an Fc region derived from a human IgGl Fc region. See, e.g., WO 2012 / 130831, Schlothauer et al., Protein Eng Des Sei 29, 457-466 (2016). In another aspect, the substitutions are L234A, L235A and D265A (LALA-DA) in an Fc region derived from a human IgGl Fc region.

[0403] In one aspect, the substitutions are S228P and L235E (SPLE) in an Fc region derived from a human IgG4 Fc region. In one aspect, the Fc region further comprises a P329G substitution. In one aspect, the substitutions are S228P, L235E and P329G (SPLE-PG) in an Fc region derived from a human IgG4 Fc region. See, e.g., WO 2012 / 130831, Schlothauer et al., Protein Eng Des Sei 29, 457-466 (2016).

[0404] In one aspect, the substitution is N297A (NA), N297G (NG) or N297Q (NQ) in an Fc region derived from a human IgGl Fc region. In one aspect, the Fc region further comprises a D265 A substitution. In one aspect, the substitutions are D265 A and N297A (DANA), or D265 A and N297G (DANG) in an Fc region derived from a human IgGl Fc region.

[0405] In one aspect, the substitutions are E233P, L234V, L235A and G236del in an Fc region derived from a human IgGl Fc region (see e.g. Armour et al., Eur. J. Immunol. 29, 2613-2624 (1999)). In one aspect, the Fc region further comprises a N297G or S267 substitution.

[0406] In one aspect, the substitutions are L234F, L235E and D265A (FEA), or L234F, L235E and P331 S (FES), in an Fc region derived from a human IgGl Fc region.

[0407] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which decrease binding to human FcRn and / or serum half-life of the antibody, e.g. substitutions at positions 253, 310 and / or 435 (Kabat EU numbering of residues). In one aspect, the substitutions are 1253 A, H310A and H435A (AAA) in an Fc region derived from a human IgGl Fc region.

[0408] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which increase binding to human FcRn and / or serum half-life of the antibody, e.g. substitutions at positions 252, 254, 256, 428 and / or 434 (Kabat EU numbering of residues). In one aspect, the substitutions are M252Y, S254T and T256E in an Fc region derived from a human IgGl Fc region (see, e.g., Dall’Acqua et al. J Biol Chem 281, 23514-23524 (2006); WO 2002 / 60919). In one aspect, the substitutions are M428L and N434S (see e.g. Zalevsky et al. Nat Biotech 28, 157-159 (2010); WO 2009 / 086320). In one aspect, the substitutions are M428L and N434A.

[0409] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which reduce binding to rheumatoid factor, e.g. substitutions at positions 424, 436, 438 and / or 440 (Kabat EU numbering of residues). In one aspect, the substitutions are Q438R and S440E (RE) (see e.g. Maeda et al. MABS 9, 844-853 (2017)). In one aspect, amino acid substitution(s) which increase FcRn binding are combined with amino acid substitution(s) which reduce binding to rheumatoid factor, as described e.g. in Maeda et al. MABS 9, 844-853 (2017). In one aspect, the substitutions are M428L, N434A and Y436T, or M428L, N434S and Y436T, in an Fc region derived from a human IgGl Fc region. In one aspect, the substitutions are N434A, Q438R, S440E, and optionally Y436T or Y436V, in an Fc region derived from a human IgGl Fc region. In one aspect, the substitutions are M428L, N434A, Q438R, S440E, and optionally Y436T or Y436V, in an Fc region derived from a human IgGl Fc region.

[0410] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which increase the antibody’s isoelectric point (pl), e.g. substitutions at positions 311 and / or 434 (Kabat EU numbering of residues). In one aspect, the substitutions are Q311R and P343R.

[0411] In one aspect, an antibody provided herein comprises an Fc region with one or more amino acid substitution(s) which increase affinity to human FcyRIIb, e.g. substitutions at positions 234, 235, 236, 238, 250, 264, 268, 295, 307, 326 and / or 330 (Kabat EU numbering of residues). In one aspect, the substitutions are L235W, G236N, H268D, Q295L, K326T and A330K, or L234Y, P238D, T250V, V264I, T307P and A330K.

[0412] For additional Fc region mutations see e.g. Abdeldaim and Schindowski, Pharmaceutics 15(10): 2402 (2023).

[0413] The C-terminus of the Fc region of an antibody provided herein may be a complete C- terminus ending with the amino acid residues PGK. The C-terminus of the Fc region may also be a shortened C-terminus in which one or two of the C terminal amino acid residues have been removed. In one aspect, the C-terminus of the Fc region is a shortened C-terminus ending with the amino acid residue P. In one aspect, the C-terminus of the Fc region is a shortened C- terminus ending with the amino acid residues PG. In one aspect, an antibody comprising an Fc region as specified herein, comprises the C-terminal glycine-lysine dipeptide (G446 and K447, Kabat EU numbering of amino acid positions). In one aspect, an antibody comprising an Fc region as specified herein, comprises a C-terminal glycine residue (G446, Kabat EU numbering of amino acid positions). d. Antibody Derivatives In one aspect, an antibody provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3- dioxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, proly propylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0414] B. Recombinant Methods and Compositions

[0415] Antibodies may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. For these methods one or more isolated nucleic acid(s) encoding an antibody are provided.

[0416] In case of a native antibody or native antibody fragment two nucleic acids are required, one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof. Such nucleic acid(s) encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chain(s) of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0417] In case of a bispecific antibody with heterodimeric heavy chains four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc-region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc-region polypeptide. The four nucleic acids can be comprised in one or more nucleic acid molecules or expression vectors. Such nucleic acid(s) encode an amino acid sequence comprising the first VL and / or an amino acid sequence comprising the first VH including the first heteromonomeric Fc-region and / or an amino acid sequence comprising the second VL and / or an amino acid sequence comprising the second VH including the second heteromonomeric Fc-region of the antibody (e.g., the first and / or second light and / or the first and / or second heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors, normally these nucleic acids are located on two or three expression vectors, i.e. one vector can comprise more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs (see, e.g., Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomeric heavy chain comprises the so-called “knob mutations” (T366W and optionally one of S354C or Y349C) and the other comprises the so-called “hole mutations” (T366S, L368A and Y407V and optionally Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73) according to EU index numbering.

[0418] In one aspect, isolated nucleic acids encoding an antibody as used in the methods as reported herein are provided.

[0419] In one aspect, a method of making an antibody that binds to CD3 comprises culturing a host cell comprising nucleic acid(s) encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0420] For recombinant production of an antibody that binds to CD3, nucleic acids encoding the antibody, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) or produced by recombinant methods or obtained by chemical synthesis.

[0421] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0422] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0423] Suitable host cells for the expression of (glycosylated) antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0424] Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).

[0425] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, e.g., in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0426] In one aspect, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). C. Assays

[0427] Antibodies that bind to CD3 provided herein may be identified, screened for, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0428] / . Binding Assays and Other Assays

[0429] In one aspect, an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.

[0430] The binding (affinity) of the antibody to a target antigen can be determined for example by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore ™ instrument (Cytiva), and receptors or target proteins such as may be obtained by recombinant expression. Alternatively, binding of antibodies to different receptors or target antigens may be evaluated using cell lines expressing the particular receptor or target antigen, for example by flow cytometry (FACS).

[0431] Specific illustrative and exemplary aspects for measuring binding activity to CD3 are described in the following.

[0432] In one aspect, the affinity to CD3 is determined by SPR at 25°C as follows: pH responsive CD3 binding is investigated using a Biacore T200 instrument (Cytiva). Biotinylated human CD3 epsilon / gamma (P1AF7199) SEQ ID NO: 251 is immobilized to a Neutravidin Sensor Chip (NAHCLM200, Xantec bioanalytics GmbH) at a surface density of approximately 50 resonance units (RU). As a running and dilution buffer, phosphate buffered saline (PBS) containing 0.05% Tween-20 was used, the pH value is adjusted to pH 6.5 and 7.4, respectively. The anti-CD3 antibodies are injected onto the surface at concentrations up to 900 nM for 90 seconds (s), dissociation is monitored for 120s. Subsequently, the surface is regenerated by injecting 10 mM Glycine pH 2.0 for 60s. Bulk refractive index differences are corrected by subtracting blank injections and by subtracting the response obtained from the reference flow cell without Neutravidin only. Curve fitting is performed using the 1 : 1 Langmuir binding model within the Biacore evaluation software.

[0433] In another aspect, the binding activity to CD3 is determined by using a cell line as follows:

[0434] The binding to human CD3 (SEQ ID NO: 211) is determined using the CD3 expressing cell line Jurkat NF AT (Jurkat_PHOPY_LUC_NFAT-RE, Promega). The cells are cultured in RPMH640 GlutaMAX with HEPES (Thermo Fischer, Cat.No. 72400-054) supplemented with 10 % FBS (Thermo Fischer, Cat.No. 16140-071), 1 mM sodium-pyruvate (Thermo Fischer, Cat.No. 11360-088), IX MEM Non-Essential Amino Acids Solution (Thermo Fischer, Cat.No. 11140035) and 200 pg / ml Hygromycin B (Thermo Fischer, Cat.no. 10687010) . Where indicated, HEK-293T (ATCC, CRL-3216) or MKN45 (ACC 409, DSMZ) cells are used as CD3 negative cell lines to assess nonspecific binding and cultured in DMEM GlutaMax (Cat.no: 31966047, Thermo Fischer) supplemented with 10 % FBS (Thermo Fischer, Cat.No. 16140-071) and RPMI1640 GlutaMAX with HEPES (Thermo Fischer, Cat.No. 72400-054) supplemented with 10 % FBS (Thermo Fischer, Cat.No. 16140-071) respectively. For binding experiments, target cells are washed once with PBS (Thermo Fisher, Cat.No. 20012-019). Adherent cell lines 293 T and MKN-45 are followed by dissociation with TrypLE Express (Thermo Fisher, Cat.No 12605036). Adherent cells and non-adherent Jurkat NF AT cells are washed once with PBS and thereafter counted with the Cedex HiRes Analyzer (Roche, Cat.no. 05650216001). CD3 negative cell lines are further stained with eFluor™ 670 proliferation dye (eBioscience, Cat No 65-0840-90). Cells are resuspended in 37°C warm PBS at a density of 1.0* 107cells / ml. Another tube with the same volume of warm PBS is prepared with eFluor™ 670 dye (5 pM, end concentration 2.5 pM). Cell suspension and PBS containing the dye are mixed in equal volumes and staining is performed for 10 minutes (min) at room temperature (RT) in the dark. FBS (l / 5th of staining volume) is added to stop the staining and cells are washed twice with excess PBS, resuspended in PBS and counted using the Cedex HiRes Analyzer (Roche, Cat.no. 05650216001). Unlabelled target cells and labeled target cells are mixed in a falcon tube at the later seeding density of 7.5*105cells / ml / cell line or 3.0*104cells / well / cell line, respectively. The cell mixture is stained for 10 min with 1 :500 diluted Zombie Aqua™ Fixable Viability dye (Biolegend, Cat.no. 423101) in PBS. Following staining the cell suspension is washed once with PBS and divided into two separate tubes for the binding at different pH conditions.

[0435] Cell binding is performed in a binding buffer containing 20mM PIPES buffer (GoldBio Cat.no. P -281-100), 154mM NaCl (Sigma Aldrich, Cat.no. 71380-M) and 1% BSA (Miltenyi, Cat.no. 130-091-376). The buffers are adjusted to pH 6.5 and pH 7.4 using either 1 M HCL (Sigma- Aldrich, Cat.no. H1758) or IM NaOH (Sigma- Aldrich, Cat.no. S2770) and an Orion VSTAR82 pH meter (Thermo Fisher).

[0436] The cell suspensions are washed once in each respective pH buffer and are then resuspended in the desired buffer volume. A target cell density of 3.0*104cells / well is seeded in 96 U bottom plate plates (TPP, Cat. No. 92097) or a 384 well plate (Thermo Fisher, Cat.no. #264573) using 40 JJ.L / well as seeding volume. The plates are centrifuged at 4 °C, 3 min at 400 x g and the supernatant is flicked off.

[0437] Upon shaking at 1200 revolutions per minute (rpm) for 30s to resuspend the pellet, 20-25 μL of primary antibody dilutions are added to each well. Serial dilution of antibodies are upfront prepared in pH 6.5 and pH 7.4 binding buffer. Primary antibodies are incubated for Ih at 4°C, protected from light. After primary antibody binding, the cells are washed two times with the respective pH buffer and centrifuged at 400 g, 3 min at 4°C. The buffer is discarded by a flick- off and cell pellets are subjected to shaking at 1200 rpm for 30s in a plate shaker. Subsequently, 20-25 μL of secondary PE-AffiniPure F(ab')2 Fragment Goat Anti-human IgG, Fc gamma Fragment Specific antibody (Jackson Immunoresearch, Cat.no. 109-116-170) diluted 300 times with PBS from stock concentration of 0.5mg / ml, is added in pH 6.5 and pH 7.4 binding buffer. The secondary antibody is incubated for 30 min at 4°C, protected from light. After another single wash with the respective pH buffer and centrifugation at 400 xg for 3 min at 4°C, cells are fixated with 1% PFA solution adjusted to pH 6.5 and pH 7.4. Following fixation the cell pellets are washed and resuspended in 15-75 μL FACS buffer (eBioscience Cat.No. 00-4222- 26). Binding assays are measured with the BD LSRFortessa cell analyzer or the iQue Screener Plus from Sartorius. The Median Fluorescence intensities are analyzed for target and control cells and normalized to their respective untreated controls at pH 6.5 and pH 7.4. The data are analyzed using FlowJo versionlO or the ForeCyte 6.2, Sartorius and plotted using the TIBCO Spotfire version 12.0.8. The MFI is subtracted for the non-treated controls at pH 7.4 and pH 6.5. Baseline-corrected MFI values are plotted against the LoglO of the antibody concentration and a 4P logistic regression model is applied to plot the dose-response graphs and calculate the EC50 using the TIBCO Spotfire version 12.0.8. Area under the curve (AUC) is calculated using an R script in TIBCO Spotfire version 12.0.8 as a sum of trapezoids between adjacent raw data points (trapezoidal rule).

[0438] 2. Activity Assays

[0439] In one aspect, assays are provided for identifying antibodies that bind to CD3 having biological activity.

[0440] Biological activity of the antibodies of the invention can be measured by various assays as described in the Examples. Biological activities may for example include the induction of proliferation of T cells, the induction of signaling in T cells, the induction of expression of activation markers in T cells, the induction of cytokine secretion by T cells, the induction of lysis of target cells such as tumor cells, and the induction of tumor regression and / or the improvement of survival.

[0441] Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0442] In certain aspects, an antibody of the invention is tested for such biological activity.

[0443] The biological activity of the antibodies that bind to CD3 and multispecific antibodies including TCBs can be determined for example by a JurkatNFAT activation assay, and primary T cell activation assays.

[0444] Specific illustrative and exemplary aspects for determining biological activity of the CD3 specific binders as bivalent or monovalent IgGs antibodies is described in the following.

[0445] 2.1 In vitro Activity Assays

[0446] In one aspect, a NF AT -responsive, stable luciferase reporter Jurkat T cell line (Jurkat PHOPY LUC NF AT-RE, Promega) referred to as Jurkat NF AT is used and crosslinked to anti-PGLALA clone VH3xVLl expressing CH0-K1 target cells (in house generated recombinant cell line expressing Chimeric antigen receptor construct in Fab format anti-PGLALA SEQ ID NO: 246). via simultaneous engagement of CD3 / TCR and PGLALA. Intracellular downstream signaling induces activation of the nuclear transcription factor Nuclear Factor of Activated T cells (NF AT) and luciferase synthesis.

[0447] Jurkat NF AT cells are cultured in RPMI1640 GlutaMAX with HEPES (Thermo Fisher, Cat.No. 72400-054) supplemented with 10 % FBS (Thermo Fisher, Cat.No. 16140-071), 1 mM sodium-pyruvate (Thermo Fisher, Cat.No. 11360-088), IX MEM Non-Essential Amino Acids Solution (Thermo Fisher, Cat.No. 11140035) and 200 pg / ml Hygromycin B (Thermo Fisher, Cat.no. 10687010)

[0448] CHO-K1 anti PGALA expressing cell line is cultured in DMEM GlutaMax (Cat.no: 31966047, Thermo Fisher) supplemented with 10 % FBS (Thermo Fisher, Cat.No. 16140-071).

[0449] Adherent target cells are washed once with PBS (Thermo Fisher, Cat.No. 20012-019) followed by dissociation with TrypLE Express (Thermo Fisher, Cat.No 12605036). The obtained cell suspension is further washed once with cell culture medium and thereafter counted with the Cedex HiRes Analyzer (Roche, Cat.no. 05650216001). Jurkat NF AT suspension cells are directly harvested and washed once with PBS prior to counting. Target and effector cell suspensions are combined in separate tubes for pH 6.5 and pH 7.4. 3*104Jurkat NFAT cells and l*104CH0-K1 anti-PGALA target cells are seeded simultaneously in 45 pl / well in a 384w white flat bottom plate (Corning, Cat.no. 353988) in pH 6.5 & pH 7.4 medium based on RPMI1640 GlutaMAX with HEPES (Thermo Fisher, Cat.No. 72400-054) supplemented with 10 % FBS (Thermo Fisher, Cat.No. 16140-071), 2 mM Calcium chloride (Sigma, Cat.no. 21115) and 20mM PIPES buffer (GoldBio Cat.no. P- 281-100) . The medium is adjusted to pH 6.5 and pH 7.4 using either 1 M HCL (Sigma- Aldrich, Cat.no. H1758) or IM NaOH (Sigma- Aldrich, Cat.no. S2770) and an Orion VSTAR82 pH meter (Thermo Fisher). Serial dilution of antibodies are upfront prepared in RPMI medium and 5 pl of antibody suspension was added to the co-culture. The mixture is incubated for 6 h at 5% CO2 in the incubator.

[0450] After 6 h, the plates are centrifuged at 400 x g for 3 min to pellet the cells and the supernatant is flicked-off. An additional wash step with 80 pl / well PBS (Thermo Fisher, Cat.No. 20012- 019) is performed, followed by addition of 15 pl / well reporter lysis buffer (Promega, Cat.no. E3971). Cell lysates are additionally frozen in the same plates to ensure complete lysis. The plates are sealed with aluminum foil and stored at -80°C for minimum 25 min or overnight.

[0451] Cell lysates are thawed at room temperature and protected from light, followed by addition of 30 pl / well of Luciferase Assay System 100 reagent (Promega, Cat.no E4450) containing the luciferase substrate. The plates are placed immediately in a Spark 10M plate reader (Tecan) to measure luminescence 500 seconds / well in Relative Luminescent Units (RLU). The luminescence background is subtracted for the non-treated controls at pH 7.4 and pH 6.5. Baseline-corrected luminescent units are plotted against the Log 10 of the antibody concentration and a 4P logistic regression model is applied to plot the dose-response graphs and calculate the EC50 values using the TIBCO Spotfire version 12.0.8.

[0452] In another aspect, biological activity of monovalent or bivalent IgG antibodies that bind to CD3 are investigated in vitro by measuring the induction of T cell activation marker CD69 on isolated primary human T cells derived from Peripheral blood mononuclear cells (PBMCs) and measured by FACS. PBMCs are obtained from our in-house cell bank isolated from buffy coats (Blutspende Zurich) via the standard density gradient procedure (Stemcell, Cat.No. 07851). Frozen PBMC stocks are thawed and rested 2 hours at 37°C. After the resting time, the PBMCs are collected and washed twice with PBS prior counting with the Cedex HiRes Analyzer (Roche, Cat.no. 05650216001). Afterwards, Pan T cells are isolated by using a commercial Pan T Cell Isolation Kit (Miltenyi, Cat. No. 130-096-535) according to the manufacturer’s instructions.

[0453] 96-U bottom TPP plates (TPP, Cat. No. 92097) or 384-V bottom plates (VWR, Cat. no. 737- 0226) are coated with 50μL or 10μL of serial diluted pH responsive monovalent or bivalent CD3 huIgGl constructs per well. The coating is done in PBS (Thermo Fisher, Cat.No. 20012- 019) for 2 hours at 37°C, 5% CO2. The molecules for coating are upfront prepared in PBS in a 8-10-point 1 :3 / 5 dilution series starting with a 50 to 1000 nM top concentration depending on the molecule. Following coating, the plates are washed thrice with 200pl of PBS per well and used immediately to plate the Pan T cells. A Pan T cell density of 6*10A4 cells / well is seeded in pH 6.5 & pH 7.4 medium respectively in a volume of 200 μL per well. The media are based on RPMH640 GlutaMAX with HEPES (Thermo Fischer, Cat.No. 72400-054) supplemented with 10 % FBS (Thermo Fischer, Cat.No. 16140-071) and 20mM PIPES buffer (GoldBio Cat.no. P-281-100). The medium is adjusted to the respective pH using either 1 M HCL (Sigma- Aldrich, Cat.no. H1758) or IM NaOH (Sigma- Aldrich, Cat.no. S2770) and an Orion VSTAR82 pH meter (ThermoFisher). The plates are placed in the incubator at 37°C, 5% CO2 for 3 days. At day 2-3, the plates are centrifuged at 400 x g for 5min to pellet the cells and the supernatant is flicked-off. The plates are shaken at 1200 rpm for 30s to resuspend the cell pellets and washed once with PBS. Subsequently, the cells are stained with 50μL of a 1 :500 diluted viability dye (LIVE / DEAD AquaTM Fixable Viability Kit, Invitrogen, Cat. No. L34957) for 15 min in the dark at room temperature. The cells are washed once with FACS buffer (eBioscience Cat.No. 00-4222-26) . The cells are subsequently stained with 50μL of an antibody cocktail diluted in FACS buffer and consisting of CD4 in BV421 (Biolegend, Cat. No. 300532), CD8 in BV711 (Biolegend, Cat. No. 301044), CD69 in PE (Biolegend, Cat. No. 310906) for 30min, at 4°C. Then, the cells are washed once with IX FACS Buffer, centrifuged 5min at 400g and resuspended in 150μL FACS buffer. Finally, the cells are acquired for FACS at the LSR Fortessa Cell Analyzer instrument (BD, Cat. No. 647800E6) using FACSDiva software. The obtained FCS files are analyzed using FlowJo software. First, the cell population is gated by using FSC and SSC. The doublets are excluded when plotting FSC-A vs FSC-H. Afterwards, the dead cells (positive for Aqua) are excluded. The CD4 and CD8 are gated using BV421 and BV711, respectively. Afterwards, the positive signals for CD69 of each cell population are quantified and exported as frequency of percent positive cells and median fluorescence intensity MFI. The readout values where mentioned, are normalized to their respective untreated controls at pH 6.5 and pH 7.4. The data are analyzed using FlowJo versionlO and plotted using the TIBCO Spotfire version 12.0.8. Percentage positive values are plotted against the LoglO of the antibody concentration and a 4P logistic regression model is applied to plot the dose-resp...

Claims

CLAIMS1. An antibody that binds to CD3, wherein the antibody binds to CD3 at neutral pH with a first KD and at an acidic pH with a second KD, wherein the first KD is greater than the second KD and the first KD and the second KD are measured by surface plasmon resonance (SPR) at 25 °C.

2. The antibody of claim 1, wherein the acidic pH ranges from 6.0 to 6.6 and the neutral pH ranges from 7.0 to 7.4, particularly wherein the acidic pH is 6.5 or 6.1 and the neutral pH is 7.0 or 7.4, more particularly wherein the acidic pH is 6.5 and the neutral pH is 7.4.

3. The antibody of claim 1 or 2, wherein the first KD is greater than the second KD by a factor of at least 2.0, at least 2.2, at least 2.8, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 4.6, at least 5.0, at least 10.0, at least 10.9, at least 14.3, at least 16.2, at least 18.0, at least 19.6, at least 20.0, at least 21.0, at least 23.0, at least 24.0, at least 25.0, at least 28.2, at least 29.7, at least 32.4, at least 33.3, at least 38.6, at least 43.5, at least 43.9, at least 52.8, at least 61.9, at least 70.0, at least 80.0, at least 90.2, at least 100.0, at least 113.2, at least 120.0, at least 130.0, at least 140.0, at least 150.0, at least 160.0, at least 170.0, at least 181.6, least 190.0, at least 200.0, at least 210.0, at least 220.0, at least 230.0, at least 236.0 or more.

4. The antibody of any one of claims 1 to 3, wherein the second KD is equal to 47 nM or less at pH 6.5 and the first KD is equal to 0.2 nM or more at pH 7.4 or wherein the second KD is equal to 4 nM or less at pH 6.5 and the first KD is equal to 9 nM or more at pH 7.4.

5. The antibody of any one of claims 1 to 4, wherein the first KD is greater than the second KD by a factor ranging from 2.0 to 236.0 or from 4.5 to 236.0.

6. The antibody of any one of claims 1 to 5, wherein the KD reflects monovalent affinity.

7. The antibody of any one of claims 1 to 6, wherein the antibody is a monoclonal antibody.

8. The antibody of any one of claims 1 to 7, wherein the antibody is a humanized antibody.

9. The antibody according to any one of claims 1 to 8, wherein the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1comprising the amino acid sequence of NX13X14IH SEQ ID NO:225, (b) CDR- H2 comprising the amino acid sequence of WIX7PGX8GX9TX10YNX11KFX12G (SEQ ID NO:226), and (c) CDR-H3 comprising the amino acid sequence of DX1X2X3X4YX5X6DY (SEQ ID NO:227), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of KSSQSLLNX22RTRKNYLA (SEQ ID NO:222), (e) CDR-L2 comprising the amino acid sequence of WASX19RX20X21 (SEQ ID NO:223), and (f) CDR-L3 comprising the amino acid sequence of X15X16SFX17X18RT (SEQ ID NO:224), wherein Xi is T, S or Q, X2is Y, V or R, X3is S or V, X4is N, Q or H, X5is F or Y, X6is F, Y or H, X7is Y, L, V, H, E, K or R, X8is D, Y, T, R or E, X9is N, D, Q or E, X10is R or K, X11is E, Q or H, X12is K, Q or E, X13is Y or E, X14is Y or H, X15is S, N or H, X16is T or N, X17is E or Q, X18is S or H, X19is T or D, X20is E or Q, X21is I or H, X22is L, H or D.

10. The antibody according to any one of claims 1 to 9, wherein the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of NX13YIH SEQ ID NO:228, (b) CDR- H2 comprising the amino acid sequence of WIX23PGDGX24TKYNEKFX25G (SEQ ID NO:229), and (c) CDR-H3 comprising the amino acid sequence of DX26X27HX28YYX29DY (SEQ ID NO:230), and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of KSSQSLLNSRTRKNYLA (SEQ ID NO:231), (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 8), and (f) CDR-L3 comprising the amino acid sequence of TX30SFILRT (SEQ ID NO:233) wherein X13is Y or E, X23is Y or E, X24is N or D, X25is E or K, X26is Q, T or H, X27is Y or V, X28is H or Q, X29is F or Y, X30is Q or E, optionally wherein X23is E when X24is D and / or X23is Y when X24is N.

11. The antibody according to any one of claims 1 to 10, wherein the antibody comprises a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 266, 272, 169 and 3, (b) CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 170 and 174, and (c) CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 273, 267, 159, 171 and 175, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ IDNO: 7 or 271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 12.

12. The antibody according to any one of claims 1 to 11, wherein the antibody comprisesA) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 169, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 170, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9;B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12;C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:159, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12;D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acidsequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12;E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 266, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:267, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; orF) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 272, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:273, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

13. An antibody that binds to CD3, wherein the antibody comprisesA) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 169, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 170, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:9;B) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 175, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12;C) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:159, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12;D) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 171, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:7, (e) CDR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO:8), and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12;E) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 266, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 174, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:267, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12; orF) a heavy chain variable domain (VH) comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 272, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:4, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:273, and a light chain variable domain (VL) comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:271, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:8, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

14. The antibody of any one of claims 9 to 13, further comprising a light chain variable domain framework FR1 sequence of SEQ ID NO:253, FR2 sequence of SEQ ID NO:254 or SEQ ID NO: 261, FR3 sequence of SEQ ID NO255:, FR4 sequence of SEQ ID NO:256, and / or a heavy chain variable domain framework FR1 sequence of SEQ ID NO:257 or SEQ ID NO:,FR2 sequence of SEQ ID NO:258, FR3 sequence of SEQ ID NO:259, and FR4 sequence of SEQ ID NO:260.

15. The antibody of any one of claims 1 to 14, wherein the antibody comprises a VH comprising an amino acid sequence ofEVQLVQSGAEVKKPGASVKVSCKASNX13YIHWVRQAPGQGLEWIGWIX23PGDGX24 TKYNEKFX25GRATLTADTSTSTAYLELSSLRSEDTAVYYCARDX26X27HX28YYX29DY WGQGTLVTVSS (SEQ ID NO: 262), and / or a VL comprising an amino acid sequence of DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRX32NYLAWYQQKPGQPPKLLIX31 WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTX30SFILRTWGQGTLVTVSS (SEQ ID NO: 263), wherein X13is Y or E, X23is Y or E, X24is N or D, X25is E or K, X26is Q, T or H, X27is Y or V, X28is H or Q, X29is F or Y, X30is Q or E and X31is H or Y, X32is K or H, optionally wherein X23is E when X24is D and / or X23is Y when X24is N and / or X31is Y when X30is Q and / or X31is H when X30is E.

16. The antibody of any one of claims 1 to 15, wherein the antibody comprisesA) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 172, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10;B) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 176, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13;C) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 160, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 206;D) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 208, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 206;E) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 268, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 269; orF) a VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 270, and / or a VL comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 265.

17. The antibody of any one of claims 1 to 16, wherein the antibody comprisesA) a VH comprising an amino acid sequence of SEQ ID NO: 172, and / or a VL comprising an amino acid sequence of SEQ ID NO: 10;B) a VH comprising an amino acid sequence of SEQ ID NO: 176, and / or a VL comprising an amino acid sequence of SEQ ID NO: 13;C) a VH comprising an amino acid sequence of SEQ ID NO: 160, and / or a VL comprising an amino acid sequence of SEQ ID NO: 206;D) a VH comprising an amino acid sequence of SEQ ID NO: 208, and / or a VL comprising an amino acid sequence of SEQ ID NO: 206;E) a VH comprising an amino acid sequence of SEQ ID NO: 268, and / or a VL comprising an amino acid sequence of SEQ ID NO: 269 ; orF) a VH comprising an amino acid sequence of SEQ ID NO: 270, and / or a VL comprising an amino acid sequence of SEQ ID NO: 265.

18. An antibody that binds to CD3, wherein the antibody comprisesA) a VH comprising an amino acid sequence of SEQ ID NO: 172, and / or a VL comprising an amino acid sequence of SEQ ID NO: 10;B) a VH comprising an amino acid sequence of SEQ ID NO: 176, and / or a VL comprising an amino acid sequence of SEQ ID NO: 13;C) a VH comprising an amino acid sequence of SEQ ID NO: 160, and / or a VL comprising an amino acid sequence of SEQ ID NO: 206;D) a VH comprising an amino acid sequence of SEQ ID NO: 208, and / or a VL comprising an amino acid sequence of SEQ ID NO: 206E) a VH comprising an amino acid sequence of SEQ ID NO: 268, and / or a VL comprising an amino acid sequence of SEQ ID NO: 269 ; orF) a VH comprising an amino acid sequence of SEQ ID NO: 270, and / or a VL comprising an amino acid sequence of SEQ ID NO: 265.

19. The antibody of any one of claims 1 to 18, wherein the antibody is an antibody fragment that binds CD3.

20. The antibody of any one of claims 1 to 19, wherein the antibody fragment is a Fab molecule.

21. The antibody of any one of claims 1 to 20, wherein the antibody is a full length IgG1antibody.

22. The antibody of any one of claims 1 to 21, comprising an Fc domain composed of a first and a second subunit.

23. The antibody of any one of claims 1 to 22, wherein the antibody is multispecific.

24. The antibody of any one of claims 1 to 23 wherein the antibody is a bispecific antibody comprising at least one domain that binds to CD3 and at least one domain that binds to an target cell antigen.

25. The antibody of claim 24, wherein the domain that binds to CD3 is a Fab molecule wherein the variable domains VL and VH or the constant domains CL and CHI, particularly the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced by each other.

26. The antibody of claim 24 or 25, wherein the domain that binds to CD3 is a Fab molecule wherein in the constant domain CL the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CHI the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).

27. The antibody of any one of claims 20 or 25 to 26, wherein the Fab molecule is a conventional Fab molecule.

28. The antibody of any one of claims 24 to 27, wherein the domain that binds to CD3 and the domain that binds to the target cell antigen are fused to each other, optionally via a peptide linker.

29. The antibody of any one of claims 24 to 28, wherein the target cell antigen is a cancer cell antigen.

30. The antibody of claim 22 , wherein the Fc domain is an IgG Fc domain, particularly an IgG1Fc domain, more particularly a human IgG1Fc domain.

31. The antibody of any one of claims 22 or 30, wherein the Fc domain is a human Fc domain.

32. The antibody of any one of claims 22, 30 or 31, wherein the Fc comprises a modification promoting the association of the first and the second subunit of the Fc domain.

33. The antibody of any one of claims 22 or 30 to 32, wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor and / or effector function.

34. The antibody of any one of claims 22 or 30 to 33, wherein the antibody comprises an Fc region derived from a human IgG1Fc region and comprising the substitutions L234A, L235A and P329G (LALA-PG) Kabat EU numbering.

35. The antibody of any one of claims 1 to 34, wherein AUC fold change pH 6.5 to pH 7.4 is at least 2.9, at least 3.0, at least 4.8, at least 4.9, at least 5.0 or at least 9.0 as measured by binding of the antibody as monovalent IgGi to human CD3 on Jurkat NF AT T cell line quantified as the percentage of CD3 positive cells at 4°C with a BD Symphony A3 cell analyzer.

36. The antibody of any one of claims 1 to 34, wherein AUC fold change pH 6.5 to pH 7.4 is at least 3.7, at least 4.0, at least 4.6, at least 4.9, at least 5.4, at least 6.0, at least 8.9 or at least1729.3 as measured by binding of the antibody as monovalent IgGl to human CD3 on JurkatNFAT T cell line quantified as the Mean Fluorescent Intensity (MFI) at 4°C with a BD Symphony A3 cell analyzer.

37. An isolated nucleic acid encoding the antibody of any of claims 1 to 36.

38. A host cell comprising the nucleic acid of claim 37.

39. A method of producing an antibody that binds to CD3 comprising culturing the host cell of claim 38 under conditions suitable for the expression of the antibody.

40. The method of claim 39, further comprising recovering the antibody from the host cell.

41. An antibody produced by the method of claim 40 or 41.

42. A pharmaceutical composition comprising the antibody of any of claims 1 to 36 or 41 and a pharmaceutically acceptable carrier.

43. The antibody of any one of claims 1 to 36 or 41 or the pharmaceutical composition of claim 42 for use as a medicament.

44. The antibody of any one of claims 1 to 36 or 41 or the pharmaceutical composition of claim 42 for use in treating cancer.

45. Use of the antibody of any one of claims 1 to 36 or 41 or the pharmaceutical composition of claim 42 in the manufacture of a medicament for treatment of cancer.

46. Use of the antibody of any one of claims 1 to 36 or 41 or the pharmaceutical composition of claim 42 in the manufacture of a medicament for pH dependent T-cell redirection and pH dependent T-cell mediated cancer cell killing.

47. A method of treating an individual having cancer comprising administering to the individual an effective amount of the antibody of any one of claims 1 to 36 or 41 or the pharmaceutical composition of claim 42.

48. A method of pH dependent T-cell redirection and T-cell mediated cancer cell killing in an individual comprising administering to the individual an effective amount of the antibody of any of claims 1 to 36 or 41 or the pharmaceutical composition of claim 42 to redirect T cells and mediate T-cells to kill cancer cells in a pH dependent manner.

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