HUMANIZED OR CHIMERATIVE CD3 ANTIBODIES

Description

Field of invention

[0001] The present invention relates to a humanized antibody binding to human CD3, compositions comprising said humanized antibody, and use of said humanized antibodies in treatment of a disease.Background

[0002] The Cluster of Differentiation 3 (CD3) has been known for many years and therefore has been subject of interest in many aspects. Specifically antibodies raised against CD3 or the T cell Receptor Complex, which CD3 is part of, are known. An in vitro characterization of recombinant chimeric CD3 isotype variants as well as a number of humanized OKT3 effector function variant antibodies has been described [1].

[0003] CD3 antibodies, e.g. muromonab-CD3, have been widely used in the treatment of acute allograft rejection. In addition, treatment with the anti-CD3 monoclonal antibody hOKT3gamma1(Ala-Ala) results in improved C-peptide responses and clinical parameters for at least 2 years after onset of type 1 diabetes in absence of continued immunosuppressive medications [2].

[0004] A promising approach to improve targeted antibody therapy is by delivering cytotoxic cells specifically to the antigen-expressing cancer cells. This concept of using T cells for efficient killing of tumor cells has been described [3]. However, initial clinical studies were rather disappointing mainly due to low efficacy, severe adverse effects (cytokine storm) and immunogenicity of the bispecific antibodies [4]. Advances in the design and application of bispecific antibodies have partially overcome the initial barrier of cytokine storm and improved clinical effectiveness without dose-limiting toxicities [5].

[0005] For example, certain bispecific antibodies targeting with one arm the antigen on the tumor cell and with the other arm for instance CD3 on T cells, and containing an active Fc fragment providing Fc receptor binding have been shown to induce tumor cell killing. Upon binding, a complex of T cells, tumor cells and effector cells that bind the antibody Fc region is potentially formed, leading to killing of the tumor cells [4]. Catumaxomab consists of a mouseIgG2a / ratIgG2b heavy chain heterodimer and has been found successful for the treatment of cancer-associated ascites after intraperitoneal application [6]. However, the mouse / rat hybrid is immunogenic [7] and cannot be applied for long-term treatment in humans. Frequent treatment-related adverse events attributed to catumaxomab included cytokine-release-related symptoms (i.e. pyrexia, nausea, vomiting, chills, tachycardia and hypotension) [8]-[9], which relate the potent polyclonal T cell activation by catumaxomab due to its active Fc fragment. Another antibody is ertumaxomab (HER2xCD3), which induces cytotoxicity in cell lines with HER2 expression. Ertumaxomab has been in Phase II clinical development for metastatic breast cancer

[10] -

[11] .

[0006] Efficacy of CD3 bispecific antibodies and other CD3 bispecific antibody-based formats is dependent on several properties of bispecific antibodies, such as the affinity of the CD3 arm and / or the affinity to the target of the second arm and the target copy number on target cells. Some CD3 bispecific antibodies show high efficacy when the CD3 affinity is low (EpCamxCD3 - Bortoletto 2002 PMID 12385030, MT103 / Blinatumomab vs TandAb - Molhoj 2007 PMID 17083975), while other CD3 bispecifics demonstrate high efficacy using a high CD3 affinity (Reusch 2015, Mabs, PMID 25875246). In some cases high CD3 affinity is required, for example when arming ex vivo expanded activated T cells from patients with a bispecific antibody comprised of an anti-CD3 targeting arm and a second arm directed at a selected tumor-associated antigen. In the latter case, CD3 affinity should be high to retain the interaction with the expanded T cell when the product is infused back into the patient to mediate cytolysis of tumor cells (Reusch 2006 Clin Cancer Res PMID 16397041). However, high affinity antibodies to CD3, in contrast to low affinity ligands, are much less effective in TCR triggering at low copy number, since they display a stoichiometry of ~1:1 and a linear dose-response curve, indicative of a single cycle rather than a serial triggering mode of T cell response (Viola 1996 Science, PMID 8658175). In other words, low affinity of CD3 arm can allow T cells to flexibly move from one target and / or target cell to the other (Hoffman 2005, PMID: 15688411).

[0007] Low CD3 affinity can potentially prevent the biased localization of the bispecific antibody to T cells (due to first encounter in circulation) and thus improve biodistribution and minimize interference with normal T cell immune responses. Depending on the target of the second arm and target copy number, the indication and / or administration route, desirable CD3 affinity can be customized to enhance a product's maximum efficacy. A panel of CD3 variants covering a range of CD3 affinity can be essential to suit these specific tailor-made needs per antibody product.

[0008] WO 2015 / 001085 discloses humanized and chimeric CD3 antibodies (pages 6 and 21-23), and bispecific antibodies comprising a first binding region of said humanized or chimeric anti-CD3 antibody and a second binding region which binds a different target than said first antigen binding region (pages 43-48). Variants of said antibodies comprising mutations in the Fc region that alter effector functions are also shown (pages 31-32). Nucleic acids, expression vectors, host cells and methods to produce said antibody using these are also provided (pages 60-64), along with pharmaceutical compositions comprising said antibody or said bispecific antibody (pages 64-65), and their use in the diagnosis and treatment of a disease (pages 68-75). This document furthermore discloses a kit for detecting the presence of CD3 antigen, or a cell expressing CD3, in a sample comprising i) an antibody or bispecific antibody according to the invention, and ii) instructions for use of the kit (pages 75-76), and an anti-idiotypic antibody which binds to the anti-CD3 antibody (pages 76-77).

[0009] WO 2014 / 108483 discloses anti-CD3 antibodies having a non-activating Fc region as compared to a wild-type protein or antibody (example 1 on pages 53-54). Antibodies can be humanised (pages 37 and 53) or chimeric (page 37). Nucleic acids, expression vectors, host cells and methods to produce said antibody using these are also provided (pages 37-40). Pharmaceutical compositions comprising said antibodies, together with the use of said antibodies or compositions in the treatment of a disease are also shown (pages 40-50). Kits for detection using said antibodies (pages 50-51) are also provided.

[0010] WO 2013 / 188693 discloses a humanized anti-CD3 antibody (page 2 and example 1 on pages 53-54). Bispecific antibodies are also shown (pages 24-25). Nucleic acids, expression vectors, host cells and methods to produce said antibody using these are also provided (pages 38-40). Use of the antibody for detection or treatment is also shown, along with kits (pages 43-53).

[0011] WO 2013 / 186613 discloses humanized monoclonal antibodies to CD3, comprising a combination of specific mutations in the variable framework regions (FRs), together with specific complementarity-determining regions (CDR) sequences, which result in improved properties, in particular biological activity and pharmaceutical stability (pages 2-3 and 12-13). Pharmaceutical compositions comprising said antibodies and methods for their use in treating diseases susceptible to amelioration through binding to CD3 are also provided (pages 32-33).

[0012] WO 2012 / 143524 discloses bispecific antibodies specific for CD3 and Her2 (page 3), wherein the Fc contains mutations at positions 366, 368, 370, 399, 405, 407 and / or 409 (pages 57-64).

[0013] Hinojosa et al., (2010) Hybridoma, Vol. 29, No. 2, pp. 115-124, discloses a human IgG1 chimeric anti-human CD3 monoclonal antibody (see abstract and page 121).

[0014] Li et al., (2010) MABS, Vol. 2, No. 4, pp 449-556, discloses a phase I clinical trial of a humanized anti-CD3 antibody containing mutations in the Fc to prevent binding to FcR in patients receiving renal allografts (see abstract and pages 449-450).

[0015] Kiewe et al., (2006) American Society of Clinical Oncology, US, Vol. 12, No. 10, pp. 3085-3091, discloses a phase I clinical trial of a bispecific anti-CD3 and anti-Her2 antibody in metastatic breast cancer (see abstract and pages 3085-3086).

[0016] CD3 antibodies cross-reactive to cynomolgus and / or rhesus monkey CD3 have been described

[12] -

[13] , however, further improvements for such cross-reactive antibodies are needed.Summary of invention

[0017] The object of the present invention is to provide humanized CD3 antibodies with an optimized affinity to CD3. Thus it is an object of the present invention to provide humanized CD3 antibodies which are optimized compared to a reference antibody such as an antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. Hence, such antibodies may have a reduced or increased affinity to CD3 compared to a reference antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. It is a further object of the present invention to provide antibodies with a lower binding affinity to CD3 than the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. The inventors found that antibodies with a reduced binding affinity to the CD3 peptide as set forth in SEQ ID NO: 402 compared to a reference antibody having the VH region sequence set forth in SEQ ID NO: 4 manitain the same or similar cytotoxic activit in vitro and in vivo compared to the reference antibody. Another object of the present invention is to provide CD3 antibodies with reduced binding affinity to CD3 compared to a reference antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8, but retaining the same cytolytic activity as the reference antibody.

[0018] The present invention provides in one aspect a humanized antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region and a variable light chain (VL) region, wherein said VL region comprises CDR1, CDR2, and CDR3 regions having the CDR sequences as set forth in SEQ ID NO:6, GTN and 7, wherein said VH region comprises the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 176, wherein the antibody is of an IgG1 isotype, and wherein said VH region has a VH sequence set forth in SEQ ID NO: 177, and wherein said VL region comprises a VL sequence selected from the group consisting of; a) a VL sequence as set forth in SEQ ID NO:8; b) a VL sequence as set forth in SEQ ID NO: 10.

[0019] The numerical numbering of positions corresponding to SEQ ID NO:4 is illustrated in figure 2. Further, the VH CDR regions have been annotated according to the IMGT definitions.

[0020] The antibody has a reduced binding affinity to human CD3 compared to the reference antibody having the VH CDR sequences set forth in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2 and CDR3 SEQ ID NO: 3.

[0021] An antibody with reduced bining affinity to a human CD3 molecule, such as a CD3 peptide e.g. SEQ ID NO:402, compared to a reference antibody may manintain the same cytolytic activity against a target cell as the reference antibody.

[0022] The antibody comprises a mutation in the position corresponding to H101 of SEQ ID NO: 4 the mutation being H101G. The amino acids in SEQ ID NO: 4 are numbered accoding to a direct numerical numbering scheme from the first amino acid to number 125 in the direction from N-terminus to the C-terminus.

[0023] That is, the inventors of the present invention in said first aspect of the invention found that a humanized antibody of said sequences had an optimized binding affinity to a CD3 peptide SEQ ID NO: 402 compared to a reference antibody such as an antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. The reference antibody specified by SEQ ID NO:4 and the VL sequence SEQ ID NO:8 has a binding affinity to the CD3 peptide of SEQ ID NO:402 of 1.5x10 -8< M as illustrated by example 7. The antibody of the present invention has a lower binding affinity to the CD3 peptide of SEQ ID NO:402 than 1.5x10 -8< M when determined by Bio-Layer Interferometry as described in Table 6 in example 7.. The binding affinity corresponds to the K D value.

[0024] CD3 may in particular be human CD3 epsilon.

[0025] In another aspect, the present invention relates to a bispecific antibody comprising a first binding region of an antibody according to the invention, and a second binding region which binds a different target than said first antigen binding region.

[0026] In another aspect, the present invention relates to an expression vector comprising (i) a nucleic acid sequence encoding a heavy chain sequence of a humanized antibody according to the invention, (ii) a nucleic acid sequence encoding a light chain sequence of a humanized antibody according to the invention, or (iii) both (i) and (ii).

[0027] In another aspect, the present invention relates to a host cell comprising an expression vector according to the invention.

[0028] In another aspect, the present invention relates to a composition comprising the antibody or bispecific antibody according to the invention.

[0029] In another aspect, the present invention relates to a pharmaceutical composition comprising the antibody or bispecific antibody according to the invention and a pharmaceutical acceptable carrier.

[0030] In another aspect, the present invention relates to the antibody or bispecific antibody, the composition, or the pharmaceutical composition according to the invention for use as a medicament.

[0031] In another aspect, the present invention relates to the antibody or bispecific antibody, the composition, or the pharmaceutical composition according to the invention for use in the treatment of a disease.

[0032] The antibody or bispecific antibody may be administered to a subject in need thereof, and may be administered by subcutaneuous or local administration.

[0033] In another aspect, the present invention relates to a method for producing an antibody or a bispecific antibody according to the invention, comprising the steps of a) culturing a host cell according to the invention, and b) purifying the antibody from the culture media.

[0034] In another aspect, the present invention relates to a diagnostic composition comprising an antibody or bispecific antibody according to any one of the embodiments as disclosed herein.

[0035] The diagnostic composition may be a companion diagnostic which is used to screen and select those patients who will benefit from treatment with the bispecific antibody.

[0036] In another aspect, the present invention relates to a method for detecting the presence of CD3 antigen, or a cell expressing CD3, in a sample comprising the steps of a) contacting the sample with an antibody or bispecific antibody according to the invention, under conditions that allow for formation of a complex between the antibody or bispecific antibody and CD3, and b) analyzing whether a complex has been formed.

[0037] In another aspect, the present invention relates to a kit for detecting the presence of CD3 antigen, or a cell expressing CD3, in a sample comprising i) an antibody or bispecific antibody according to the invention, and ii) instructions for use of the kit.

[0038] In another aspect, the present invention relates to an anti-idiotypic antibody or a pair of anti-idiotypic antibodies which bind to an antibody according to the invention.Brief description of figures

[0039] Figure 1: Heat plot with binding ratios of mutant versus wt UniTE-huCD3-H1L1-T41K molecules. Ratios above 1 indicate stronger binding than wt, while ratios below 1 indicate weaker binding than wt. Binding was determined on Freestyle 293-F cells transfected with CD3 / TCR-LC13. Figure 2: Alignment of selected CD3 affinity variants in the generated library with mutations in the VH. CDRs are underlined in the humanized wild type sequence (SEQ ID NO:4) HuCD3-H1. Highlighted amino acids are the substitutions. Figure 3: T cell binding curves of selected VH affinity variants of humanized CD3 (UniTE-huCD3-H1L1-T41K) antibodies as determined by flow cytometry Affinity variants depicted cover a broad range of T cell binding capacity between the wild type response and undetectable response. Figure 4: T cell binding curves of selected VH affinity variants of humanized CD3 (UniTE-huCD3-H1L1-T41K) antibodies as determined by flow cytometry showing a very low, undetectable T cell binding. Figure 5: T cell binding curves of selected VH affinity variants of humanized CD3 (BisG1-huCD3-H1L1-X-FEAL / 1014-Herceptin-FEAR) antibodies as determined by flow cytometry. Affinity variants depicted cover a broad range of T cell binding capacity between the wild type response and undetectable response . Figure 6: Cytotoxicity of CD3 affinity variants on solid tumor cell lines measured by alamar blue assay. (A) NCI-N87 cells, Effector cells (T cells): Tumor cell (NCI-N87 cell) ratio = 3:1, 48 hours of incubation, n=2 donors (B) SKOV3 cells, T cells: SKOV3 cell ratio = 4:1 , 48 hours of incubation, n=2 donors (C) MDA-MB-231 cells, T cells : MDA-MB-231 cell ratio = 8:1 , 48 hours of incubation, n=2 donors. The tested affinity variants depicted cover a broad range of cytotoxicity between wild type response and no observed cytotoxicity for all tested tumor cell lines. Figure 7: Cytotoxicity of CD3 affinity variants on a hematological (Daudi) cell line measured by chromium release assay. T cell: Daudi celll ratio = 10:1, 24 hours of incubation, 1 donor. The tested affinity variants depicted cover a broad range of cytotoxicity between wild type response and no observed cytotoxicity for the tested tumor cell line. Figure 8: Cytotoxicity of CD3xHER2 bispecific antibodies in a NCI-N87, human PBMC co-engraftment model in NOD-SCID mice. HLA-A-matched human unstimulated PBMCs, as a source of human T cells, were co-inoculated with NCI-N87 tumor cells in NOD-SCID mice at two different dose levels of CD3 affinity antibodies (0.5 and 0.05 mg / kg). Humanized WT CD3 (huCD3) and 4 different CD3 affinity variants (N57E, H101K, S110A, Y114M) were tested. (A) Average tumor volume followed over time after treatment with 0.05 mg / kg of antibody (n=4 per group). (B) Average tumor volume followed over time after treatment with 0.5 mg / kg of antibody (n=4 per group). (C) Average tumor volume at day 44 after treatment with 0.05 mg / kg of antibody at day 0 (n=4 per group). (D) Average tumor volume at day 44 after treatment with 0.5 mg / kg of antibody at day 0 (n=4 per group). Statistics has been performed on data for C and D. Detailed description

[0040] In one aspect, the present invention relates to a humanized antibody binding to human CD3 with an optimized affinity to CD3. Thus it is an object of the present invention to provide humanized CD3 antibodies which are optimized compared to a reference antibody such as the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. It is a further object of the invention to provide antibodies with optimized in vivo efficacy compared to a reference antibody such as the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. It is a further object of the present invention to provide antibodies with a lower binding affinity to CD3 than the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8.

[0041] In one aspect the invention relaes to a humanized antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region and a variable light chain (VL) region, wherein said VL region comprises CDR1, CDR2, and CDR3 regions having the CDR sequences as set forth in SEQ ID NO:6, GTN and 7, wherein said VH region comprises the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 176, wherein the antibody is of an IgG1 isotype, and wherein said VH region has a VH sequence set forth in SEQ ID NO: 177, and wherein said VL region comprises a VL sequence selected from the group consisting of; c) a VL sequence as set forth in SEQ ID NO:8; d) a VL sequence as set forth in SEQ ID NO:10.

[0042] The amino acids in SEQ ID NO: 4 are numbered accoding to a direct numerical numbering scheme from the first amino acid to number 125 in the direction from N-terminus to the C-terminus. The numerical numbering of positions corresponding to SEQ ID NO: 4 is illustrated in figure 2. Further, The CDR regions have been annotated according to the IMGT defnitions.

[0043] The antibody of the invention has a reduced binding affinity to human CD3 compared to the reference antibody having the VH CDR sequences set forth in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2 and CDR3 SEQ ID NO: 3.

[0044] An antibody with reduced bining affinity to a CD3 molecule, such a CD3 peptide e.g. SEQ ID NO:402, compared to a reference antibody may manintain the same cytolytic activity against a target cell as the reference antibody.

[0045] The antibody of the invention comprises a mutation in the position H101. Position H101 is in accordance to SEQ ID NO: 4. The mutation is H101G.

[0046] In one embodiment of the invention the antibody comprises a mutation in the position G105 . Position G105 is in accordance to SEQ ID NO: 4. In one embodiment the mutation is G105P.

[0047] The reference antibody specified by SEQ ID NO:4 and the VL sequence SEQ ID NO:8 has a binding affinity to the CD3 peptide of SEQ ID NO:402 corresponding to a K D value of 1.5x10 -8< M as illustrated by example 7.

[0048] The antibody of the present invention has a lower binding affinity to the CD3 peptide of SEQ ID NO:402 than 1.5x10 -8< M when determined by Bio-Layer Interferometry as described in Example 7.

[0049] The term "antibody" as used herein is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or time sufficient for the antibody to recruit an effector activity). The binding region (or binding domain which may also be used herein, both terms having the same meaning) which interacts with an antigen, comprises variable regions of both the heavy and light chains of the immunoglobulin molecule. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells and T cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. As indicated above, the term antibody as used herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that retain the ability to specifically interact, such as bind, to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H 1 domains, or a monovalent antibody as described in WO2007059782 (Genmab A / S); (ii) F(ab') 2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the V H and C H 1 domains; and (iv) a Fv fragment consisting essentially of the V L and V H domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, V L and V H , are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present invention are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.

[0050] The term "immunoglobulin heavy chain", "heavy chain of an immunoglobulin" or "heavy chain" as used herein is intended to refer to one of the chains of an immunoglobulin. A heavy chain is typically comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) which defines the isotype of the immunoglobulin. The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. The heavy chain constant region may further comprise a hinge region. The term "immunoglobulin" as used herein is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized (see for instance

[14] ). Within the structure of the immunoglobulin (e.g. IgG), the two heavy chains are inter-connected via disulfide bonds in the so-called "hinge region". Equally to the heavy chains each light chain is typically comprised of several regions; a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region typically is comprised of one domain, CL. Furthermore, the VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see

[15] ). CDR sequences may be determined by use of the method provided by IMGT

[16] -

[17] .

[0051] The term "isotype" as used herein, refers to the immunoglobulin (sub)class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f) [SEQ ID NO:407]) that is encoded by heavy chain constant region genes. Thus, in one embodiment, the antibody comprises a heavy chain of an immunoglobulin of the IgG1 class or any allotype thereof. Further, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.

[0052] The term "chimeric antibody" as used herein, refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by antibody engineering. "Antibody engineering" is a generic term used for different kinds of modifications of antibodies, and which is a well-known process for the skilled person. In particular, a chimeric antibody may be generated by using standard DNA techniques as described in

[18] . Thus, the chimeric antibody may be a genetically engineered recombinant antibody. Some chimeric antibodies may be both genetically or an enzymatically engineered. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody according to the present invention may be performed by other methods than described herein. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity. They may typically contain non-human (e.g. murine) variable regions, which are specific for the antigen of interest, and human constant antibody heavy and light chain domains. The terms "variable region" or "variable domains" as used in the context of chimeric antibodies, refers to a region which comprises the CDRs and framework regions of both the heavy and light chains of the immunoglobulin.

[0053] The term "humanized antibody" as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see

[19] -

[20] ). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.

[0054] The humanized antibody according to any aspect or embodiment of the present invention may be termed "humanized CD3 antibody", "humanized antibody of the invention", "CD3 antibody", or "CD3 antibody of the invention", which all have the same meaning and purpose unless otherwise contradicted by context.

[0055] The amino acid sequence of an antibody of non-human origin is distinct from antibodies of human origin, and therefore a non-human antibody is potentially immunogenic when administered to human patients. However, despite the non-human origin of the antibody, its CDR segments are responsible for the ability of the antibody to bind to its target antigen and humanization aims to maintain the specificity and binding affinity of the antibody. Thus, humanization of non-human therapeutic antibodies is performed to minimize its immunogenicity in man while such humanized antibodies at the same time maintain the specificity and binding affinity of the antibody of non-human origin.

[0056] The term "binding region" as used herein, refers to a region of an antibody which is capable of binding to any molecule, such as a polypeptide, e.g. present on a cell, bacterium, or virion.

[0057] The term "binding" as used herein, refers to the binding of an antibody to a predetermined antigen or target to which binding typically is with an affinity corresponding to a K D of about 10 -6< M or less, e.g. 10 -7< M or less, such as about 10 -8< M or less, such as about 10 -9< M or less, about 10 -10< M or less, or about 10 -11< M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a K D that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The degree with which the affinity is lower is dependent on the K D of the antibody, so that when the K D of the antibody is very low (that is, the antibody is highly specific), then the degree with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold. The term "K D " (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0058] The term "human CD3" as used herein, refers to the human Cluster of Differentiation 3 protein which is part of the T cell co-receptor protein complex and is composed of four distinct chains. CD3 is also found in other species, and thus, the term "CD3" may be used herein and is not limited to human CD3 unless contradicted by context. In mammals, the complex contains a CD3γ (gamma) chain (human CD3γ chain Swissprot P09693, or cynomolgus monkey CD3γ Swissprot Q95LI7), a CD3δ (delta) chain (human CD3δ Swissprot P04234, or cynomolgus monkey CD3δ Swissprot Q95LI8), two CD3ε (epsilon) chains (human CD3ε Swissprot P07766; or cynomolgus CD3ε Swissprot Q95LI5), rhesus CD3ε (Swissprot G7NCB9), and a CD3ζ-chain (zeta) chain (human CD3ζ Swissprot P20963, cynomolgus monkey CD3ζ Swissprot Q09TK0). These chains associate with a molecule known as the T cell receptor (TCR) and generate an activation signal in T lymphocytes. The TCR and CD3 molecules together comprise the TCR complex.

[0059] It is within the knowledge of the skilled person that amino acid sequences referred to as Swissprot numbers include a signal peptide which is removed after translation of the protein. Thus, proteins, such as CD3, present on cell surfaces do not include the signal peptide. In particular, the amino acid sequences listed in Table 1 do not contain such signal peptide. Such proteins as listed in Table 1 may be termed "mature proteins". Thus, SEQ ID NO:398 shows the amino acid sequence of mature human CD3δ (delta), SEQ ID NO:399 shows the amino acid sequence of mature human CD3ε (epsilon), SEQ ID NO:403 shows the amino acid sequence of mature cynomolgus CD3ε, and SEQ ID NO:404 shows the amino acid sequence of mature rhesus CD3ε. Thus, the term "mature" as used herein, refers to a protein which does not comprise any signal or leader sequence.

[0060] It is well-known that signal peptide sequence homology, length, and the cleavage site position, varies significantly between different proteins. Signal peptides may be determined by different methods, e.g. SEQ ID NO:399 of the present invention has been determined according to the SignalP application (available on http: / / www.cbs.dtu.dk / services / SignalP / ).

[0061] In a particular embodiment, the humanized antibody of the present invention binds the epsilon chain of CD3, such as the epsilon chain of human CD3 (SEQ ID NO:399). In yet another particular embodiment, the humanized antibody binds an epitope within amino acids 1-27 of the N-terminal part of human CD3ε (epsilon) (SEQ ID NO:402). In such a particular embodiment, the antibody may even further cross-react with other non-human primate species, such as cynomolgus monkeys (cynomolgus CD3 epsilon SEQ ID NO:403) and / or rhesus monkeys (rhesus CD3 epsilon SEQ ID NO:404).

[0062] The term "sequence identity" as used in the context of the present invention, refers to the percent identity between two sequences as a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller

[21] . In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch algorithm

[22] . Multiple alignments are preferably performed using the Clustal W algorithm

[23] (as used e.g., in Vector NTI Advance ®< software version 11.5; Invitrogen Inc.).

[0063] The VH region, as illustrated in the sequence table 1 of the present document, consists of 125 amino acid sequence. Thus a second VH sequence consisting of 125 amino acids whereof 124 amino acid postions are identical with the one of the first VH sequences listed above has 99,2% sequence identity with said first VH sequence. A second sequence consisting of 125 amino acids whereof 120 amino acid positons are identical with one of the first VH sequences listed above have 96% sequence identity with said first VH sequence. A second sequence consisting of 125 amino acids whereof 115 amino acid postions are identical with one of the first VH sequences listed above have 92% sequence identity with said first VH sequence.

[0064] In one embodiment of the invention the antibody is a full-length antibody.

[0065] The humanized antibody according to the present invention may be generated by comparison of the heavy and light chain variable region amino acid sequences against a database of human germline variable region sequences in order to identify the heavy and light chain human sequence with the appropriate degree of homology for use as human variable framework regions. A series of humanized heavy and light chain variable regions may be designed by grafting, e.g. the murine, CDRs onto the framework regions (identified as described above) and, if necessary, by back-mutation (mutation of one or more of the human amino acid residues in the framework regions back to the non-human amino acid at the specific position(s)) to the specific murine sequence of residues identified which may be critical to the restoration of the antibody binding efficiency. Variant sequences with the lowest incidence of potential T cell epitopes may then be selected as determined by application of in silico technologies; iTope ™< and TCED ™< (

[24] ,

[25] , and

[26] ). Furthermore, the humanized antibodies according to the present invention may also be "deimmunized". Deimmmunization may be desired, as within a protein sequence, such as a humanized antibody according to the present invention, the presence of human T cell epitopes may increase the immunogenicity risk profile as they have the potential to activate helper T cells. Such activation of helper T cells may be avoided by deimmunization. Deimmunization may be performed by introducing a mutation in the amino acid sequence of the humanized antibody in order to remove the T cell epitopes without significantly reducing the binding affinity of the antibody.

[0066] Thus, the humanized antibody may be produced by a method comprising the steps of (i) comparing the non-human full variable heavy chain sequence and / or the full variable light chain sequence to a database of human germline sequences, (ii) selecting the human germline sequence having the highest homology to the non-human sequence to obtain a humanized sequence, (iii) optimizing the humanized sequence by back-mutation(s) if required, and (iv) expressing the sequence in a suitable expression system.

[0067] Thus, a full-length antibody according to the present invention may be produced by a method comprising the steps of (i) comparing the non-human variable heavy chain sequence and the variable light chain sequences to a database of human germline sequences, (ii) selecting the human germline sequence having the highest homology to the non-human sequence, (iii) grafting of the non-human CDRs in to the selected human germline to obtain a humanized sequence, (iv) optimizing the humanized sequences by back-mutation(s) if required, (v) identifying constant heavy and light chain sequences, and (vi) expressing the complete heavy chain sequences and complete light chain sequences in suitable expression systems. A full-length antibody according to the present invention may, thus, be produced as described in Example 1. It is within the knowledge of the skilled person to produce a full-length antibody when starting out from either CDR sequences or full variable region sequences. Thus, the skilled person would know how to generate a full-length antibody according to the present invention.

[0068] The term "complete heavy chain sequences" as used herein, refers to a sequence consisting of variable heavy chain and constant heavy chain sequences.

[0069] The term "complete light chain sequences" as used herein, refers to a sequence consisting of variable light chain and constant light chain sequences.

[0070] Back-mutation(s) may be introduced by standard DNA mutagenesis. Such standard techniques for DNA mutagenesis are described in

[18] . Alternatively, use of commercially available kits such as Quickchange ™< Site-Directed Mutagenesis Kit (Stratagene), or the desired back-mutations may be introduced by de novo DNA synthesis.

[0071] Thus, in one embodiment, the antibody is a humanized antibody.

[0072] Chimeric antibodies may be generated by substituting all constant region sequences of a non-human (such as murine) antibody with constant region sequences of human origin. Thus, fully non-human variable region sequences are maintained in the chimeric antibody. Thus, a chimeric antibody may be produced by a method comprising the step of expressing the non-human variable heavy chain (SEQ ID NO:405), non-human variable light chain sequences (SEQ ID NO:406), human constant heavy chain and human constant light chain sequences in suitable expression systems, and thereby generating a full-length chimeric antibody. Alternative methods may be used. Such methods of producing a chimeric antibody is within the knowledge of the skilled person, and thus, the skilled person would know how to produce a chimeric antibody according to the present invention. Thus to make a chimeric antibody one would introduce the mutations according to the invention in the non-human (such as murine) VH or VL sequence.

[0073] In one embodiment, the antibody is a full-length antibody. The term "full-length antibody" as used herein, refers to an antibody (e.g., a parent or variant antibody) which contains all heavy and light chain constant and variable domains correspond to those that are normally found in a wild-type antibody of that isotype.

[0074] The antibody may comprise an Fc region comprising a first and a second immunoglobulin heavy chain.

[0075] The term "Fc region" as used herein, refers to a region comprising, in the direction from the N- to C-terminal, at least a hinge region, a CH2 region and a CH3 region. An Fc region may further comprise a CH1 region at the N-terminal end of the hinge region.

[0076] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the Eu numbering as set forth in Kabat.

[0077] Unless otherwise stated or contradicted by context, the amino acids of the constant region sequences are herein numbered according to the Eu-index of numbering (described in

[27] ) and may be termed "according to the Eu numbering as set forth in Kabat", "Eu numbering according to Kabat", or "according to the Eu numbering system".

[0078] The term "CH1 region" or "CH1 domain" as used herein, refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering system. However, the CH1 region may also be any of the other subtypes as described herein.

[0079] The term "CH2 region" or "CH2 domain" as used herein, refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering system. However, the CH2 region may also be any of the other subtypes as described herein.

[0080] The term "CH3 region" or "CH3 domain" as used herein, refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering system. However, the CH3 region may also be any of the other subtypes as described herein.

[0081] In one embodiment, the isotype of the immunoglobulin heavy chain is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The immunoglobulin heavy chain may be any allotype within each of the immunoglobulin classes, such as IgG1m(f) (SEQ ID NO:407). Thus, in one particular embodiment, the isotype of the immunoglobulin heavy chains is an IgG1, or any allotype thereof, such as IgG1m(f) (SEQ ID NO:407).

[0082] When targeting the antigen CD3 which is part of the T cell Receptor (TCR), the T cell specific mechanisms of cell killing is desirable. Other effector functions, e.g. complement activation, may not be wanted, and therefore, reduction of effector functions is desirable. C1q binding is the first step in the complement cascade, and therefore serves as an indicator for complement-dependent cytotoxicity (CDC) capacity of antibodies. If binding of C1q to the antibody can be avoided, activation of the complement cascade can be avoided as well.

[0083] Thus, the antibody may comprise an Fc region which has been modified so that binding of C1q to said antibody is reduced compared to a wild-type antibody by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99,9% or 100%, wherein C1q binding is determined by ELISA. In a prefered embodiement the antibody comprises an Fc region, which has been modiefied so that the binding of C1q to said antibody is reduced compared to a wild-type antibody by at least 99% to a 100%, wherein C1q binding is determined by ELISA.

[0084] The term "modified" as used herein, refers to the amino acid sequence of an Fc region which is not identical to the amino acid sequence of a wild-type Fc region. I.e. amino acid residues in specific positions of the wild-type Fc region have been substituted, deleted or inserted in order to alter, for example, the binding site for C1q, binding site for other effector molecules or binding to Fc Receptors (FcRs). Such modification(s) of the amino acid sequence may be prepared by substituting one or more amino acids with a conservative amino acid or may be prepared by substituting one or more amino acids with an alternative amino acid which is physically and / or functionally similar to the amino acid present in the wild-type. Substitutions may also be prepared by substituting with a non-conservative amino acid.

[0085] In the context of the present invention, amino acids may be described as conservative or non-conservative amino acids, and may therefore be classified accordingly. Amino acid residues may also be divided into classes defined by alternative physical and functional properties. Thus, classes of amino acids may be reflected in one or both of the following tables:Amino acid residue of conservative class

[0086] Acidic ResiduesD and EBasic ResiduesK, R, and HHydrophilic Uncharged ResiduesS, T, N, and QAliphatic Uncharged ResiduesG, A, V, L, and INon-polar Uncharged ResiduesC, M, and PAromatic ResiduesF, Y, and W Alternative Physical and Functional Classifications of Amino Acid Residues

[0087] Alcohol group-containing residuesS and TAliphatic residuesI, L, V, and MCycloalkenyl-associated residuesF, H, W, and YHydrophobic residuesA, C, F, G, H, I, L, M, R, T, V, W, and YNegatively charged residuesD and EPolar residuesC, D, E, H, K, N, Q, R, S, and TPositively charged residuesH, K, and RSmall residuesA, C, D, G, N, P, S, T, and VVery small residuesA, G, and SResidues involved in turn formationA, C, D, E, G, H, K, N, Q, R, S, P, and TFlexible residuesQ, T, K, S, G, P, D, E, and R

[0088] In the context of the present invention, a substitution in an antibody, such as a humanized antibody, is indicated as: Original amino acid - position - substituted amino acid; Referring to the well-recognized nomenclature for amino acids, the three letter code, or one letter code, is used, including the codes Xaa and X to indicate any amino acid residue. Accordingly, the notation "L234F" or "Leu234Phe" means, that the antibody comprises a substitution of Leucine with Phenylalanine in amino acid position 234.

[0089] Substitution of an amino acid at a given position to any other amino acid is referred to as: Original amino acid - position; or e.g. "L234".

[0090] For a modification where the original amino acid(s) and / or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the more than one amino acid may be separated by "," or " / ". E.g. the substitution of Leucine for Phenylalanine, Arginine, Lysine or Tryptophan in position 234 is: "Leu234Phe,Arg,Lys,Trp" or "Leu234Phe / Arg / Lys / Trp" or "L234F,R,K,W" or "L234F / R / K / W" or "L234 to F, R, K or W" Such designation may be used interchangeably in the context of the invention but have the same meaning and purpose.

[0091] Furthermore, the term "a substitution" embraces a substitution into any one of the other nineteen natural amino acids, or into other amino acids, such as non-natural amino acids. For example, a substitution of amino acid L in position 234 includes each of the following substitutions: 234A, 234C, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P, and 234Y. This is, by the way, equivalent to the designation 234X, wherein the X designates any amino acid other than the original amino acid. These substitutions can also be designated L234A, L234C, etc., or L234A,C,etc., or L234A / C / etc. The same applies by analogy to each and every position mentioned herein, to specifically include herein any one of such substitutions.

[0092] The antibody according to the invention may also comprise a deletion of an amino acid residue. Such deletion may be denoted "del", and includes, e.g., writing as L234del. Thus, in such embodiments, the Leucine in position 234 has been deleted from the amino acid sequence.

[0093] The terms "amino acid" and "amino acid residue" may herein be used interchangeably.

[0094] Binding to CD3 may be binding to full length CD3 such as CD3 present on a T cell. Alternatively, binding to CD3 may be binding to a CD3 peptide e.g. as set forth in SEQ ID NO: 402. Binding to the CD3 peptide and whether or not any further mutations may modify binding to CD3 can be deterimined by Bio-Layer Inerferometry as disclosed in Example 7.

[0095] In one embodiment, the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

[0096] The term "C1q binding" as used herein, refers to the binding of C1q to an antibody, when said antibody is bound to its antigen. The term "bound to its antigen" as used herein, refers to binding of an antibody to its antigen both in vivo and in vitro.

[0097] The term "reduced" as used herein when referring to C1q binding, refers to the ability of the antibody according to the invention to reduce, minimize or even completely inhibit the binding of C1q to the antibody when compared to the C1q binding to a wild-type antibody.

[0098] The term "reduced" or "reducing" as used herein or any variation thereof when used in relation to binding affinity of an antibody bining to human CD3, referes to a binding affinity that is lower when compared to a reference binding affinity. In this context, the reference binding affinity may be the binding affinity of reference antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8 when binding to the CD3 peptide as SEQ ID NO: 402 and determined by Bio-Layer Interferomerty as described in example 7.

[0099] The term "binding affinity" as used herein referes to the binding of an antibody to a predetermined antigen or target to which binding typically is with an affinity corresponding to a K D . The term "K D " (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0100] The term "wild-type antibody" as used herein, in relation to use in comparison assays of an antibody according to the present invention, refers to an antibody which is identical to the antibody to be tested except for not being inert. In this context, the term "inert" refers to a modified Fc region having reduced or no binding of C1q, i.e. where C1q binding is determined by ELISA; reduced or no Fc-mediated T cell proliferation as determined in a PBMC based functional assay, i.e. T cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay; and / or reduced or no Fc-mediated CD69 expression as determined in a PBMC-based functional assay. Thus, the wild-type antibody comprises the naturally occurring amino acids in the immunoglobulin heavy chains, i.e. an antibody which does not comprise any amino acid modifications which may alter or reduce the ability of the antibody to interact with e.g. C1q, Fc Receptors or the like. Thus, such a wild-type antibody will remain an activating antibody which is able to bind e.g. C1q. A wild-type antibody and an antibody of the present invention may comprise other amino acid modifications than those affecting the antibody's ability of inducing effector functions, in order to make the antibody a bispecific antibody or the like.

[0101] The term "ELISA" as used herein refers to enzyme-linked immunosorbent assay which is a test that uses antibodies and color change to identify a substance. A first specific antibody is attached to the plate surface. Thereby the protein from a sample is added wherein binding to said first specific antibody is tested. A second antibody binding the antibody from the sample is added. The second antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. The concept of the ELISA method is well-known within the art and various ways of performing an ELISA are contemplated to be part of a method to evaluate the antibody according to the invention

[0102] Specifically, the ability of an antibody according to the present invention to bind C1q may be determined by ELISA comprising the steps of (i) coating said antibody on a 96-well plate, (ii) adding 3% serum, (iii) adding an anti-human C1q antibody, (iv) developing the plate, and (v) measuring OD 405 nm. Thus, in one embodiment, the antibody comprises an Fc region which has been modified so that binding of C1q to said antibody is reduced compared to a wild-type antibody by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein C1q binding is determined by ELISA comprising the steps of (i) coating said antibodies on a 96-well plate, (ii) adding 3% serum, (iii) adding an anti-human C1q, (iv) developing the plate, and (v) measuring OD 405 nm.

[0103] The terms "Fc Receptor" or "FcR" as used herein, refers to a protein found on the surface of certain cells. FcRs bind to the Fc region of antibodies. There are several different types of FcRs which are classified based on the type of antibody they recognize. E.g. Fcy (gamma) Receptors bind to antibodies of the IgG class.

[0104] The terms "Fcy Receptor", "Fc gamma Receptor" or "FcyR" as used herein, refers to a group of Fc Receptors belonging to the immunoglobulin superfamily and is the most important Fc receptors for inducing phagocytosis of opsonized (coated) microbes. This family includes several members, FcyRI (CD64), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRIIIa (CD16a), FcyRIIIb (CD16b), which differ in their antibody affinities due to their different molecular structure.

[0105] Fc-mediated effector functions form part of the biological activity of human immunoglobulin G (IgG) molecules. Examples of such effector functions include e.g. antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) which are triggered by the binding of various effector molecules to the Fc region. In the context of the present invention, "Fc binding", "Fc Receptor binding", "FcR binding", and "binding of an antibody Fc region to FcR" refers to the binding of the Fc region to an Fc Receptor (FcR) or an effector molecule. The terms "FcyR binding" and "FcyRI binding" refer to binding to or with an Fc region to the Fc gamma Receptor and Fc gamma Receptor I, respectively. When a CD3 antibody binds T cells, the wild-type Fc region of the CD3 antibody binds to FcRs present on other cells, e.g. monocytes, which leads to non-specific, Fc-mediated activation of the T cell. Such non-specific, Fc-mediated activation of T cells may be undesired. T cells may also be activated by targeted, or target-specific, T cell activation. Such targeted T cell activation may be highly desirable for the treatment of a range of indications, such as cancer. The term "targeted T cell activation" as used herein, refers to directing the T cells to specific cells, such as tumor cells by use of a bispecific antibody comprising a first binding region binding a specific target, such as a tumor target on a tumor cell, and a second binding region binding a T cell specific target, such as CD3. Thus, targeting of T cells to specific cells, e.g. tumor cells, may be facilitated by use of a bispecific antibody, wherein one of the binding regions binds CD3 present on the T cell and the other binding region binds a target specific antigen, e.g. on a tumor cell. Although, non-specific, Fc-mediated T cells activation may still be possible and therefore such undesired non-specific, Fc-mediated T cell activation via Fc-mediated cross-linking should be avoided and may be disabled by making the Fc region inert for such activity. Thereby, interaction between said inert Fc region with Fc Receptors present is prevented.

[0106] An antibody according to the present invention may comprise modifications in the Fc region. When an antibody comprises such modifications it may become an inert, or non-activating, antibody. The term "inertness", "inert" or "non-activating" as used herein, refers to an Fc region which is at least not able to bind any Fcy Receptors, induce Fc-mediated cross-linking via FcRs, or induce FcR-mediated cross-linking of target antigens via the Fc region, or is not able to bind C1q. The inertness of an Fc region of a humanized CD3 antibody is advantageously tested using the antibody in a monospecific format although an inert Fc region so identified can be used in bispecific or other multispecific CD3 antibodies.

[0107] Several variants can be constructed to make the Fc region of an antibody inactive for interactions with Fc gamma Receptors and C1q for therapeutic antibody development. Examples of such variants are described herein.

[0108] Thus, the antibody may comprise an Fc region which has been modified so that said antibody mediates reduced Fc-mediated T cell proliferation compared to a wild-type antibody by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein said T cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0109] The term " reduce" when referring to T cell proliferation, refers to the ability of the antibody according to the invention to reduce, minimize or even completely inhibit the proliferation of T cells when compared to the proliferation of T cells bound by a wild-type antibody. The ability of an antibody to reduce T cell proliferation may be evaluated by a PBMC-based functional assay. The assay may be performed with human PBMCs. Alternatively, the assay may be performed with cynomolgus PBMCs. In another alternative, the assay may be performed with rhesus PBMCs. Since the antibody according to the present invention is cross-reactive, a PBMC-based assay as herein described may be performed with any species PBMCs to show reduction of T cell proliferation as long as the species PBMC used are within the cross-reactivity spectra of the antibodies, e.g. human, cynomolgus or rhesus monkeys.

[0110] The term "peripheral blood mononuclear cell (PBMC)-based functional assay" as used herein refers to an assay used for evaluating a functional feature of the antibody of the present invention, such as the ability of said antibody to affect T cell proliferation or CD69 expression, wherein the only cells present are peripheral blood mononuclear cells. Thus, T cell proliferation may be measured by a method comprising the steps of incubating PBMCs with antibody in the range of 1-1000 ng / mL at 37°C in a 5% (vol / vol) CO 2 humidified incubator for three days, adding a chemical compound, such as BrdU, which is incorporated into the DNA of proliferating cells, incubating for five hrs., pelleting cells, drying cells, optionally storing the cells at 4°C, coating cells to ELISA plates, incubating with anti-BrdU-peroxidase for 90 min at room temperature, developing for about 30 min with 1 mg / mL 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), adding 100 µL 2% oxalic acid to stop the reaction, and measuring absorbance at 405 nm in a suitable microplate reader.

[0111] The term "proliferation" as used herein, refers to cell growth in the context of cell division.

[0112] The term "BrdU" as used herein, refers to 5-bromo-2'-deoxyuridine, which is a homologue to thymidine. When BrdU is added to cell culture for a limited period of time (e.g. 4 hours) it will be incorporated into the DNA of proliferating cells. After fixing the cells, detection of incorporated BrdU may be performed in an ELISA using anti-BrdU-peroxidase. BrdU incorporation is therefore a measure for proliferation.

[0113] The antibody may comprise an Fc region which has been modified so that said antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% when compared to a wild-type antibody wherein said Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0114] In particular, the term "reduce" when referring to expression level of the T cell activation marker CD69, refers to a reduction in expression level of CD69 when compared to expression level of CD69 when the T cell is bound by a wild-type antibody bound to CD3 and interacting with an Fc receptor. An antibody's ability to reduce expression of CD69 may be evaluated by a PBMC-based functional. Thus, expression of CD69 may be measured by a method comprising the steps of incubating PBMCs with an antibody in the range of 1-1000 ng / mL at 37°C in a 5% (vol / vol) CO 2 humidified incubator for 16-24 hrs, washing the cells, staining the cells at 4°C with a mouse anti-human CD28-PE and mouse-anti-human CD69-APC antibody, and determining CD69-expression on CD28 positive cells by flow cytometry.

[0115] The term "CD69" as used herein, refers to Cluster of Differentiation 69 which is a human transmembrane C-Type lectin protein encoded by the CD69 gene. Activation of T lymphocytes and natural killer (NK) cells, both in vivo and in vitro, induces expression of CD69. CD69 function as a signal transmitting receptor involved in cellular activation events including proliferation, functions as a signal-transmitting receptor in lymphocytes, including natural killer cells and platelets, and the induction of specific genes.

[0116] The term "peripheral blood mononuclear cell (PBMC)-based functional assay" as used herein refers to an assay used for evaluating a functional feature of the antibody of the present invention, such as the ability of said antibody to affect T cell proliferation or CD69 expression, wherein the only cells present are peripheral blood mononuclear cells. A PBMC-based functional assay comprises the steps of (i) incubating PBMCs with an antibody at 37°C in a 5% (vol / vol) CO 2 humidified incubator for about 16-24 hrs, (ii) washing the cells, (iii) staining the cells at 4°C with a mouse anti-human CD28-PE and mouse-anti-human CD69-APC antibody, and (iv) determining the CD69 expression on CD28 positive cells by flow cytometry, when CD69 expression is evaluated.

[0117] Amino acids in the Fc region that play a dominant role in the interactions with C1q and the Fc Gamma Receptors may be modified. Examples of amino acid positions that may be modified include positions L234, L235 and P331. Combinations thereof, such as L234F / L235E / P331S, can cause a profound decrease in binding to human CD64, CD32A, CD16 and C1q.

[0118] Hence, the amino acid in at least one position corresponding to L234, L235 and P331, may be A, A and S, respectively ([1],

[28] ). Also, L234F and L235E amino acid substitutions can result in Fc regions with abrogated interactions with Fc Gamma Receptors and C1q (

[29] -

[30] ). Hence, in one embodiment, the amino acids in the positions corresponding to L234 and L235, may be F and E, respectively. A D265A amino acid substitution can decrease binding to all Fc gamma Receptors and prevent ADCC (

[31] ). Hence, the amino acid in the position corresponding to D265 may be A. Binding to C1q can be abrogated by mutating positions D270, K322, P329, and P331. Mutating these positions to either D270A or K322A or P329A or P331A can make the antibody deficient in CDC activity (

[32] ). Hence, the amino acids in at least one position corresponding to D270, K322, P329 and P331, may be A, A, A, and A, respectively.

[0119] An alternative approach to minimize the interaction of the Fc region with Fc gamma Receptors and C1q is by removal of the glycosylation site of an antibody. Mutating position N297 to e.g. Q, A, and E removes a glycosylation site which is critical for IgG-Fc gamma Receptor interactions. Hence, the amino acid in a position corresponding to N297, may be G, Q, A or E (

[33] ). Another alternative approach to minimize interaction of the Fc region with Fc gamma Receptors may be obtained by the following mutations; P238A, A327Q, P329A or E233P / L234V / L235A / G236del (

[31] ).

[0120] Alternatively, human IgG2 and IgG4 subclasses are considered naturally compromised in their interactions with C1q and Fc gamma Receptors although, interactions with Fcy Receptors (Fc gamma Receptors) were reported (

[34] -

[35] ). Mutations abrogating these residual interactions can be made in both isotypes, resulting in reduction of unwanted side-effects associated with FcR binding. For IgG2, these include L234A and G237A, and for IgG4, L235E. Hence, the amino acid in a position corresponding to L234 and G237 in a human IgG2 heavy chain, may be A and A, respectively.The amino acid in a position corresponding to L235 in a human IgG4 heavy chain, may be E.

[0121] Other approaches to further minimize the interaction with Fc gamma Receptors and C1q in IgG2 antibodies include those described in

[36] and

[37] .

[0122] The hinge region of the antibody can also be of importance with respect to interactions with Fc gamma Receptors and complement (

[38] -

[39] ). Accordingly, mutations in or deletion of the hinge region can influence effector functions of an antibody.

[0123] The term "cross-linking" as used herein, refers to the indirect bridging of antibody Fab arm(s) (monovalently or bivalently) bound to the target antigen by FcR-bearing cell through binding to the antibody Fc region. Thus, an antibody which binds its target antigen on target antigen-bearing cells may cross-link with another cell expressing FcRs.

[0124] The term "unspecific killing" as used herein, refers to the killing of cells by the cytotoxic function of T cells or other effector cells, through tumor target antigen-independent activation of said cells. Thus, by unspecific killing is meant that the tumor-target bearing cells may be killed by e.g. cytotoxic T cells and not by the antibody binding the tumor target by e.g. induction of CDC.

[0125] A non-activating Fc region may be obtained by modifying one or more of at least five specific amino acid positions in the Fc region. In one embodiment, the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

[0126] Thus, in one embodiment, the antibody comprises a first and a second immunoglobulin heavy chain, wherein in at least one of said first and second immunoglobulin heavy chains one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are not L, L, D, N, and P, respectively.

[0127] Disclosed herein is an antibody, wherein in both the first and second heavy chains one or more amino acids in the position corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are not L, L, D, N, and P, respectively.

[0128] Also disclosed herein is an antibody, wherein in at least one of the first and second heavy chains one or more amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain, are not L, L and D, respectively, and the amino acids in the positions corresponding to N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0129] The term "amino acid corresponding to positions" as used herein refers to an amino acid position number in a human IgG1 heavy chain. Unless otherwise stated or contradicted by context, the amino acids of the constant region sequences are herein numbered according to the Eu-index of numbering (described in

[27] ). Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or at least 95% identity to a human IgG1 heavy chain. It is considered well-known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present invention.

[0130] In the context of the present invention, the amino acid may be defined as described above.

[0131] The term "the amino acid is not" or similar wording when referring to amino acids in a heavy chain is to be understood to mean that the amino acid is any other amino acid than the specific amino acid mentioned. For example, the amino acid in the position corresponding to L234 in a human IgG1 heavy chain is not L, means that the amino acid may be any of the other naturally or non-naturally occurring amino acids than L.

[0132] In one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain, is not D.

[0133] Disclosed herein is an antibody, wherein in at least one of the first and second heavy chains the amino acid in the position corresponding to D265 in a human IgG1 heavy chain, is not D, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0134] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to position D265 in a human IgG1 heavy chain is hydrophobic or polar amino acids.

[0135] The term "hydrophobic" as used herein in relation to an amino acid residue, refers to an amino acid residue selected from the group consisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0136] The term "polar" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of; C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human heavy chain is selected from the group consisting of: C, E, H, K, N, Q, R, S, and T.

[0137] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is an aliphatic uncharged, aromatic or acidic amino acid.

[0138] The term "aliphatic uncharged" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: A, G, I, L, and V. Thus, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group consisting of: A, G, I, L, and V.

[0139] The term "aromatic" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: F, T, and W. Thus, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group consisting of: F, T, and W.

[0140] The term "acidic" as used herein in relation to amino acid residues, refers to any amino acid residue chosen from the group consisting of: D and E. Thus, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group consisting of; D and E.

[0141] In at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group consisting of: A, E, F, G, I, L, T, V, and W.

[0142] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain, is not D.

[0143] Disclosed herein is an antibody, wherein in both the first and second heavy chains the amino acid in the position corresponding to D265 in a human IgG1 heavy chain, is not D, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0144] In both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be hydrophobic or polar amino acid.

[0145] The term "hydrophobic" as used herein in relation to an amino acid residue, refers to an amino acid residue selected from the group consisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group of amino acids consisting of; A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0146] The term "polar" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of; C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human heavy chain is selected from the group consisting of; C, E, H, K, N, Q, R, S, and T. Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0147] In both said first and second heavy chains the amino acids in the positions corresponding to position D265 in a human heavy chain may be selected from the group consisting of; C, E, H, K, N, Q, R, S, and T.

[0148] In both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be aliphatic uncharged, aromatic or acidic amino acids.

[0149] The term "aliphatic uncharged" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: A, G, I, L, and V. Thus, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group consisting of; A, G, I, L, and V.

[0150] The term "aromatic" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: F, T, and W. Thus, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group consisting of; F, T, and W.

[0151] The term "acidic" as used herein in relation to amino acid residues, refers to any amino acid residue chosen from the group consisting of: D and E. Thus, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group consisting of; D and E.

[0152] In both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group consisting of: A, E, F, G, I, L, T, V, and W.

[0153] In further embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position N297 in a human IgG1 heavy chain, is not N.

[0154] Disclosed herein is an antibody, wherein in at least one of the first and second heavy chains the amino acid in the position corresponding to N297 in a human IgG1 heavy chain, is not N, and the amino acid in the position corresponding to position P331 in a human IgG1 heavy chain, is P.

[0155] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acid in the position corresponding to positions N297 in a human IgG1 heavy chain, is not N.

[0156] Disclosed herein is an antibody, wherein in both the first and second heavy chains the amino acid in the position corresponding to N297 in a human IgG1 heavy chain, is not N, and the amino acid in the position corresponding to position P331 in a human IgG1 heavy chain, is P.

[0157] In further embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are not L and L, respectively.

[0158] Disclosed herein is an antibody, wherein in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain, are not L and L, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0159] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of; A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V.

[0160] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0161] The term "hydrophobic" as used herein in relation to an amino acid residue, refers to an amino acid residue selected from the group consisting of: A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain may each be selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.

[0162] The term "polar" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of; C, D, E, H, K, N, Q, R, S, and T. Thus, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain may each be selected from the group of amino acids consisting of: C, D, E, H, K, N, Q, R, S, and T.

[0163] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0164] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are not L and L, respectively.

[0165] Disclosed herein is an antibody, wherein in both the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain, are not L and L, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0166] In both said first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain may be hydrophobic or polar amino acids.

[0167] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.

[0168] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group of amino acids consisting of: C, D, E, H, K, N, Q, R, S, and T.

[0169] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0170] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0171] The term "aliphatic uncharged" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: A, G, I, L, and V. Thus, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain may each be selected from the group consisting of: A, G, I, and V.

[0172] The term "aromatic" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: F, T, and W. Thus, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain may each be selected from the group consisting of: F, T, and W.

[0173] The term "acidic" as used herein in relation to amino acid residues, refers to any amino acid residue chosen from the group consisting of: D and E. Thus, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain may each be selected from the group consisting of: D and E.

[0174] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of: A, D, E, F, G, I, T, V, and W.

[0175] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E; or A and A, respectively.

[0176] Disclosed herein is an antibody, wherein in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain, are F and E; or A and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0177] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E; or A and A, respectively.

[0178] Disclosed herein is an antibody, wherein in both the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain, are F and E; or A and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0179] In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E, respectively.

[0180] In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E, respectively.

[0181] In one embodiment, in at least one of said first and second heavy chains at least the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are A and A, respectively.

[0182] Disclosed herein is an antibody, wherein in both said first and second heavy chains at least the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are A and A, respectively.

[0183] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively.

[0184] Disclosed herein is an antibody, wherein in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain, are not L, L and D, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0185] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of; A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V, and W, and the amino acid corresponding to position D265 is selected from the group consisting of; A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, and W.

[0186] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0187] The term "hydrophobic" as used herein in relation to an amino acid residue, refers to an amino acid residue selected from the group consisting of: A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain may each be selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.

[0188] The term "polar" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: C, D, E, H, K, N, Q, R, S, and T. Thus, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain may each be selected from the group of amino acids consisting of: C, D, E, H, K, N, Q, R, S, and T, the amino acid in the position corresponding to position D265 in a human heavy chain may be selected from the group consisting of: C, E, H, K, N, Q, R, S, and T.

[0189] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of: A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0190] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0191] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.

[0192] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group of amino acids consisting of: C, D, E, H, K, N, Q, R, S, and T, the amino acid in the position corresponding to position D265 in a human heavy chain is selected from the group consisting of: C, E, H, K, N, Q, R, S, and T.

[0193] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of: A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0194] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0195] The term "aliphatic uncharged" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: A, G, I, L, and V. Thus, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain may be selected from the group consisting of: A, G, I, L, and V, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain may each be selected from the group consisting of: A, G, I, and V.

[0196] The term "aromatic" as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: F, T, and W. Thus, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain may be each selected from the group consisting of: F, T, and W.

[0197] The term "acidic" as used herein in relation to amino acid residues, refers to any amino acid residue chosen from the group consisting of: D and E. Thus, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain may each be selected from the group consisting of: D and E.

[0198] Disclosed herein is an antibody, wherein in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, E, F, G, I, L, T, V, and W, and the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of: A, D, E, F, G, I, T, V, and W.

[0199] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively.

[0200] Disclosed herein is an antibody, wherein in both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0201] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0202] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of: A, G, I, L, and V, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of: A, G, I, and V.

[0203] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are each selected from the group consisting of: D and E.

[0204] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of: A, E, F, G, I, L, T, V, and W, and the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of: A, D, E, F, G, I, T, V, and W.

[0205] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively.

[0206] Disclosed herein is an antibody, wherein in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0207] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively.

[0208] Disclosed herein is an antibody, wherein in both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

[0209] In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0210] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0211] In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are A, A, and A, respectively.

[0212] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are A, A, and A, respectively.

[0213] In another embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are F, E, A, Q, and S, respectively.

[0214] Disclosed herein is an antibody, wherein in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are F, E, A, Q, and S, respectively.

[0215] Disclosed herein is an antibody, wherein the antibodycomprises a VH sequence of SEQ ID NOs 177 , a VL sequence as set out in SEQ ID NO:8, and in at least one, or both of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively. The present disclosure provides anti CD3 antibodies with reduced affinity to human CD3 epsilon compared to a reference antibody comprising the VH and VL sequences as set out in SEQ ID NO:4 and 8, and where the antibodies further comprises a non-activating Fc region.

[0216] Disclosed herein is an antibody, wherein the antibody comprises a VH sequence as set out in SEQ ID NO: 177, a VL sequence as set out in SEQ ID NO:10, and in at least one, or both of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively. The present disclosure provides anti CD3 antibodies with reduced affinity to human CD3 epsilon compared to a reference antibody comprising the VH and VL sequences as set out in SEQ ID NO:4 and 8, and where the antibodies further comprises a non-activating Fc region and a VL region that allows for enhanced production.

[0217] Disclosed herein is an antibody, wherein the human IgG1 heavy chain has the IgG1m(f) sequence as set out in SEQ ID NO:407. Further disclosed is an antibody, wherein the amino acids in positons corresponding to postions L234, L235, and D265 in a human IgG1m(f) as set out in SEQ ID NO:407, are F, E, and A, respectively.

[0218] Disclosed herein is an antibody, wherein the human IgG1 heavy chain has the IgG1m(f) sequence as set out in SEQ ID NO:409.

[0219] Disclosed herein is an antibody, which comprises the human IgLC2 / IgLC3 constant domain lambda light chain of SEQ ID NO:408.

[0220] The antibody may be modified in the light chain (LC) and / or heavy chain (HC) to increase the expression level and / or production yield. The antibodies according to the invention may be modified in the light chain (LC). Such modifications are known in the art and may be performed according to the methods described in e.g. Zheng, L., Goddard, J.-P., Baumann, U., & Reymond, J.-L. (2004). Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis. Journal of Molecular Biology, 341(3), 807-14. doi:10.1016 / j.jmb.2004.06.014.

[0221] Disclosed herein is an antibody, which is modified in the VH region and / or the VL region to to reduce the affinity of the antibody. This may be advantageous in some settings and lead to increased efficacy. In particular low affinity of the CD3 arm may have an impact on the motility of T cells in circulation and at tumor site thus leading to better engagement of T cells with tumor cells, cf. MølhØj et al, Molecular Immunology 44 (2007). In particular this may be useful in bispecific formats, in which a CD3 antibody is used as one of the binding arms. Modifications that lead to reduced antibody affinity are known in the art, see for example Webster et al. Int J Cancer Suppl. 1988;3:13-6.

[0222] Disclosed herein is a multispecific antibody comprising at least a first binding region of an antibody according to any aspect or embodiment herein described, and one or more binding regions which binds one or more different targets than the first binding region. Such a multispecific antibody may be a bispecific antibody.

[0223] Thus, in one aspect, the present invention relates to a bispecific antibody comprising a first binding region of an antibody according to any aspect or embodiment herein described, and a second binding region which binds a different target than the first binding region.

[0224] The term "multispecific antibody" refers to an antibody having specificities for at least two different, such as at least three, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types.

[0225] The term "bispecific antibody" refers to an antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types.

[0226] In one embodiment, the bispecific antibody comprises a first and a second heavy chain.

[0227] The embodiments relating to modification of the Fc region and embodiments relating to specific amino acid substitutions are contemplated to be part of any bispecific antibody according to the invention. Thus, at least one of the first and second heavy chains may comprise one or more amino acids modified as defined in any embodiment herein described, such as those described to in relation to providing an inert Fc region. Disclosed herein is an antibody, wherein both said first and second heavy chains comprise one or more amino acids modified as defined in any embodiment herein described, such as those described to in relation to providing an inert Fc region. Accordingly, the bispecific antibody may comprise an Fc region modified according to any aspect or embodiment herein described; or at least one of said first and second heavy chains may comprise one or more amino acids modified as defined in any aspect or embodiment herein described.

[0228] Examples of bispecific antibody molecules which may be used in the present invention comprise (i) a single antibody that has two arms comprising different antigen-binding regions, (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-Ig ™< ), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (

[40] ); (iv) a chemically-linked bispecific (Fab')2 fragment; (v) a TandAb ®< , which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (vii) a so called "dock and lock" molecule (Dock-and-Lock ®< ), based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (viii) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody.

[0229] The bispecific antibody of the present invention may be a diabody, a cross-body, or a bispecific antibody obtained via a controlled Fab arm exchange, e.g. DuoBody ®< (such as described in

[41] ) as those described in the present invention.

[0230] Examples of different classes of bispecific antibodies include but are not limited to (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; (v) Fab fusion molecules, wherein different Fab-fragments are fused together, fused to heavy-chain constant-domains, Fc-regions or parts thereof; and (vi) ScFv-and diabody-based and heavy chain antibodies (e.g., domain antibodies, Nanobodies ®< ) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, Nanobodies ®< ) are fused to each other or to another protein or carrier molecule fused to heavy-chain constant-domains, Fc-regions or parts thereof.

[0231] Examples of IgG-like molecules with complementary CH3 domains molecules include but are not limited to the Triomab ®< (Trion Pharma / Fresenius Biotech,

[42] ), the Knobs-into-Holes (Genentech,

[43] ), CrossMAbs (Roche,

[44] ) and the electrostatically-matched (Amgen,

[45] -

[46] ; Chugai,

[47] ; Oncomed,

[48] ), the LUZ-Y (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG-body and PIG-body (Pharmabcine, WO2010134666, WO2014081202), the Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono,

[49] ), the Biclonics (Merus, WO2013157953), FcΔAdp (Regeneron,

[50] ), bispecific IgG1 and IgG2 (Pfizer / Rinat,

[51] ), Azymetric scaffold (Zymeworks / Merck,

[52] ), mAb-Fv (Xencor,

[53] ), bivalent bispecific antibodies (Roche, WO2009080254) and DuoBody ®< molecules (Genmab A / S,

[41] ).

[0232] Examples of recombinant IgG-like dual targeting molecules include but are not limited to Dual Targeting (DT)-Ig (GSK / Domantis, WO2009058383), Two-in-one Antibody (Genentech, Bostrom, et al 2009. Science 323, 1610-1614), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star,

[54] ), Zybodies ™< (Zyngenia, LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), approaches with common light chain (Crucell / Merus,

[55] ), κλBodies (NovImmune, WO2012023053) and CovX-body ®< (CovX / Pfizer, Doppalapudi, V.R., et al 2007. Bioorg. Med. Chem. Lett. 17,501-506).

[0233] Examples of IgG fusion molecules include but are not limited to Dual Variable Domain (DVD)-Ig ™< (Abbott,

[56] ), Dual domain double head antibodies (Unilever; Sanofi Aventis,

[57] ), IgG-like Bispecific (ImClone / Eli Lilly, Lewis etal. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ, Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb (Zymogenetics, WO2010111625), HERCULES (Biogen Idec,

[0234]

[58] ), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc,

[59] ) and TvAb (Roche,

[59] ,

[60] ).

[0235] Examples of Fc fusion molecules include but are not limited to ScFv / Fc Fusions (Academic Institution, Pearce et al Biochem Mol Biol Int. 1997 Sep;42(6):1179-88.), SCORPION (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100 th Annual meeting 2009 (Abstract # 5465); Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART ™< ) (MacroGenics,

[62] ,

[63] ) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine - China).

[0236] Examples of Fab fusion bispecific antibodies include but are not limited to F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock ®< (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).

[0237] Examples of ScFv-, diabody-based and domain antibodies include but are not limited to Bispecific T Cell Engager (BiTE ®< ) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART ™< ) (MacroGenics), Single-chain Diabody (Academic, Lawrence FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75), dual targeting nanobodies ®< (Ablynx, Hmila et al., FASEB J. 2010), dual targeting heavy chain only domain antibodies.

[0238] It is further contemplated that any monospecific antibody fulfilling the assay conditions herein described may form the basis of a bispecific antibody. I.e. a bispecific antibody wherein one of the binding regions binds CD3 may originate from any monospecific CD3 antibody tested in the functional assays and fulfilling the requirements stated herein. Such a bispecific antibody may be provided by the methods described in

[41] .

[0239] The bispecific antibody of the invention may comprise a first Fc-region comprising a first CH3 region, and a second Fc-region comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab).

[0240] Thus, in a particular embodiment, each of said first and second heavy chain comprises at least a hinge region, a CH2 and CH3 region, wherein in said first heavy chain at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and in said second heavy chain at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and wherein said first and said second heavy chains are not substituted in the same positions. In this context the term "substituted", refers to that the amino acid in a specific amino acid position has been substituted with another naturally or non-naturally occurring amino acid. Thus, a "substituted" amino acid in a position corresponding to the position in a human IgG1 heavy chain means the amino acid at the particular position is different from the naturally occurring amino acid in an IgG1 heavy chain.

[0241] Disclosed herein is an antibody, wherein in said first heavy chain the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is not K, L or M, and optionally the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is F, and in said second heavy chain at least one of the amino acids in the positions corresponding to a position selected from the group consisting of: T366, L368, K370, D399, F405, and Y407 in a human IgG1 heavy chain has been substituted.

[0242] Disclosed herein is an antibody, wherein in said first heavy chain the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is not K, L or M, and in said second heavy chain the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is not F and optionally the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is K.

[0243] Disclosed herein is an antibody, wherein in said first heavy chain, the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is not F, R, and G, and in said second heavy chain the amino acids in the positions corresponding to a position selected form the group consisting of: T366, L368, K370, D399, Y407, and K409 in a human IgG1 heavy chain has been substituted.

[0244] Disclosed herein is an antibody, wherein the amino acid in position corresponding to K409 in a human IgG1 heavy chain is not K, L or M in said first heavy chain, and the amino acid in position corresponding to F405 in a human IgG1 heavy chain is not F.

[0245] In a further embodiment, the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in said first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in said second heavy chain, or vice versa.

[0246] Disclosed herein is an antibody, wherein the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in the first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in the second heavy chain.

[0247] The humanized CD3 antibody of the invention may contain in at least one of the first and second heavy chain one or more of the inactivating substitutions as disclosed in any one of the above embodiments, such as L234F, L235E, and D265A; and that the amino acid in the position corresponding to F405 may not be F. The humanized CD3 antibody of the invention may contain in at least one of the first and second heavy chain one or more of the inactivating substitutions as disclosed in any one of the above embodiments, such as L234F, L235E, and D265A; and a further substitution in the K409 position, such as K409R. In particularthe humanized CD3 antibody of the invention may contain in both the first and second heavy chain one or more of the inactivating substitutions as disclosed in any one of the above embodiments, such as L234F, L235E, and D265A; and a substitution in the F405 position, such as F405L. The humanized CD3 antibody of the invention may contain in both the first and second heavy chain one or more of the inactivating substitutions as disclosed in any one of the above embodiments, such as L234F, L235E, and D265A; and a further substitution in the K409 position, such as K409R. Such antibodies are useful for generating a bispecific antibody.

[0248] Accordingly, in at least one of the first and second heavy chains the amino acids in the positions corresponding to position L234, L235, and D265 in a human IgG1 heavy chain may be F, E, and A, respectively, the amino acid in the position corresponding to F405 in a human IgG1 heavy chain may be L in the first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain may be R in the second heavy chain.

[0249] In at least one of the first and second heavy chains the amino acids in the positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain may be F, E, A, N, and P respectively, the amino acid in the position corresponding to F405 in a human IgG1 heavy chain may be L in the first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain may be R in the second heavy chain.

[0250] In at least one of the first and second heavy chains the amino acids in the positions corresponding to position L234, L235, and D265 in a human IgG1 heavy chain may be F, E, and A, respectively, the amino acid in the position corresponding to K409 in a human IgG1 heavy chain may be R in the first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain may be L in the second heavy chain.

[0251] In at least one of the first and second heavy chains the amino acids in the positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain may be F, E, A, N, and P respectively, the amino acid in the position corresponding to K409 in a human IgG1 heavy chain may be R in the first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain may be L in the second heavy chain.

[0252] In both the first and second heavy chains the amino acids in the positions corresponding to position L234, L235, and D265 in a human IgG1 heavy chain may be F, E, and A, respectively, the amino acid in the position corresponding to F405 in a human IgG1 heavy chain may be L in the first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain may be R in the second heavy chain.

[0253] In both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain may be F, E, A, N, and P respectively, the amino acid in the position corresponding to F405 in a human IgG1 heavy chain may be L in the first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain may be R in the second heavy chain.

[0254] In in both the first and second heavy chains the amino acids in the positions corresponding to position L234, L235, and D265 in a human IgG1 heavy chain may be F, E, and A, respectively, the amino acid in the position corresponding to K409 in a human IgG1 heavy chain may be R in the first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain may be L in the second heavy chain.

[0255] In both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain may be F, E, A, N, and P respectively, the amino acid in the position corresponding to K409 in a human IgG1 heavy chain may be R in the first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain may be L in the second heavy chain.

[0256] As described herein, T cell recruitment to specific target cells, such as cancer or tumor cells, provides a way of killing the target cells. T cell mediated killing may be obtained by a bispecific antibody targeting CD3 with the first binding region and another target with the second binding region. Thus, the first binding region may be according to any embodiments described herein for the humanized CD3 antibody, and the second binding region may bind a different target than the first binding region. It is to be understood that when the antibody is a bispecific antibody, at least one half of the antibody, i.e. one of the pair of heavy and light chains of the antibody, is a humanized antibody as herein described. Thus, one half of the bispecific antibody may be a humanized antibody binding CD3 according to the present invention and the other half may be humanized, chimeric, fully non-human or fully human binding a second target. Thus, the antibody may comprise a first and a second heavy chain, a first and second light chain, wherein said first heavy and said first light chains are humanized and are connected via disulfide bridges forming a first binding region; and said second heavy and light chains are fully human and are connected via disulfide bridges forming a second binding region, wherein said first binding region is according to any aspect or embodiment herein described, and said second binding region binds a different target. The antibody may comprise a first and a second heavy chain, a first and second light chain, wherein said first heavy and said first light chains are humanized and are connected via disulfide bridges forming a first binding region; and said second heavy and light chains are humanized and are connected via disulfide bridges forming a second binding region, wherein said first binding region is according to any aspect or embodiment herein described, and said second binding region binds a different epitope of CD3 than said first binding region.

[0257] The term "disulfide bridges" as used herein refers to the covalent bond between two Cysteine residues, i.e. said interaction may also be designated a Cys-Cys interaction.

[0258] The term "target" as used herein, refers to a molecule to which the binding region of the antibody according to the invention binds. When used in the context of the binding of an antibody the term includes any antigen towards which the raised antibody is directed.

[0259] The first heavy and the first light chains may be humanized and connected via disulfide bridges forming a first binding region; and the second heavy and light chains may be fully human and connected via disulfide bridges forming a second binding region, wherein the first binding region is according to any aspect or embodiment herein described, and the second binding region binds a different target; and wherein in at least one of the first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0260] The first heavy and the first light chains may be humanized and connected via disulfide bridges forming a first binding region; and the second heavy and light chains may be fully human and connected via disulfide bridges forming a second binding region, wherein the first binding region is according to any aspect or embodiment herein described, and the second binding region binds a different epitope of CD3 than the first binding region; and wherein in at least one of the first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0261] The first heavy and the first light chains may be humanized and connected via disulfide bridges forming a first binding region; and the second heavy and light chains may be fully human and connected via disulfide bridges forming a second binding region, wherein the first binding region is according to any aspect or embodiment herein described, and the second binding region binds a different target; and wherein in both the first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

[0262] The first heavy and the first light chains may be humanized and connected via disulfide bridges forming a first binding region; and the second heavy and light chains may be fully human and connected via disulfide bridges forming a second binding region, wherein the first binding region is according to any aspect or embodiment herein described, and the second binding region binds a different epitope of CD3 than the first binding region; and wherein in both the first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.Expression vectors, and host cells

[0263] In one embodiment, the invention provides an expression vector comprising (i) a nucleic acid sequence encoding a heavy chain sequence of a humanized antibody according to the invention; and (ii) a nucleic acid sequence encoding a light chain sequence of a humanized antibody according to the invention. Thus, the expression vector may comprise one or more nucleic acid constructs or nucleic acid sequences according to any aspect or embodiment herein described.

[0264] The expression vector may further comprise a nucleic acid sequence encoding the constant region of a light chain, a heavy chain or both light and heavy chains of an antibody, e.g. a human antibody.

[0265] Such expression vectors as described above may be used for recombinant production of antibodies of the invention.

[0266] An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a humanized CD3 antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in for instance

[64] ), a compacted nucleic acid vector (as described in for instance

[65] and / or

[66] ), a plasmid vector such as pBR322, pUC 19 / 18, or pUC 118 / 119, a "midge" minimally-sized nucleic acid vector (as described in for instance

[67] ), or as a precipitated nucleic acid vector construct, such as a CaPO 4 -< -precipitated construct (as described in for instance

[68] ,

[69] ,

[70] , and

[71] ). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance

[72] and

[73] ).

[0267] The vector may be suitable for expression of the humanized CD3 antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (

[74] ), pET vectors (Novagen, Madison WI) and the like.

[0268] An expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in:

[75] and

[76] ).

[0269] A nucleic acid construct and / or vector may also comprise a nucleic acid sequence encoding a secretion / localization sequence, which can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides, organelle-targeting sequences (e. g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization / anchor sequences (e. g., stop transfer sequences, GPI anchor sequences), and the like which are well-known in the art.

[0270] In an expression vector of the invention, humanized CD3 antibody-encoding nucleic acids may comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e. g., human CMV IE promoter / enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as selectable marker, and / or a convenient cloning site (e.g., a polylinker). Nucleic acid constructs and / or vectors may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE (the skilled person will recognize that such terms are actually descriptors of a degree of gene expression under certain conditions).

[0271] The humanized CD3 antibody-encoding expression vector may be positioned in and / or delivered to the host cell or host animal via a viral vector.

[0272] Such expression vectors may be used for recombinant production of humanized CD3 antibodies.

[0273] In one aspect, the invention provides a host cell comprising an expression vector according to the invention.

[0274] In one aspect, the humanized CD3 antibodies of any aspect or embodiment described herein are provided by use of recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell which produces the antibody. Accordingly, the invention provides a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell, which produces a humanized CD3 antibody or immunoglobulin as defined herein. Examples of host cells include yeast, bacterial and mammalian cells, such as CHO or HEK-293 cells. For example, the host cell may comprise a nucleic acid sequence stably integrated into the cellular genome that comprises a sequence coding for expression of a humanized CD3 antibody described herein.The host cell may comprise a non-integrated nucleic acid sequence, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of a humanized CD3 antibody described herein.

[0275] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which an expression vector or nucleic acid construct or sequence has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, eukaryotic host cells, such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi.

[0276] In a further aspect, the invention relates to a method for producing a humanized CD3 antibody of the invention, said method comprising the steps of a) culturing a host cell of the invention as described herein above, and b) purifying the antibody of the invention from the culture media.

[0277] The nucleotide sequence encoding a sequence of a humanized CD3 antibody may further encode a second moiety, such as a therapeutic polypeptide. Exemplary therapeutic polypeptides are described elsewhere herein. Disclosed herein is a method for producing a humanized CD3 antibody fusion protein, said method comprising the steps of a) culturing a host cell comprising an expression vector comprising such a nucleotide sequence, and b) retrieving and / or purifying the humanized CD3 antibody fusion protein from the culture media. Compositions

[0278] In one aspect, the invention provides a composition comprising the antibody or bispecific antibody according to any aspect and embodiment herein described.

[0279] In one aspect, the invention provides a pharmaceutical composition comprising the antibody or bispecific antibody as defined in any one of the aspects and embodiments herein described, and a pharmaceutically acceptable carrier.

[0280] The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in

[77] .

[0281] The pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the humanized antibody of the present invention and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the chosen compound or pharmaceutical composition of the present invention (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.)) on antigen binding.

[0282] A pharmaceutical composition of the present invention may also include diluents, fillers, salts, buffers, detergents (e. g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition.

[0283] The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0284] The pharmaceutical composition may be administered by any suitable route and mode. Suitable routes of administering a humanized antibody of the present invention in vivo and in vitro are well known in the art and may be selected by those of ordinary skill in the art.

[0285] A pharmaceutical composition of the present invention is administered parenterally.

[0286] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-orbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.

[0287] The pharmaceutical composition may be administered by intravenous or subcutaneous injection or infusion.

[0288] In a preferred embodiment the pharmaceutical compostion is administered subcutaneous.

[0289] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption-delaying agents, and the like that are physiologically compatible with a humanized antibody of the present invention.

[0290] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical arts.

[0291] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated. When referring to the "active compound" it is contemplated to also refer to the humanized antibody according to the present invention.

[0292] Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0293] Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0294] Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.

[0295] The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The humanized antibody of the present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and micro-encapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly-orthoesters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art (see e.g.,

[78] ).

[0296] The humanized antibody of the present invention may be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated. Other active or therapeutic compounds may also be incorporated into the compositions.

[0297] Pharmaceutical compositions for injection must typically be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, micro-emulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be an aqueous or a non-aqueous solvent or dispersion medium containing for instance water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0298] Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum-drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.Therapeutic applications

[0299] In another aspect, the present invention relates to a humanized antibody, or pharmaceutical composition of the invention as defined in any aspect or embodiment herein described, for use as a medicament.

[0300] In another aspect, the present invention relates to a humanized antibody, or pharmaceutical composition of the invention as defined in any aspect or embodiment herein described, for use in the treatment of a disease.

[0301] The bispecific antibody, the composition, and the pharmaceutical composition may be for use in the treatment of a disease.

[0302] In one embodiment of the present invention, the bispecific antibody, the composition or the pharmaceutical composition is for use for the treatment of a disease, wherein the disease is cancer, an infectious disease, or autoimmune diseases.

[0303] The humanized antibody or pharmaceutical composition of the invention can be used as in the treatment of any cancer wherein the effector mechanisms of cytotoxic T cells are desired. For example, the humanized antibody may be administered to cells in culture, e.g., in vitro or ex vivo, or to human subjects, e.g. in vivo, to treat or prevent disorders such as cancer, inflammatory or autoimmune disorders. As used herein, the term "subject" is typically a human which respond to the humanized antibody, or pharmaceutical composition. Subjects may for instance include human patients having disorders that may be corrected or ameliorated by modulating a target function or by leading to killing of the cell, directly or indirectly.

[0304] In another aspect, the present invention relates to the use as defined in any aspect or embodiments herein described wherein the humanized antibody is a bispecific antibody specifically binding to both CD3 and a cancer-specific target, or a target that is overexpressed in cancer or associated with cancer, such as HER2, CD19, EpCAM, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2 or MCSP (or HMW-MAA), CD20 and wherein the disease is cancer, such as breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer and squamous cell carcinoma of the head & neck, cervical cancer, pancreatic cancer, testis cancer, malignant melanoma, a soft-tissue cancer (e.g., synovial sarcoma), an indolent or aggressive form of B-cell lymphoma, chronic lymphatic leukemia or acute lymphatic leukemia.

[0305] The efficient dosages and dosage regimens for the humanized antibody depend on the disease or condition to be treated and may be determined by the persons skilled in the art.

[0306] A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of the humanized antibody employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the humanized antibody which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above.

[0307] For example, an "effective amount" for therapeutic use may be measured by its ability to stabilize the progression of disease. The ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition may be evaluated by examining the ability of the humanized antibody to inhibit cell growth or to induce cytotoxicity by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound, i.e. a therapeutic humanized antibody, or pharmaceutical composition according to the invention, may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0308] An exemplary, non-limiting range for a therapeutically effective amount of a humanized antibody of the invention is about 0.001-30 mg / kg, such as about 0.001-20 mg / kg, such as about 0.001-10 mg / kg, such as about 0.001-5 mg / kg, for example about 0.001-2 mg / kg, such as about 0.001-1 mg / kg, for instance about 0.001, about 0.01, about 0.1, about 1, about 5, about 8, about 10, about 12, about 15, about 18 mg / kg.

[0309] Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target.

[0310] Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0311] The efficacy of the treatment may be monitored during the therapy, e.g. at predefined points in time.

[0312] If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In another embodiment, the humanized antibody, or pharmaceutical composition is administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.

[0313] While it is possible for a humanized antibody of the present invention to be administered alone, it is preferable to administer the humanized antibody as a pharmaceutical composition as described above.

[0314] An effective dose of a humanized antibody of the invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. Alternatively, an effective dose of a humanized antibody of the invention may be administered every second, third or fourth week.

[0315] The humanized antibody may be administered by infusion in a weekly dosage of calculated by mg / m 2< . Such dosages can, for example, be based on the mg / kg dosages provided above according to the following: dose (mg / kg) x 70: 1.8. Such administration may be repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours.The humanized antibody may be administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.

[0316] The humanized antibody may be administered in a weekly dosage of calculated as a fixed dose for up to 8 times, such as from 4 to 6 times when given once a week. Such regimen may be repeated one or more times as necessary, for example, after 6 months or 12 months. Such fixed dosages can, for example, be based on the mg / kg dosages provided above, with a body weight estimate of 70 kg. The dosage may be determined or adjusted by measuring the amount of humanized antibody of the present invention in the blood upon administration by for instance taking out a biological sample and using anti-idiotypic antibodies which target the binding region of the humanized antibodies of the present invention.

[0317] In one embodiment, the humanized antibody may be administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.

[0318] A humanized antibody may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and / or reduce the risk of recurrence when a cancer is in remission.

[0319] Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0320] A humanized antibody may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and / or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in patients wherein it is difficult to locate a tumor that is known to be present due to other biological factors.Diagnostic applications

[0321] The humanized antibody of the invention may also be used for diagnostic purposes, using a composition comprising a humanized antibody as described herein. Accordingly, the invention provides diagnostic methods and compositions using the humanized antibodies described herein. Such methods and compositions can be used for purely diagnostic purposes, such as detecting or identifying a disease, as well as for monitoring of the progress of therapeutic treatments, monitoring disease progression, assessing status after treatment, monitoring for recurrence of disease, evaluating risk of developing a disease, and the like.

[0322] The humanized antibody of the present invention may be used ex vivo, such as in diagnosing a disease in which cells expressing a specific target of interest and to which the humanized antibody binds, are indicative of disease or involved in the pathogenesis, by detecting levels of the target or levels of cells which express the target of interest on their cell surface in a sample taken from a patient. This may be achieved, for example, by contacting the sample to be tested, optionally along with a control sample, with the humanized antibody according to the invention under conditions that allow for binding of the antibody to the target. Complex formation can then be detected (e.g., using an ELISA). When using a control sample along with the test sample, the level of humanized antibody or antibody-target complex is analyzed in both samples and a statistically significant higher level of humanized antibody or antibody-target complex in the test sample indicates a higher level of the target in the test sample compared with the control sample.

[0323] Examples of conventional immunoassays in which humanized antibodies of the present invention can be used include, without limitation, ELISA, RIA, FACS assays, plasmon resonance assays, chromatographic assays, tissue immunohistochemistry, Western blot, and / or immunoprecipitation.

[0324] In one embodiment, the invention relates to a method for detecting the presence of a target, or a cell expressing the target, in a sample comprising: contacting the sample with a humanized antibody of the invention under conditions that allow for binding of the humanized antibody to the target in the sample; and analyzing whether a complex has been formed. Typically, the sample is a biological sample.

[0325] The sample may be a tissue sample known or suspected of containing a specific target and / or cells expressing the target. For example, in situ detection of the target expression may be accomplished by removing a histological specimen from a patient, and providing the humanized antibody of the present invention to such a specimen. The humanized antibody may be provided by applying or by overlaying the humanized antibody to the specimen, which is then detected using suitable means. It is then possible to determine not only the presence of the target or target-expressing cells, but also the distribution of the target or target-expressing cells in the examined tissue (e.g., in the context of assessing the spread of cancer cells). Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) may be modified in order to achieve such in situ detection.

[0326] In the above assays, the humanized antibody can be labeled with a detectable substance to allow bound antibody to be detected. Alternatively, bound (primary) specific humanized antibody may be detected by an antibody which is labeled with a detectable substance and which binds to the primary specific humanized antibody. Furthermore, in the above assays, a diagnostic composition comprising an antibody or bispecific antibody according to any aspect or embodiments herein described may be used. Thus, in one aspect, the present invention relates to a diagnostic composition comprising an antibody or bispecific antibody according to any aspect or embodiment herein described.

[0327] The level of target in a sample can also be estimated by a competition immunoassay utilizing target standards labeled with a detectable substance and an unlabeled target-specific humanized antibody. In this type of assay, the biological sample, the labeled target standard(s) and the target-specific humanized antibody are combined, and the amount of labeled target standard bound to the unlabeled target-specific humanized antibody is determined. The amount of target in the biological sample is inversely proportional to the amount of labeled target standard bound to the target-specific humanized antibody.

[0328] Suitable labels for the target-specific humanized antibody, secondary antibody and / or target standard used in in vitro diagnostic techniques include, without limitation, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125< I, 131< I, 35< S, and 3< H.

[0329] The target-specific humanized antibody of the invention may be used in the in vivo imaging of target-expressing tissues such as tumors. For in vivo methods, antibody fragments such as, e.g., (Fab') 2 , Fab and Fab' fragments, are particularly advantageous because of their rapid distribution kinetics.

[0330] In vivo imaging can be performed by any suitable technique. For example, a target-specific humanized antibody (e.g., an antibody or a fragment) labeled with 99< Tc, 131< I, 111< In or other gamma-ray emitting isotope may be used to image target-specific antibody accumulation or distribution in target-expressing tissues such as tumors with a gamma scintillation camera (e.g., an Elscint Apex 409ECT device), typically using low-energy, high resolution collimator or a low-energy all-purpose collimator. Alternatively, labeling with 89< Zr, 76< Br, 18< F or other positron-emitting radionuclide may be used to image target-specific humanized antibody, or antibody fragment distribution in tumors using positron emission tomography (PET). The images obtained by the use of such techniques may be used to assess biodistribution of target in a patient, mammal, or tissue, for example in the context of using target as a biomarker for the presence of cancer / tumor cells. Variations on this technique may include the use of magnetic resonance imaging (MRI) to improve imaging over gamma camera techniques. Conventional immunoscintigraphy methods and principles are described in, e.g.,

[79] ,

[80] , and

[81] . Moreover, such images may also, or alternatively, serve as the basis for surgical techniques to remove tumors. Furthermore, such in vivo imaging techniques may allow for the identification and localization of a tumor in a situation where a patient is identified as having a tumor (due to the presence of other biomarkers, metastases, etc.), but the tumor cannot be identified by traditional analytical techniques.

[0331] In one aspect, the present invention relates to a diagnostic composition comprising an antibody or bispecific antibody according to the invention.

[0332] In a further aspect, the invention relates to a kit for detecting the presence of target antigen or a cell expressing the target, in a sample, comprising: a target-specific humanized antibody of the invention; and instructions for use of the kit.

[0333] Provided herein is a kit for detecting the presence of a CD3 antigen, or a cell expressing CD3, in a sample comprising the steps of; a) contacting the sample with an antibody or bispecific antibody according to the invention, under conditions that allow for formation of a complex between the antibody or bispecific antibody and CD3; and b) analyzing whether a complex has been formed.

[0334] Provided herein is a kit for diagnosis of cancer comprising a container comprising a target-specific humanized antibody, and one or more reagents for detecting binding of the target-specific humanized antibody to the target. Reagents may include, for example, fluorescent tags, enzymatic tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions, wherein the enzymatic reactions produce a product that may be visualized. In one embodiment, the present invention provides a diagnostic kit comprising one or more target-specific humanized antibodies of the present invention in labeled or unlabeled form in suitable container(s), reagents for the incubations for an indirect assay, and substrates or derivatizing agents for detection in such an assay, depending on the nature of the label. Control reagent(s) and instructions for use also may be included.

[0335] Diagnostic kits may also be supplied for use with a target-specific humanized antibody, such as a labeled target-specific antibody, for the detection of the presence of the target in a tissue sample or host. In such diagnostic kits, as well as in kits for therapeutic uses described elsewhere herein, a target-specific humanized antibody typically may be provided in a lyophilized form in a container, either alone or in conjunction with additional antibodies specific for a target cell or peptide. Typically, a pharmaceutically acceptable carrier (e.g., an inert diluent) and / or components thereof, such as a Tris, phosphate, or carbonate buffer, stabilizers, preservatives, biocides, inert proteins, e.g., serum albumin, or the like, also are included (typically in a separate container for mixing) and additional reagents (also typically in separate container(s)). In certain kits, a secondary antibody capable of binding to the target-specific humanized antibody, which typically is present in a separate container, is also included. The second antibody is typically conjugated to a label and formulated in a manner similar to the target-specific humanized antibody of the present invention. Using the methods described above and elsewhere herein, target-specific humanized antibodies may be used to define subsets of cancer / tumor cells and characterize such cells and related tumor tissues.Anti-idiotypic antibodies

[0336] In a further aspect, the invention relates to an anti-idiotypic antibody which binds to a humanized antibody of the invention as described herein or a bispecific antibody according to the invention.

[0337] An anti-idiotypic (Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An anti-Id antibody may be prepared by immunizing an animal of the same species and genetic type as the source of a CD3 monoclonal antibody with the monoclonal antibody to which an anti-Id is being prepared. The immunized animal typically can recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). Such antibodies are described in for instance US 4,699,880. Such antibodies are further features of the present invention.

[0338] An anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. An anti-anti-Id antibody may be epitopically identical to the original monoclonal antibody, which induced the anti-Id antibody. Thus, by using antibodies to the idiotypic determinants of a monoclonal antibody, it is possible to identify other clones expressing antibodies of identical specificity. Anti-Id antibodies may be varied (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein with respect to CD3-specific antibodies of the present invention. For example, a monoclonal anti-Id antibody may be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Sera from these mice typically will contain anti-anti-Id antibodies that have the binding properties similar, if not identical, to an original / parent CD3 antibody.Sequences

[0339] Table 1 SEQ ID NO: Clone name Sequence SEQ ID NO:1VH-huCD3-H1 CDR1GFTFNTYASEQ ID NO:2VH-huCD3-H1 CDR2IRSKYNNYATSEQ ID NO:3VH-huCD3-H1 CDR3VRHGNFGNSYVSWFAYSEQ ID NO:4VH-huCD3-H1SEQ ID NO:5VH-huCD3-H1SEQ ID NO:6VL-huCD3-L1 CDR1TGAVTTSNYVL-huCD3-L1 CDR2GTNSEQ ID NO:7VL-huCD3-L1 CDR3ALWYSNLWVSEQ ID NO:8VL-huCD3-L1SEQ ID NO:9VL-huCD3-L1SEQ ID NO:6VL-huCD3-L1-T41K CDR1TGAVTTSNYVL-huCD3-L1-T41K CDR2GTNSEQ ID NO:7VL-huCD3-L1-T41K CDR3ALWYSNLWVSEQ ID NO:10VL-huCD3-L1-T41KSEQ ID NO:11VL-huCD3-L1-T41KSEQ ID NO:12HC_N30A CDR1GFTFATYASEQ ID NO:2HC_N30A CDR2IRSKYNNYATSEQ ID NO:3HC_N30A CDR3VRHGNFGNSYVSWFAYSEQ ID NO:13HC_N30ASEQ ID NO:14HC_N30C CDR1GFTFCTYASEQ ID NO:2HC_N30C CDR2IRSKYNNYATSEQ ID NO:3HC_N30C CDR3VRHGNFGNSYVSWFAYSEQ ID NO:15HC_N30CSEQ ID NO:16HC_N30D CDR1GFTFDTYASEQ ID NO:2HC_N30D CDR2IRSKYNNYATSEQ ID NO:3HC_N30D CDR3VRHGNFGNSYVSWFAYSEQ ID NO:17HC_N30DSEQ ID NO:18HC_N30F CDR1GFTFFTYASEQ ID NO:2HC_N30F CDR2IRSKYNNYATSEQ ID NO:3HC_N30F CDR3VRHGNFGNSYVSWFAYSEQ ID NO:19HC_N30FSEQ ID NO:20HC_N30G CDR1GFTFGTYASEQ ID NO:2HC_N30G CDR2IRSKYNNYATSEQ ID NO:3HC_N30G CDR3VRHGNFGNSYVSWFAYSEQ ID NO:21HC_N30GSEQ ID NO:22HC_N30H CDR1GFTFHTYASEQ ID NO:2HC_N30H CDR2IRSKYNNYATSEQ ID NO:3HC_N30H CDR3VRHGNFGNSYVSWFAYSEQ ID NO:23HC_N30HSEQ ID NO:24HC_N30K CDR1GFTFKTYASEQ ID NO:2HC_N30K CDR2IRSKYNNYATSEQ ID NO:3HC_N30K CDR3VRHGNFGNSYVSWFAYSEQ ID NO:25HC_N30KSEQ ID NO:26HC_N30L CDR1GFTFLTYASEQ ID NO:2HC_N30L CDR2IRSKYNNYATSEQ ID NO:3HC_N30L CDR3VRHGNFGNSYVSWFAYSEQ ID NO:27HC_N30LSEQ ID NO:28HC_N30P CDR1GFTFPTYASEQ ID NO:2HC_N30P CDR2IRSKYNNYATSEQ ID NO:3HC_N30P CDR3VRHGNFGNSYVSWFAYSEQ ID NO:29HC_N30PSEQ ID NO:30HC_N30Q CDR1GFTFQTYASEQ ID NO:2HC_N30Q CDR2IRSKYNNYATSEQ ID NO:3HC_N30Q CDR3VRHGNFGNSYVSWFAYSEQ ID NO:31HC_N30QSEQ ID NO:32HC_N30R CDR1GFTFRTYASEQ ID NO:2HC_N30R CDR2IRSKYNNYATSEQ ID NO:3HC_N30R CDR3VRHGNFGNSYVSWFAYSEQ ID NO:33HC_N30RSEQ ID NO:34HC_N30T CDR1GFTFTTYASEQ ID NO:2HC_N30T CDR2IRSKYNNYATSEQ ID NO:3HC_N30T CDR3VRHGNFGNSYVSWFAYSEQ ID NO:35HC_N30TSEQ ID NO:36HC_N30V CDR1GFTFVTYASEQ ID NO:2HC_N30V CDR2IRSKYNNYATSEQ ID NO:3HC_N30V CDR3VRHGNFGNSYVSWFAYSEQ ID NO:37HC_N30VSEQ ID NO:38HC_N30W CDR1GFTFWTYASEQ ID NO:2HC_N30W CDR2IRSKYNNYATSEQ ID NO:3HC_N30W CDR3VRHGNFGNSYVSWFAYSEQ ID NO:39HC_N30WSEQ ID NO:40HC_T31A CDR1GFTFNAYASEQ ID NO:2HC_T31A CDR2IRSKYNNYATSEQ ID NO:3HC_T31A CDR3VRHGNFGNSYVSWFAYSEQ ID NO:41HC_T31ASEQ ID NO:42HC_T31C CDR1GFTFNCYASEQ ID NO:2HC_T31C CDR2IRSKYNNYATSEQ ID NO:3HC_T31C CDR3VRHGNFGNSYVSWFAYSEQ ID NO:43HC_T31CSEQ ID NO:44HC_T31D CDR1GFTFNDYASEQ ID NO:2HC_T31D CDR2IRSKYNNYATSEQ ID NO:3HC_T31D CDR3VRHGNFGNSYVSWFAYSEQ ID NO:45HC_T31DSEQ ID NO:46HC_T31E CDR1GFTFNEYASEQ ID NO:2HC_T31E CDR2IRSKYNNYATSEQ ID NO:3HC_T31E CDR3VRHGNFGNSYVSWFAYSEQ ID NO:47HC_T31ESEQ ID NO:48HC_T31F CDR1GFTFNFYASEQ ID NO:2HC_T31F CDR2IRSKYNNYATSEQ ID NO:3HC_T31F CDR3VRHGNFGNSYVSWFAYSEQ ID NO:49HC_T31FSEQ ID NO:50HC_T31H CDR1GFTFNHYASEQ ID NO:2HC_T31H CDR2IRSKYNNYATSEQ ID NO:3HC_T31H CDR3VRHGNFGNSYVSWFAYSEQ ID NO:51HC_T31HSEQ ID NO:52HC_T31L CDR1GFTFNLYASEQ ID NO:2HC_T31L CDR2IRSKYNNYATSEQ ID NO:3HC_T31L CDR3VRHGNFGNSYVSWFAYSEQ ID NO:53HC_T31LSEQ ID NO:54HC_T31M CDR1GFTFNMYASEQ ID NO:2HC_T31M CDR2IRSKYNNYATSEQ ID NO:3HC_T31M CDR3VRHGNFGNSYVSWFAYSEQ ID NO:55HC_T31MSEQ ID NO:56HC_T31N CDR1GFTFNNYASEQ ID NO:2HC_T31N CDR2IRSKYNNYATSEQ ID NO:3HC_T31N CDR3VRHGNFGNSYVSWFAYSEQ ID NO:57HC_T31NSEQ ID NO:58HC_T31P CDR1GFTFNPYASEQ ID NO:2HC_T31P CDR2IRSKYNNYATSEQ ID NO:3HC_T31P CDR3VRHGNFGNSYVSWFAYSEQ ID NO:59HC_T31PSEQ ID NO:60HC_T31Q CDR1GFTFNQYASEQ ID NO:2HC_T31Q CDR2IRSKYNNYATSEQ ID NO:3HC_T31Q CDR3VRHGNFGNSYVSWFAYSEQ ID NO:61HC_T31QSEQ ID NO:62HC_T31W CDR1GFTFNWYASEQ ID NO:2HC_T31W CDR2IRSKYNNYATSEQ ID NO:3HC_T31W CDR3VRHGNFGNSYVSWFAYSEQ ID NO:63HC_T31WSEQ ID NO:64HC_T31Y CDR1GFTFNYYASEQ ID NO:2HC_T31Y CDR2IRSKYNNYATSEQ ID NO:3HC_T31Y CDR3VRHGNFGNSYVSWFAYSEQ ID NO:65HC_T31YSEQ ID NO:66HC_Y32A CDR1GFTFNTAASEQ ID NO:2HC_Y32A CDR2IRSKYNNYATSEQ ID NO:3HC_Y32A CDR3VRHGNFGNSYVSWFAYSEQ ID NO:67HC_Y32ASEQ ID NO:68HC_Y32C CDR1GFTFNTCASEQ ID NO:2HC_Y32C CDR2IRSKYNNYATSEQ ID NO:3HC_Y32C CDR3VRHGNFGNSYVSWFAYSEQ ID NO:69HC_Y32CSEQ ID NO:70HC_Y32F CDR1GFTFNTFASEQ ID NO:2HC_Y32F CDR2IRSKYNNYATSEQ ID NO:3HC_Y32F CDR3VRHGNFGNSYVSWFAYSEQ ID NO:71HC_Y32FSEQ ID NO:72HC_Y32G CDR1GFTFNTGASEQ ID NO:2HC_Y32G CDR2IRSKYNNYATSEQ ID NO:3HC_Y32G CDR3VRHGNFGNSYVSWFAYSEQ ID NO:73HC_Y32GSEQ ID NO:74HC_Y32H CDR1GFTFNTHASEQ ID NO:2HC_Y32H CDR2IRSKYNNYATSEQ ID NO:3HC_Y32H CDR3VRHGNFGNSYVSWFAYSEQ ID NO:75HC_Y32HSEQ ID NO:76HC_Y32I CDR1GFTFNTIASEQ ID NO:2HC_Y32I CDR2IRSKYNNYATSEQ ID NO:3HC_Y32I CDR3VRHGNFGNSYVSWFAYSEQ ID NO:77HC_Y32ISEQ ID NO:78HC_Y32K CDR1GFTFNTKASEQ ID NO:2HC_Y32K CDR2IRSKYNNYATSEQ ID NO:3HC_Y32K CDR3VRHGNFGNSYVSWFAYSEQ ID NO:79HC_Y32KSEQ ID NO:80HC_Y32L CDR1GFTFNTLASEQ ID NO:2HC_Y32L CDR2IRSKYNNYATSEQ ID NO:3HC_Y32L CDR3VRHGNFGNSYVSWFAYSEQ ID NO:81HC_Y32LSEQ ID NO:82HC_Y32M CDR1GFTFNTMASEQ ID NO:2HC_Y32M CDR2IRSKYNNYATSEQ ID NO:3HC_Y32M CDR3VRHGNFGNSYVSWFAYSEQ ID NO:83HC_Y32MSEQ ID NO:84HC_Y32N CDR1GFTFNTNASEQ ID NO:2HC_Y32N CDR2IRSKYNNYATSEQ ID NO:3HC_Y32N CDR3VRHGNFGNSYVSWFAYSEQ ID NO:85HC_Y32NSEQ ID NO:86HC_Y32P CDR1GFTFNTPASEQ ID NO:2HC_Y32P CDR2IRSKYNNYATSEQ ID NO:3HC_Y32P CDR3VRHGNFGNSYVSWFAYSEQ ID NO:87HC_Y32PSEQ ID NO:88HC_Y32Q CDR1GFTFNTQASEQ ID NO:2HC_Y32Q CDR2IRSKYNNYATSEQ ID NO:3HC_Y32Q CDR3VRHGNFGNSYVSWFAYSEQ ID NO:89HC_Y32QSEQ ID NO:90HC_Y32R CDR1GFTFNTRASEQ ID NO:2HC_Y32R CDR2IRSKYNNYATSEQ ID NO:3HC_Y32R CDR3VRHGNFGNSYVSWFAYSEQ ID NO:91HC_Y32RSEQ ID NO:92HC_Y32S CDR1GFTFNTSASEQ ID NO:2HC_Y32S CDR2IRSKYNNYATSEQ ID NO:3HC_Y32S CDR3VRHGNFGNSYVSWFAYSEQ ID NO:93HC_Y32SSEQ ID NO:94HC_Y32T CDR1GFTFNTTASEQ ID NO:2HC_Y32T CDR2IRSKYNNYATSEQ ID NO:3HC_Y32T CDR3VRHGNFGNSYVSWFAYSEQ ID NO:95HC_Y32TSEQ ID NO:96HC_Y32V CDR1GFTFNTVASEQ ID NO:2HC_Y32V CDR2IRSKYNNYATSEQ ID NO:3HC_Y32V CDR3VRHGNFGNSYVSWFAYSEQ ID NO:97HC_Y32VSEQ ID NO:98HC_Y32W CDR1GFTFNTWASEQ ID NO:2HC_Y32W CDR2IRSKYNNYATSEQ ID NO:3HC_Y32W CDR3VRHGNFGNSYVSWFAYSEQ ID NO:99HC_Y32WSEQ ID NO:1HC_N57A CDR1GFTFNTYASEQ ID NO:100HC_N57A CDR2IRSKYNAYATSEQ ID NO:3HC_N57A CDR3VRHGNFGNSYVSWFAYSEQ ID NO:101HC_N57ASEQ ID NO:1HC_N57C CDR1GFTFNTYASEQ ID NO:102HC_N57C CDR2IRSKYNCYATSEQ ID NO:3HC_N57C CDR3VRHGNFGNSYVSWFAYSEQ ID NO:103HC_N57CSEQ ID NO:1HC_N57D CDR1GFTFNTYASEQ ID NO:104HC_N57D CDR2IRSKYNDYATSEQ ID NO:3HC_N57D CDR3VRHGNFGNSYVSWFAYSEQ ID NO:105HC_N57DSEQ ID NO:1HC_N57E CDR1GFTFNTYASEQ ID NO:106HC_N57E CDR2IRSKYNEYATSEQ ID NO:3HC_N57E CDR3VRHGNFGNSYVSWFAYSEQ ID NO:107HC_N57ESEQ ID NO:1HC_N57F CDR1GFTFNTYASEQ ID NO:108HC_N57F CDR2IRSKYNFYATSEQ ID NO:3HC_N57F CDR3VRHGNFGNSYVSWFAYSEQ ID NO:109HC_N57FSEQ ID NO:1HC_N57G CDR1GFTFNTYASEQ ID NO:110HC_N57G CDR2IRSKYNGYATSEQ ID NO:3HC_N57G CDR3VRHGNFGNSYVSWFAYSEQ ID NO:111HC_N57GSEQ ID NO:1HC_N57I CDR1GFTFNTYASEQ ID NO:112HC_N57I CDR2IRSKYNIYATSEQ ID NO:3HC_N57I CDR3VRHGNFGNSYVSWFAYSEQ ID NO:113HC_N57ISEQ ID NO:1HC_N57K CDR1GFTFNTYASEQ ID NO:114HC_N57K CDR2IRSKYNKYATSEQ ID NO:3HC_N57K CDR3VRHGNFGNSYVSWFAYSEQ ID NO:115HC_N57KSEQ ID NO:1HC_N57L CDR1GFTFNTYASEQ ID NO:116HC_N57L CDR2IRSKYNLYATSEQ ID NO:3HC_N57L CDR3VRHGNFGNSYVSWFAYSEQ ID NO:117HC_N57LSEQ ID NO:1HC_N57M CDR1GFTFNTYASEQ ID NO:118HC_N57M CDR2IRSKYNMYATSEQ ID NO:3HC_N57M CDR3VRHGNFGNSYVSWFAYSEQ ID NO:119HC_N57MSEQ ID NO:1HC_N57P CDR1GFTFNTYASEQ ID NO:120HC_N57P CDR2IRSKYNPYATSEQ ID NO:3HC_N57P CDR3VRHGNFGNSYVSWFAYSEQ ID NO:121HC_N57PSEQ ID NO:1HC_N57Q CDR1GFTFNTYASEQ ID NO:122HC_N57Q CDR2IRSKYNQYATSEQ ID NO:3HC_N57Q CDR3VRHGNFGNSYVSWFAYSEQ ID NO:123HC_N57QSEQ ID NO:1HC_N57R CDR1GFTFNTYASEQ ID NO:124HC_N57R CDR2IRSKYNRYATSEQ ID NO:3HC_N57R CDR3VRHGNFGNSYVSWFAYSEQ ID NO:125HC_N57RSEQ ID NO:1HC_N57T CDR1GFTFNTYASEQ ID NO:126HC_N57T CDR2IRSKYNTYATSEQ ID NO:3HC_N57T CDR3VRHGNFGNSYVSWFAYSEQ ID NO:127HC_N57TSEQ ID NO:1HC_N57V CDR1GFTFNTYASEQ ID NO:128HC_N57V CDR2IRSKYNVYATSEQ ID NO:3HC_N57V CDR3VRHGNFGNSYVSWFAYSEQ ID NO:129HC_N57VSEQ ID NO:1HC_N57W CDR1GFTFNTYASEQ ID NO:130HC_N57W CDR2IRSKYNWYATSEQ ID NO:3HC_N57W CDR3VRHGNFGNSYVSWFAYSEQ ID NO:131HC_N57WSEQ ID NO:1HC_N57Y CDR1GFTFNTYASEQ ID NO:132HC_N57Y CDR2IRSKYNYYATSEQ ID NO:3HC_N57Y CDR3VRHGNFGNSYVSWFAYSEQ ID NO:133HC_N57YSEQ ID NO:1HC_A59C CDR1GFTFNTYASEQ ID NO:134HC_A59C CDR2IRSKYNNYCTSEQ ID NO:3HC_A59C CDR3VRHGNFGNSYVSWFAYSEQ ID NO:135HC_A59CSEQ ID NO:1HC_A59D CDR1GFTFNTYASEQ ID NO:136HC_A59D CDR2IRSKYNNYDTSEQ ID NO:3HC_A59D CDR3VRHGNFGNSYVSWFAYSEQ ID NO:137HC_A59DSEQ ID NO:1HC_A59E CDR1GFTFNTYASEQ ID NO:138HC_A59E CDR2IRSKYNNYETSEQ ID NO:3HC_A59E CDR3VRHGNFGNSYVSWFAYSEQ ID NO:139HC_A59ESEQ ID NO:1HC_A59F CDR1GFTFNTYASEQ ID NO:140HC_A59F CDR2IRSKYNNYFTSEQ ID NO:3HC_A59F CDR3VRHGNFGNSYVSWFAYSEQ ID NO:141HC_A59FSEQ ID NO:1HC_A59G CDR1GFTFNTYASEQ ID NO:142HC_A59G CDR2IRSKYNNYGTSEQ ID NO:3HC_A59G CDR3VRHGNFGNSYVSWFAYSEQ ID NO:143HC_A59GSEQ ID NO:1HC_A59H CDR1GFTFNTYASEQ ID NO:144HC_A59H CDR2IRSKYNNYHTSEQ ID NO:3HC_A59H CDR3VRHGNFGNSYVSWFAYSEQ ID NO:145HC_A59HSEQ ID NO:1HC_A59I CDR1GFTFNTYASEQ ID NO:146HC_A59I CDR2IRSKYNNYITSEQ ID NO:3HC_A59I CDR3VRHGNFGNSYVSWFAYSEQ ID NO:147HC_A59ISEQ ID NO:1HC_A59K CDR1GFTFNTYASEQ ID NO:148HC_A59K CDR2IRSKYNNYKTSEQ ID NO:3HC_A59K CDR3VRHGNFGNSYVSWFAYSEQ ID NO:149HC_A59KSEQ ID NO:1HC_A59L CDR1GFTFNTYASEQ ID NO:150HC_A59L CDR2IRSKYNNYLTSEQ ID NO:3HC_A59L CDR3VRHGNFGNSYVSWFAYSEQ ID NO:151HC_A59LSEQ ID NO:1HC_A59M CDR1GFTFNTYASEQ ID NO:152HC_A59M CDR2IRSKYNNYMTSEQ ID NO:3HC_A59M CDR3VRHGNFGNSYVSWFAYSEQ ID NO:153HC_A59MSEQ ID NO:1HC_A59N CDR1GFTFNTYASEQ ID NO:154HC_A59N CDR2IRSKYNNYNTSEQ ID NO:3HC_A59N CDR3VRHGNFGNSYVSWFAYSEQ ID NO:155HC_A59NSEQ ID NO:1HC_A59P CDR1GFTFNTYASEQ ID NO:156HC_A59P CDR2IRSKYNNYPTSEQ ID NO:3HC_A59P CDR3VRHGNFGNSYVSWFAYSEQ ID NO:157HC_A59PSEQ ID NO:1HC_A59Q CDR1GFTFNTYASEQ ID NO:158HC_A59Q CDR2IRSKYNNYQTSEQ ID NO:3HC_A59Q CDR3VRHGNFGNSYVSWFAYSEQ ID NO:159HC_A59QSEQ ID NO:1HC_A59R CDR1GFTFNTYASEQ ID NO:160HC_A59R CDR2IRSKYNNYRTSEQ ID NO:3HC_A59R CDR3VRHGNFGNSYVSWFAYSEQ ID NO:161HC_A59RSEQ ID NO:1HC_A59S CDR1GFTFNTYASEQ ID NO:162HC_A59S CDR2IRSKYNNYSTSEQ ID NO:3HC_A59S CDR3VRHGNFGNSYVSWFAYSEQ ID NO:163HC_A59SSEQ ID NO:1HC_A59V CDR1GFTFNTYASEQ ID NO:164HC_A59V CDR2IRSKYNNYVTSEQ ID NO:3HC_A59V CDR3VRHGNFGNSYVSWFAYSEQ ID NO:165HC_A59VSEQ ID NO:1HC_A59W CDR1GFTFNTYASEQ ID NO:166HC_A59W CDR2IRSKYNNYWTSEQ ID NO:3HC_A59W CDR3VRHGNFGNSYVSWFAYSEQ ID NO:167HC_A59WSEQ ID NO:1HC_A59Y CDR1GFTFNTYASEQ ID NO:168HC_A59Y CDR2IRSKYNNYYTSEQ ID NO:3HC_A59Y CDR3VRHGNFGNSYVSWFAYSEQ ID NO:169HC_A59YSEQ ID NO:1HC_H101A CDR1GFTFNTYASEQ ID NO:2HC_H101A CDR2IRSKYNNYATSEQ ID NO:170HC_H101A CDR3VRAGNFGNSYVSWFAYSEQ ID NO:171HC_H101ASEQ ID NO:1HC_H101C CDR1GFTFNTYASEQ ID NO:2HC_H101C CDR2IRSKYNNYATSEQ ID NO:172HC_H101C CDR3VRCGNFGNSYVSWFAYSEQ ID NO:173HC_H101CSEQ ID NO:1HC_H101F CDR1GFTFNTYASEQ ID NO:2HC_H101F CDR2IRSKYNNYATSEQ ID NO:174HC_H101F CDR3VRFGNFGNSYVSWFAYSEQ ID NO:175HC_H101FSEQ ID NO:1HC_H101G CDR1GFTFNTYASEQ ID NO:2HC_H101G CDR2IRSKYNNYATSEQ ID NO:176HC_H101G CDR3VRGGNFGNSYVSWFAYSEQ ID NO:177HC_H101GSEQ ID NO:1HC_H1011 CDR1GFTFNTYASEQ ID NO:2HC_H1011 CDR2IRSKYNNYATSEQ ID NO:178HC_H1011 CDR3VRIGNFGNSYVSWFAYSEQ ID NO:179HC_H1011SEQ ID NO:1HC_H101K CDR1GFTFNTYASEQ ID NO:2HC_H101K CDR2IRSKYNNYATSEQ ID NO:180HC_H101K CDR3VRKGNFGNSYVSWFAYSEQ ID NO:181HC_H101KSEQ ID NO:1HC_H101L CDR1GFTFNTYASEQ ID NO:2HC_H101L CDR2IRSKYNNYATSEQ ID NO:182HC_H101L CDR3VRLGNFGNSYVSWFAYSEQ ID NO:183HC_H101LSEQ ID NO:1HC_H101N CDR1GFTFNTYASEQ ID NO:2HC_H101N CDR2IRSKYNNYATSEQ ID NO:184HC_H101N CDR3VRNGNFGNSYVSWFAYSEQ ID NO:185HC_H101NSEQ ID NO:1HC_H101P CDR1GFTFNTYASEQ ID NO:2HC_H101P CDR2IRSKYNNYATSEQ ID NO:186HC_H101P CDR3VRPGNFGNSYVSWFAYSEQ ID NO:187HC_H101PSEQ ID NO:1HC_H101Q CDR1GFTFNTYASEQ ID NO:2HC_H101Q CDR2IRSKYNNYATSEQ ID NO:188HC_H101Q CDR3VRQGNFGNSYVSWFAYSEQ ID NO:189HC_H101QSEQ ID NO:1HC_H101R CDR1GFTFNTYASEQ ID NO:2HC_H101R CDR2IRSKYNNYATSEQ ID NO:190HC_H101R CDR3VRRGNFGNSYVSWFAYSEQ ID NO:191HC_H101RSEQ ID NO:1HC_H101S CDR1GFTFNTYASEQ ID NO:2HC_H101S CDR2IRSKYNNYATSEQ ID NO:192HC_H101S CDR3VRSGNFGNSYVSWFAYSEQ ID NO:193HC_H101SSEQ ID NO:1HC_H101T CDR1GFTFNTYASEQ ID NO:2HC_H101T CDR2IRSKYNNYATSEQ ID NO:194HC_H101T CDR3VRTGNFGNSYVSWFAYSEQ ID NO:195HC_H101TSEQ ID NO:1HC_H101V CDR1GFTFNTYASEQ ID NO:2HC_H101V CDR2IRSKYNNYATSEQ ID NO:196HC_H101V CDR3VRVGNFGNSYVSWFAYSEQ ID NO:197HC_H101VSEQ ID NO:1HC_H101W CDR1GFTFNTYASEQ ID NO:2HC_H101W CDR2IRSKYNNYATSEQ ID NO:198HC_H101W CDR3VRWGNFGNSYVSWFAYSEQ ID NO:199HC_H101WSEQ ID NO:1HC_H101Y CDR1GFTFNTYASEQ ID NO:2HC_H101Y CDR2IRSKYNNYATSEQ ID NO:200HC_H101Y CDR3VRYGNFGNSYVSWFAYSEQ ID NO:201HC_H101YSEQ ID NO:1HC_G105A CDR1GFTFNTYASEQ ID NO:2HC_G105A CDR2IRSKYNNYATSEQ ID NO:202HC_G105A CDR3VRHGNFANSYVSWFAYSEQ ID NO:203HC_G105ASEQ ID NO:1HC_G105C CDR1GFTFNTYASEQ ID NO:2HC_G105C CDR2IRSKYNNYATSEQ ID NO:204HC_G105C CDR3VRHGNFCNSYVSWFAYSEQ ID NO:205HC_G105CSEQ ID NO:1HC_G105E CDR1GFTFNTYASEQ ID NO:2HC_G105E CDR2IRSKYNNYATSEQ ID NO:206HC_G105E CDR3VRHGNFENSYVSWFAYSEQ ID NO:207HC_G105ESEQ ID NO:1HC_G105F CDR1GFTFNTYASEQ ID NO:2HC_G105F CDR2IRSKYNNYATSEQ ID NO:208HC_G105F CDR3VRHGNFFNSYVSWFAYSEQ ID NO:209HC_G105FSEQ ID NO:1HC_G105H CDR1GFTFNTYASEQ ID NO:2HC_G105H CDR2IRSKYNNYATSEQ ID NO:210HC_G105H CDR3VRHGNFHNSYVSWFAYSEQ ID NO:211HC_G105HSEQ ID NO:1HC_G105I CDR1GFTFNTYASEQ ID NO:2HC_G105I CDR2IRSKYNNYATSEQ ID NO:212HC_G105I CDR3VRHGNFINSYVSWFAYSEQ ID NO:213HC_G105ISEQ ID NO:1HC_G105L CDR1GFTFNTYASEQ ID NO:2HC_G105L CDR2IRSKYNNYATSEQ ID NO:214HC_G105L CDR3VRHGNFLNSYVSWFAYSEQ ID NO:215HC_G105LSEQ ID NO:1HC_G105M CDR1GFTFNTYASEQ ID NO:2HC_G105M CDR2IRSKYNNYATSEQ ID NO:216HC_G105M CDR3VRHGNFMNSYVSWFAYSEQ ID NO:217HC_G105MSEQ ID NO:1HC_G105N CDR1GFTFNTYASEQ ID NO:2HC_G105N CDR2IRSKYNNYATSEQ ID NO:218HC_G105N CDR3VRHGNFNNSYVSWFAYSEQ ID NO:219HC_G105NSEQ ID NO:1HC_G105P CDR1GFTFNTYASEQ ID NO:2HC_G105P CDR2IRSKYNNYATSEQ ID NO:220HC_G105P CDR3VRHGNFPNSYVSWFAYSEQ ID NO:221HC_G105PSEQ ID NO:1HC_G105Q CDR1GFTFNTYASEQ ID NO:2HC_G105Q CDR2IRSKYNNYATSEQ ID NO:222HC_G105Q CDR3VRHGNFQNSYVSWFAYSEQ ID NO:223HC_G105QSEQ ID NO:1HC_G105R CDR1GFTFNTYASEQ ID NO:2HC_G105R CDR2IRSKYNNYATSEQ ID NO:224HC_G105R CDR3VRHGNFRNSYVSWFAYSEQ ID NO:225HC_G105RSEQ ID NO:1HC_G105S CDR1GFTFNTYASEQ ID NO:2HC_G105S CDR2IRSKYNNYATSEQ ID NO:226HC_G105S CDR3VRHGNFSNSYVSWFAYSEQ ID NO:227HC_G105SSEQ ID NO:1HC_G105T CDR1GFTFNTYASEQ ID NO:2HC_G105T CDR2IRSKYNNYATSEQ ID NO:228HC_G105T CDR3VRHGNFTNSYVSWFAYSEQ ID NO:229HC_G105TSEQ ID NO:1HC_G105V CDR1GFTFNTYASEQ ID NO:2HC_G105V CDR2IRSKYNNYATSEQ ID NO:230HC_G105V CDR3VRHGNFVNSYVSWFAYSEQ ID NO:231HC_G105VSEQ ID NO:1HC_G105W CDR1GFTFNTYASEQ ID NO:2HC_G105W CDR2IRSKYNNYATSEQ ID NO:232HC_G105W CDR3VRHGNFWNSYVSWFAYSEQ ID NO:233HC_G105WSEQ ID NO:1HC_G105Y CDR1GFTFNTYASEQ ID NO:2HC_G105Y CDR2IRSKYNNYATSEQ ID NO:234HC_G105Y CDR3VRHGNFYNSYVSWFAYSEQ ID NO:235HC_G105YSEQ ID NO:1HC_S110A CDR1GFTFNTYASEQ ID NO:2HC_S110A CDR2IRSKYNNYATSEQ ID NO:236HC_S110A CDR3VRHGNFGNSYVAWFAYSEQ ID NO:237HC_S110ASEQ ID NO:1HC_S110C CDR1GFTFNTYASEQ ID NO:2HC_S110C CDR2IRSKYNNYATSEQ ID NO:238HC_S110C CDR3VRHGNFGNSYVCWFAYSEQ ID NO:239HC_S110CSEQ ID NO:1HC_S110E CDR1GFTFNTYASEQ ID NO:2HC_S110E CDR2IRSKYNNYATSEQ ID NO:240HC_S110E CDR3VRHGNFGNSYVEWFAYSEQ ID NO:241HC_S110ESEQ ID NO:1HC_S110F CDR1GFTFNTYASEQ ID NO:2HC_S110F CDR2IRSKYNNYATSEQ ID NO:242HC_S110F CDR3VRHGNFGNSYVFWFAYSEQ ID NO:243HC_S110FSEQ ID NO:1HC_S110G CDR1GFTFNTYASEQ ID NO:2HC_S110G CDR2IRSKYNNYATSEQ ID NO:244HC_S110G CDR3VRHGNFGNSYVGWFAYSEQ ID NO:245HC_S110GSEQ ID NO:1HC_S110H CDR1GFTFNTYASEQ ID NO:2HC_S110H CDR2IRSKYNNYATSEQ ID NO:246HC_S110H CDR3VRHGNFGNSYVHWFAYSEQ ID NO:247HC_S110HSEQ ID NO:1HC_S110K CDR1GFTFNTYASEQ ID NO:2HC_S110K CDR2IRSKYNNYATSEQ ID NO:248HC_S110K CDR3VRHGNFGNSYVKWFAYSEQ ID NO:249HC_S110KSEQ ID NO:1HC_S110L CDR1GFTFNTYASEQ ID NO:2HC_S110L CDR2IRSKYNNYATSEQ ID NO:250HC_S110L CDR3VRHGNFGNSYVLWFAYSEQ ID NO:251HC_S110LSEQ ID NO:1HC_S110N CDR1GFTFNTYASEQ ID NO:2HC_S110N CDR2IRSKYNNYATSEQ ID NO:252HC_S110N CDR3VRHGNFGNSYVNWFAYSEQ ID NO:253HC_S110NSEQ ID NO:1HC_S110P CDR1GFTFNTYASEQ ID NO:2HC_S110P CDR2IRSKYNNYATSEQ ID NO:254HC_S110P CDR3VRHGNFGNSYVPWFAYSEQ ID NO:255HC_S110PSEQ ID NO:1HC_S110Q CDR1GFTFNTYASEQ ID NO:2HC_S110Q CDR2IRSKYNNYATSEQ ID NO:256HC_S110Q CDR3VRHGNFGNSYVQWFAYSEQ ID NO:257HC_S110QSEQ ID NO:1HC_S110R CDR1GFTFNTYASEQ ID NO:2HC_S110R CDR2IRSKYNNYATSEQ ID NO:258HC_S110R CDR3VRHGNFGNSYVRWFAYSEQ ID NO:259HC_S110RSEQ ID NO:1HC_S110T CDR1GFTFNTYASEQ ID NO:2HC_S110T CDR2IRSKYNNYATSEQ ID NO:260HC_S110T CDR3VRHGNFGNSYVTWFAYSEQ ID NO:261HC_S110TSEQ ID NO:1HC_S110W CDR1GFTFNTYASEQ ID NO:2HC_S110W CDR2IRSKYNNYATSEQ ID NO:262HC_S110W CDR3VRHGNFGNSYVWWFAYSEQ ID NO:263HC_S110WSEQ ID NO:1HC_S110Y CDR1GFTFNTYASEQ ID NO:2HC_S110Y CDR2IRSKYNNYATSEQ ID NO:264HC_S110Y CDR3VRHGNFGNSYVYWFAYSEQ ID NO:265HC_S110YSEQ ID NO:1HC_Y114A CDR1GFTFNTYASEQ ID NO:2HC_Y114A CDR2IRSKYNNYATSEQ ID NO:266HC_Y114A CDR3VRHGNFGNSYVSWFAASEQ ID NO:267HC_Y114ASEQ ID NO:1HC_Y114C CDR1GFTFNTYASEQ ID NO:2HC_Y114C CDR2IRSKYNNYATSEQ ID NO:268HC_Y114C CDR3VRHGNFGNSYVSWFACSEQ ID NO:269HC_Y114CSEQ ID NO:1HC_Y114E CDR1GFTFNTYASEQ ID NO:2HC_Y114E CDR2IRSKYNNYATSEQ ID NO:270HC_Y114E CDR3VRHGNFGNSYVSWFAESEQ ID NO:271HC_Y114ESEQ ID NO:1HC_Y114F CDR1GFTFNTYASEQ ID NO:2HC_Y114F CDR2IRSKYNNYATSEQ ID NO:272HC_Y114F CDR3VRHGNFGNSYVSWFAFSEQ ID NO:273HC_Y114FSEQ ID NO:1HC_Y114G CDR1GFTFNTYASEQ ID NO:2HC_Y114G CDR2IRSKYNNYATSEQ ID NO:274HC_Y114G CDR3VRHGNFGNSYVSWFAGSEQ ID NO:275HC_Y114GSEQ ID NO:1HC_Y114H CDR1GFTFNTYASEQ ID NO:2HC_Y114H CDR2IRSKYNNYATSEQ ID NO:276HC_Y114H CDR3VRHGNFGNSYVSWFAHSEQ ID NO:277HC_Y114HSEQ ID NO:1HC_Y114I CDR1GFTFNTYASEQ ID NO:2HC_Y114I CDR2IRSKYNNYATSEQ ID NO:278HC_Y114I CDR3VRHGNFGNSYVSWFAISEQ ID NO:279HC_Y114ISEQ ID NO:1HC_Y114K CDR1GFTFNTYASEQ ID NO:2HC_Y114K CDR2IRSKYNNYATSEQ ID NO:280HC_Y114K CDR3VRHGNFGNSYVSWFAKSEQ ID NO:281HC_Y114KSEQ ID NO:1HC_Y114L CDR1GFTFNTYASEQ ID NO:2HC_Y114L CDR2IRSKYNNYATSEQ ID NO:282HC_Y114L CDR3VRHGNFGNSYVSWFALSEQ ID NO:283HC_Y114LSEQ ID NO:1HC_Y114M CDR1GFTFNTYASEQ ID NO:2HC_Y114M CDR2IRSKYNNYATSEQ ID NO:284HC_Y114M CDR3VRHGNFGNSYVSWFAMSEQ ID NO:285HC_Y114MSEQ ID NO:1HC_Y114N CDR1GFTFNTYASEQ ID NO:2HC_Y114N CDR2IRSKYNNYATSEQ ID NO:286HC_Y114N CDR3VRHGNFGNSYVSWFANSEQ ID NO:287HC_Y114NSEQ ID NO:1HC_Y114P CDR1GFTFNTYASEQ ID NO:2HC_Y114P CDR2IRSKYNNYATSEQ ID NO:288HC_Y114P CDR3VRHGNFGNSYVSWFAPSEQ ID NO:289HC_Y114PSEQ ID NO:1HC_Y114Q CDR1GFTFNTYASEQ ID NO:2HC_Y114Q CDR2IRSKYNNYATSEQ ID NO:290HC_Y114Q CDR3VRHGNFGNSYVSWFAQSEQ ID NO:291HC_Y114QSEQ ID NO:1HC_Y114R CDR1GFTFNTYASEQ ID NO:2HC_Y114R CDR2IRSKYNNYATSEQ ID NO:292HC_Y114R CDR3VRHGNFGNSYVSWFARSEQ ID NO:293HC_Y114RSEQ ID NO:1HC_Y114S CDR1GFTFNTYASEQ ID NO:2HC_Y114S CDR2IRSKYNNYATSEQ ID NO:294HC_Y114S CDR3VRHGNFGNSYVSWFASSEQ ID NO:295HC_Y114SSEQ ID NO:1HC_Y114T CDR1GFTFNTYASEQ ID NO:2HC_Y114T CDR2IRSKYNNYATSEQ ID NO:296HC_Y114T CDR3VRHGNFGNSYVSWFATSEQ ID NO:297HC_Y114TSEQ ID NO:1HC_Y114V CDR1GFTFNTYASEQ ID NO:2HC_Y114V CDR2IRSKYNNYATSEQ ID NO:298HC_Y114V CDR3VRHGNFGNSYVSWFAVSEQ ID NO:299HC_Y114VSEQ ID NO:1HC_Y114W CDR1GFTFNTYASEQ ID NO:2HC_Y114W CDR2IRSKYNNYATSEQ ID NO:300HC_Y114W CDR3VRHGNFGNSYVSWFAWSEQ ID NO:301HC_Y114WSEQ ID NO:302LC_T31A CDR1TGAVTASNYLC_T31A CDR2GTNSEQ ID NO:7LC_T31A CDR3ALWYSNLWVSEQ ID NO:303LC_T31ASEQ ID NO:304LC_T31D CDR1TGAVTDSNYLC_T31D CDR2GTNSEQ ID NO:7LC_T31D CDR3ALWYSNLWVSEQ ID NO:305LC_T31DSEQ ID NO:306LC_T31E CDR1TGAVTESNYLC_T31E CDR2GTNSEQ ID NO:7LC_T31E CDR3ALWYSNLWVSEQ ID NO:307LC_T31ESEQ ID NO:308LC_T31F CDR1TGAVTFSNYLC_T31F CDR2GTNSEQ ID NO:7LC_T31F CDR3ALWYSNLWVSEQ ID NO:309LC_T31FSEQ ID NO:310LC_T31G CDR1TGAVTGSNYLC_T31G CDR2GTNSEQ ID NO:7LC_T31G CDR3ALWYSNLWVSEQ ID NO:311LC_T31GSEQ ID NO:312LC_T31H CDR1TGAVTHSNYLC_T31H CDR2GTNSEQ ID NO:7LC_T31H CDR3ALWYSNLWVSEQ ID NO:313LC_T31HLC_T31I CDR2GTNSEQ ID NO:314LC_T31K CDR1TGAVTKSNYLC_T31K CDR2GTNSEQ ID NO:7LC_T31K CDR3ALWYSNLWVSEQ ID NO:315LC_T31KSEQ ID NO:316LC_T31L CDR1TGAVTLSNYLC_T31L CDR2GTNSEQ ID NO:7LC_T31L CDR3ALWYSNLWVSEQ ID NO:317LC_T31LSEQ ID NO:318LC_T31M CDR1TGAVTMSNYLC_T31M CDR2GTNSEQ ID NO:7LC_T31M CDR3ALWYSNLWVSEQ ID NO:319LC_T31MSEQ ID NO:320LC_T31N CDR1TGAVTNSNYLC_T31N CDR2GTNSEQ ID NO:7LC_T31N CDR3ALWYSNLWVSEQ ID NO:321LC_T31NSEQ ID NO:322LC_T31P CDR1TGAVTPSNYLC_T31P CDR2GTNSEQ ID NO:7LC_T31P CDR3ALWYSNLWVSEQ ID NO:323LC_T31PSEQ ID NO:324LC_T31Q CDR1TGAVTQSNYLC_T31Q CDR2GTNSEQ ID NO:7LC_T31Q CDR3ALWYSNLWVSEQ ID NO:325LC_T31QSEQ ID NO:326LC_T31R CDR1TGAVTRSNYLC_T31R CDR2GTNSEQ ID NO:7LC_T31R CDR3ALWYSNLWVSEQ ID NO:327LC_T31RLC_T31S CDR2GTNSEQ ID NO:328LC_T31V CDR1TGAVTVSNYLC_T31V CDR2GTNSEQ ID NO:7LC_T31V CDR3ALWYSNLWVSEQ ID NO:329LC_T31VSEQ ID NO:330LC_T31Y CDR1TGAVTYSNYLC_T31Y CDR2GTNSEQ ID NO:7LC_T31Y CDR3ALWYSNLWVSEQ ID NO:331LC_T31YSEQ ID NO:6LC_L92A CDR1TGAVTTSNYLC_L92A CDR2GTNSEQ ID NO:332LC_L92A CDR3AAWYSNLWVSEQ ID NO:333LC_L92ASEQ ID NO:6LC_L92C CDR1TGAVTTSNYLC_L92C CDR2GTNSEQ ID NO:334LC_L92C CDR3ACWYS N LWVSEQ ID NO:335LC_L92CSEQ ID NO:6LC_L92D CDR1TGAVTTSNYLC_L92D CDR2GTNSEQ ID NO:336LC_L92D CDR3ADWYSNLWVSEQ ID NO:337LC_L92DSEQ ID NO:6LC_L92E CDR1TGAVTTSNYLC_L92E CDR2GTNSEQ ID NO:338LC_L92E CDR3AEWYSNLWVSEQ ID NO:339LC_L92ESEQ ID NO:6LC_L92F CDR1TGAVTTSNYLC_L92F CDR2GTNSEQ ID NO:340LC_L92F CDR3AFWYSNLWVSEQ ID NO:341LC_L92FSEQ ID NO:6LC_L92G CDR1TGAVTTSNYLC_L92G CDR2GTNSEQ ID NO:342LC_L92G CDR3AGWYSNLWVSEQ ID NO:343LC_L92GSEQ ID NO:6LC_L92I CDR1TGAVTTSNYLC_L92I CDR2GTNSEQ ID NO:344LC_L92I CDR3AIWYSNLWVSEQ ID NO:345LC_L92ISEQ ID NO:6LC_L92K CDR1TGAVTTSNYLC_L92K CDR2GTNSEQ ID NO:346LC_L92K CDR3AKWYSNLWVSEQ ID NO:347LC_L92KSEQ ID NO:6LC_L92M CDR1TGAVTTSNYLC_L92M CDR2GTNSEQ ID NO:348LC_L92M CDR3AMWYSNLWVSEQ ID NO:349LC_L92MSEQ ID NO:6LC_L92N CDR1TGAVTTSNYLC_L92N CDR2GTNSEQ ID NO:350LC_L92N CDR3ANWYSNLWVSEQ ID NO:351LC_L92NSEQ ID NO:6LC_L92P CDR1TGAVTTSNYLC_L92P CDR2GTNSEQ ID NO:352LC_L92P CDR3APWYSNLWVSEQ ID NO:353LC_L92PSEQ ID NO:6LC_L92R CDR1TGAVTTSNYLC_L92R CDR2GTNSEQ ID NO:354LC_L92R CDR3ARWYSNLWVSEQ ID NO:355LC_L92RSEQ ID NO:6LC_L92S CDR1TGAVTTSNYLC_L92S CDR2GTNSEQ ID NO:356LC_L92S CDR3ASWYSNLWVSEQ ID NO:357LC_L92SSEQ ID NO:6LC_L92T CDR1TGAVTTSNYLC_L92T CDR2GTNSEQ ID NO:358LC_L92T CDR3ATWYSNLWVSEQ ID NO:359LC_L92TSEQ ID NO:6LC_L92V CDR1TGAVTTSNYLC_L92V CDR2GTNSEQ ID NO:360LC_L92V CDR3AVWYSNLWVSEQ ID NO:361LC_L92VSEQ ID NO:6LC_L92W CDR1TGAVTTSNYLC_L92W CDR2GTNSEQ ID NO:362LC_L92W CDR3AWWYSNLWVSEQ ID NO:363LC_L92WSEQ ID NO:6LC_L92Y CDR1TGAVTTSNYLC_L92Y CDR2GTNSEQ ID NO:364LC_L92Y CDR3AYWYSNLWVSEQ ID NO:365LC_L92YSEQ ID NO:6LC_L97D CDR1TGAVTTSNYLC_L97D CDR2GTNSEQ ID NO:366LC_L97D CDR3ALWYSNDWVSEQ ID NO:367LC_L97DSEQ ID NO:6LC_L97E CDR1TGAVTTSNYLC_L97E CDR2GTNSEQ ID NO:368LC_L97E CDR3ALWYS N EWVSEQ ID NO:369LC_L97ESEQ ID NO:6LC_L97F CDR1TGAVTTSNYLC_L97F CDR2GTNSEQ ID NO:370LC_L97F CDR3ALWYSNFWVSEQ ID NO:371LC_L97FSEQ ID NO:6LC_L97G CDR1TGAVTTSNYLC_L97G CDR2GTNSEQ ID NO:372LC_L97G CDR3ALWYSNGWVSEQ ID NO:373LC_L97GSEQ ID NO:6LC_L97H CDR1TGAVTTSNYLC_L97H CDR2GTNSEQ ID NO:374LC_L97H CDR3ALWYSNHWVSEQ ID NO:375LC_L97HSEQ ID NO:6LC_L97K CDR1TGAVTTSNYLC_L97K CDR2GTNSEQ ID NO:376LC_L97K CDR3ALWYS N KWVSEQ ID NO:377LC_L97KSEQ ID NO:6LC_L97M CDR1TGAVTTSNYLC_L97M CDR2GTNSEQ ID NO:378LC_L97M CDR3ALWYSNMWVSEQ ID NO:379LC_L97MSEQ ID NO:6LC_L97N CDR1TGAVTTSNYLC_L97N CDR2GTNSEQ ID NO:380LC_L97N CDR3ALWYSNNWVSEQ ID NO:381LC_L97NSEQ ID NO:6LC_L97P CDR1TGAVTTSNYLC_L97P CDR2GTNSEQ ID NO:382LC_L97P CDR3ALWYSNPWVSEQ ID NO:383LC_L97PSEQ ID NO:6LC_L97Q CDR1TGAVTTSNYLC_L97Q CDR2GTNSEQ ID NO:384LC_L97Q CDR3ALWYSNQWVSEQ ID NO:385LC_L97QLC_L97R CDR2GTNSEQ ID NO:6LC_L97S CDR1TGAVTTSNYLC_L97S CDR2GTNSEQ ID NO:386LC_L97S CDR3ALWYSNSWVSEQ ID NO:387LC_L97SSEQ ID NO:6LC_L97T CDR1TGAVTTSNYLC_L97T CDR2GTNSEQ ID NO:388LC_L97T CDR3ALWYSNTWVSEQ ID NO:389LC_L97TSEQ ID NO:6LC_L97V CDR1TGAVTTSNYLC_L97V CDR2GTNSEQ ID NO:390LC_L97V CDR3ALWYSNVWVSEQ ID NO:391LC_L97VSEQ ID NO:6LC_L97W CDR1TGAVTTSNYLC_L97W CDR2GTNSEQ ID NO:392LC_L97W CDR3ALWYSNWWVSEQ ID NO:393LC_L97WSEQ ID NO:6LC_L97Y CDR1TGAVTTSNYLC_L97Y CDR2GTNSEQ ID NO:394LC_L97Y CDR3ALWYSNYWVSEQ ID NO:395LC_L97YSEQ ID NO:396Mature huTRASEQ ID NO:397Mature huTRB 3SEQ ID NO:398Mature huCD3δSEQ ID NO:399Mature huCD3εSEQ ID NO:400Mature huCD3γSEQ ID NO:401Mature huCD3ζSEQ ID NO:402Mature CD3ε27-GSKaSEQ ID NO:403Mature cyno CD3ε (epsilon)SEQ ID NO:404Mature rhesus CD3ε (epsilon)SEQ ID NO:405Parent murine VH SEQ ID NO:406Parent murine VL SEQ ID NO:407IgG1m(f) heavy chain constant region SEQ ID NO:408Human IgLC2 / IgLC3 constant domain SEQ ID NO:409IgG1m(f) heavy chain constant region with FEA mutationsSEQ ID NO:410Primer CMV P f (MAR5)GCTTCGCGATGTACGGGCCAGATATACSEQ ID NO:411TK pA r(MAR1)

[0340] The CDR regions have been annotated according to the IMGT definitions.Examples Example 1 - Generation of humanized CD3 antibodies and non-activating antibody variants Humanization of CD3 antibodies

[0341] Humanization of a murine CD3 antibody (US 8,236,308, described herein as IgG1-CD3) was performed by Antitope (Cambridge, UK) using their improved version of the germline humanization (CDR-grafting) technology (EP 0 629 240). Using this technology, 1 different VH chain (SEQ ID NO:4) and 2 different VL chains (SEQ ID NO:8, 10 ) were designed. By combining these 1 VH with the 2 VL chains, 2 different antibodies were generated. The humanized variants are described herein as huCD3. Thus, humanized variants comprising a VH and a VL according to the invention, are described as, e.g., IgG1-huCD3-H1L1 meaning that said specific variant is of the IgG1 isotype, is a humanized CD3 and comprises the VH amino acid sequence termed "H1" and is defined according to SEQ ID NO:4, and the VL amino acid sequence termed "L1" and is defined according to SEQ ID NO: 8. Thus, H1 refers to the variable heavy chain region VH1, L1 refers to the variable light chain region VL1, and so forth.

[0342] In particular, the variants IgG1-huCD3-H1L1 (humanized CD3 comprising the VH1 sequence set forth in SEQ ID NO:4 and the VL1 sequence set forth in SEQ ID NO:8), IgG1-huCD3-L1-T41K (humanized CD3 comprising the VH1 sequence set forth in SEQ ID NO:4 and the VL sequence set forth in SEQ ID NO:10.b12 antibody

[0343] In some of the examples the antibody b12, a HIV-1 gp120 specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5;230(3):812-23.) was used as a negative control, and is termed "IgG1-b12".Expression

[0344] Antibodies were expressed as IgG1,κ or IgG1,λ with or without the non-activating mutations described below and with a mutation in the CH3 domain enabling the generation of bispecific antibodies by the method described below. Plasmid DNA mixtures encoding both heavy and light chain of antibodies were transiently transfected to Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer.Purification of antibodies

[0345] Culture supernatant was filtered over 0.2 µm dead-end filters, loaded on 5 mL MabSelect SuRe columns (GE Health Care) and eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluate was immediately neutralized with 2M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B.Braun). Alternatively, subsequent to purification, the eluate was loaded on a HiPrep Desalting column and the antibody was exchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B.Braun) buffer. After dialysis or exchange of buffer, samples were sterile filtered over 0.2 µm dead-end filters. Purity was determined by SDS-PAGE and concentration was measured by absorbance at 280 nm. Purified antibodies were stored at 2-8°C.Example 2: Generation of mutant library

[0346] Point mutations were generated by random mutagenesis performed using the Quick change mutagenesis kit (Stratagene, according to the manufacturer's instructions and the HC (p33HGTE-huCD3-H1) and LC (p33L-huCD3-L1-T41K) expression plasmids as templates. The HC plasmid encodes for the monovalent UniBody -TE format as described in WO2011110642. Each selected position was randomized by using primers containing a NNS codon at the selected position (N=G, A, T or C and S = G or C). Mutant libraries were transformed to OneShot DH5alpha (Invitrogen) according to manufacturer's instructions.Colony picking and LEE PCR

[0347] For each mutated position 96 clones were individually picked into 50 µL LEE (linear expression element) PCR buffer (5 µL 10x AccuPrime PCR buffer 1, 44.6 µL water (B.Braun) , 0.1 µL CMV P f (MAR5) and 0.1 µL Tk pA r (MAR1) primers (100 µM stock), 0.2 µL Accuprime Taq (Invitrogen) to amplify the expression cassette from the expression plasmid (promoter up to poly A). LEE PCRs were performed by incubating the mixtures 2' 94 °C, [30" 94 °C, 30" 55 0C, 5' 68 °C]35x, 10' 72 °C and storage at 4 °C until further use.

[0348] Each library (12) of 96 colonies was sequenced using Sanger sequencing (Beckman Coulter Genomics, UK). Table 2: Primer sequences for LEE PCRPrimer namePrimer SequenceCMV P f (MAR5)GCTTCGCGATGTACGGGCCAGATATACTK pA r(MAR1)GGATACCCCCTAGAGCCCCAGCTGCGCAGATCTGCTATGGC Example 3: Expression of mutant library and IgG quantification

[0349] Of each mutant (12x96 in total) 1.11 µL HC and 1.11 µL LC LEE PCR product were diluted in 2.78 µL water. The 5 µL DNA dilution was used to transfect a single well in a 96 well plate.

[0350] Per well 0.4 µL ExpiFectamine ™< 293 (Invitrogen, US) and 4.6 µL Opti-MEM (Gibco, US) were mixed and incubated for 5 minutes at room temperature. Next, the Fectin / Opti-MEM mix was added to the 5 µL DNA dilution and incubated for 30 minutes at room temperature. Finally, 8.3 µL of the Fectin / Opti-MEM / DNA mix was added to 117.5 µL Expi293F ™< cells. During all procedures, the plates with Expi293F ™< cells were shaken to keep the cells in suspension. After transfection, cells were incubated at 37 °C / 8% CO2 for 5 days.

[0351] Five days post transfection, the supernatant was harvested. Antibody concentration in supernatant was measured by BioLayer Interferometry using the Octet RED (ForteBio, US).Example 4: Generation of CD3 / TCR-LC13 screening library

[0352] Freestyle 293-F cells (Invitrogen, US) were co-transfected with expression constructs encoding the human alpha and beta chains of the TCR (SEQ ID NO: 396 and SEQ ID NO: 397 respectively), human CD3δ (SEQ ID NO: 398), human CD3ε (SEQ ID NO: 399), human CD3γ (SEQ ID NO: 400) and human CD3ζ (SEQ ID NO: 401). The signal peptide sequence is excluded in these sequences. Transfection was performed according to manufacturer's instructions (Invitrogen, US). One day post transfection, cells were frozen until further use.Example 5: Screening of affinity mutants Homogeneous assay (Dose Response)

[0353] Based on the sequence data, mutants were selected where sequence traces showed high PHRED scores, indicative for the absence of multiple mutations. Per mutation, multiple redundant clones were selected when available.

[0354] The binding of recombinantly produced UniBody molecules in cell culture supernatant was determined by homogeneous antigen specific binding assays using Fluorometric Micro volume Assay Technology (FMAT; Applied Biosystems, Foster City, CA, USA). In the assay test design samples were analyzed in dose response for binding of antibodies or monovalent antibody molecules to CD3 / TCR-LC13 (Freestyle 293-F cells transiently expressed human CD3 and human T cell receptor (TCR); produced as described above) and Freestyle 293-F wild-type cells (negative control which does not express human TCR). IgG levels for sample normalization prior to dose response binding were measured using an Octet instrument (Fortebio, Menlo Park, USA).

[0355] Dilution series of samples were added to the cells to allow binding to CD3. Subsequently, binding of monovalent antibody molecules was detected using a fluorescent conjugate (Goat anti-Human IgG Fc gamma-Alexa647; Jackson ImmunoResearch). The CD3 specific humanized mouse antibody IgG1-HuM291-F405L (produced in Freestyle 293-F cells) and monovalent anibody UniTE-huCD3-H1L1-LT41K were used as a positive control and ChromPure Human IgG, whole molecule (Jackson ImmunoResearch) was used as negative control. The samples were scanned using an Applied Biosystems 8200 Cellular Detection System (8200 CDS) and total fluorescence over sample concentration was used as readout. Samples were stated positive when counts were higher than 50 and counts x fluorescence (total florescence) was at least three times higher than the negative control.Heatmap

[0356] From the Homogeneous Dose Response screen, the binding curves were fitted using a 4 parameter sigmoidal model. From the fit, the maximal binding for every mutant was determined. Per mutant, the average maximal binding was calculated, and depicted as a ratio between average maximal over wt binding as shown in Figure 1.Alignment

[0357] Selected HC mutants generated in these libraries are aligned and depicted in Figure 2. The CDR regions have been annotated according to the IMGT definitions. Numbering of the sequence is annotated according to a direct numbering scheme.Example 6 - Generation of bispecific antibodies by 2-MEA-induced Fab-arm exchange

[0358] The bispecific antibodies according to the invention may be generated by use of the methods disclosed in WO2011131746 and WO2013060867 (Genmab).

[0359] By way of example a mutation in position F405L may be introduced in one parental antibody of IgG1 isotype, and a mutation in position K409R may be introduced in the other parental antibody of IgG1 isotype.

[0360] These two parental antibodies, each at a final concentration of 0.5 mg / mL (equimolar concentration), may be incubated under reducing conditions with 25 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 µL Tris-EDTA (TE) at 37°C for 90 min. The reduction reaction is stopped when the reducing agent 2-MEA is removed by using spin columns (Microcon centrifugal filters, 30k, Millipore) according to the manufacturer's protocol.

[0361] The bispecific antibodies may be filtered over 0.2 µm dead-end filters and the absorbance at 280 nm (A280) of the bispecific antibodies may be measured to determine the final concentration thereof.Example 7: Binding data

[0362] For all binding assays below a selected panel of heavy chain variants of huCD3-H1L1 were tested in different formats: Table 3: Selected affinity variants of huCD3-H1L1 in monovalent antibody-TE formatSelected UniTE-huCD3 HC affinity variantsUniTE-huCD3-H1L1-LT41K (WT)UniTE-huCD3-H1L1-T31M-LT41KUniTE-huCD3-H1L1-T31P-LT41KUniTE-huCD3-H1L1-Y32A-LT41KUniTE-huCD3-H1L1-N57E-LT41KUniTE-huCD3-H1L1-H101F-LT41KUniTE-huCD3-H1L1-H101G-LT41KUniTE-huCD3-H1L1-H101I-LT41KUniTE-huCD3-H1L1-H101K-LT41KUniTE-huCD3-H1L1-H101L-LT41KUniTE-huCD3-H1L1-H101N-LT41KUniTE-huCD3-H1L1-G105P-LT41KUniTE-huCD3-H1L1-S110A-LT41KUniTE-huCD3-H1L1-S110G-LT41KUniTE-huCD3-H1L1-Y114M-LT41KUniTE-huCD3-H1L1-Y114R-LT41KUniTE-huCD3-H1L1-Y114V-LT41K Octet binding affinity determination of the CD3 affinity mutants in monovalent antibody-TE format

[0363] Affinities of a selected panel of affinity VH variants (Table 3) were determined using Bio-Layer Interferometry on a ForteBio Octet HTX. Anti-human Fc Capture (AHC) biosensors (ForteBio, Portsmouth, UK; cat no. 18-5060) were loaded for 600 s with the CD3 affinity mutants in monovalent antibody-TE format (2 µg / mL), aiming at a loading response of 0.4 nm. Antibodies of the UniBody-TE format were used to specifically measure the monovalent interaction affinity between the CD3 affinity mutants and the CD3ε27-GSKa ligand. After a baseline (150 s) the association (1000 s) and dissociation (1000 s) of CD3ε27-GSKa (100 and 1000 nM) was determined. The CD3ε27-GSKa protein consists of the human CD3ε peptide (aa1-27) fused to the N-terminus of a kappa LC (SEQ ID NO: 402). For calculations, the theoretical molecular mass of CD3ε27-GSKa based on the amino acid sequence was used, i.e. 27.1 kDa. Experiments were carried out while shaking at 1000 rpm and at 30°C.

[0364] Data was analyzed with ForteBio Data Analysis Software v8.1, using the 1:1 model and a global full fit with 1000 s association time and 200 s dissociation time. Data traces were corrected by subtraction of a reference curve (CD3 affinity mutant without CD3ε27-GSKa), the Y-axis was aligned to the last 5 s of the baseline, and interstep correction as well as Savitzky-Golay filtering was applied. Table 4: Equilibrium dissociation constant (KD) for selected variantsAntibody IDKD (M)1 / KD (M -1< )UniTE-huCD3-H1L1-N57E-LT41K2.4x10 -8< 4.1x10 7< uniTE-huCD3-H1L1-LT41K3.4x10 -8< 3.0x10 7< UniTE-huCD3-H1L1-G105P-LT41K3.5x10 -8< 2.8x10 7< UniTE-huCD3-H1L1-T31P-LT41K4.9x10 -8< 2.0x10 7< UniTE-huCD3-H1L1-S110G-LT41K6.1x10 -8< 1.6x10 7< UniTE-huCD3-H1L1-Y114V-LT41K7.5x10 -8< 1.3x10 7< UniTE-huCD3-H1L1-Y114M-LT41K7.6x10 -8< 1.3x10 7< UniTE-huCD3-H1L1-T31M-LT41K2.0x10 -7< 5.1x10 6< UniTE-huCD3-H1L1-H101N-LT41K2.0x10 -7< 5.1x10 6< UniTE-huCD3-H1L1-H101I-LT41K2.2x10 -7< 4.6x10 6< UniTE-huCD3-H1L1-S110A-LT41K3.2x10 -7< 3.1x10 6< UniTE-huCD3-H1L1-H101G-LT41K9.7x10 -7< 1.0x10 6< UniTE-huCD3-H1L1-H101L-LT41KndndUniTE-huCD3-H1L1-Y32A-LT41KndndUniTE-huCD3-H1L1-H101K-LT41KndndUniTE-huCD3-H1L1-T31A-LT41KndndUniTE-huCD3-H1L1-H101F-LT41KndndUniTE-huCD3-H1L1-Y114R-LT41Kndndnd = not determined T cell binding of affinity variants of humanized CD3 (UniTE-huCD3-H1L1-LT41K) on flow cytometry (FACS)

[0365] T cell binding of purified VH affinity variants of humanized CD3 (IgG1-huCD3-H1L1) antibodies was determined using Fluorescence-Activated Cell Sorting on a FACSCanto 752 (BD Biosciences). T cells were isolated from a buffy coat fraction of anti-coagulated human donor blood samples and resuspended in PBS / 0.1% BSA / 0.02% azide at 1.8 x 10E6 cells / mL. 50 µL of T cell suspension and 50 µL of the antibody dilutions were combined in a 96 well plate on ice, incubated for 30 min at 4°C and washed twice with PBS / 0.1% BSA / 0.02% azide. Next, 50 µL of secondary antibody, R-Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab')2 (109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1 / 200 in PBS / 0.1% BSA / 0.02% azide, was added for staining, the mixture was incubated for 30 min at 4°C and subsequently washed twice with PBS / 0.1% BSA / 0.02% azide. The cells were resuspended in 120 µL PBS / 0.1% BSA / 0.02% azide and PE geometric Mean Fluorescence Intensity was measured. Binding curves were analyzed using non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA) and apparent affinity (KD) was derived from the concentration at half-maximal binding. Figure 4 shows binding curves of affinity variants of humanized CD3 (UniTE-huCD3-H1L1-LT41K) and Figure 5 shows the binding curves of low affinity variants of humanized CD3 (UniTE-huCD3-H1L1-LT41K). Table 5: Summary of binding data of CD3 affinity mutants in monovalent antibody-TE formatantibody IDTcell binding average at 5000ng / ml (Geometric MFI)Max CD3 / TCRLC13 (RFU)KD (M)UniTE-huCD3-H1L1-LT41K1320620305493.4x10 -8< UniTE-huCD3-H1L1-G105P-LT41K1052626282283.5x10 -8< UniTE-huCD3-H1L1-T31A-LT41K102401836187UniTE-huCD3-H1L1-N57E-LT41K849217888512.4x10 -8< UniTE-huCD3-H1L1-Y114R-LT41K61971599412UniTE-huCD3-H1L1-Y114V-LT41K602518074857.5x10 -8< UniTE-huCD3-H1L1-T31P-LT41K428617183174.9x10 -8< UniTE-huCD3-H1L1-Y114M-LT41K383017717147.6x10 -8< UniTE-huCD3-H1L1-T31M-LT41K191915395062.0x10 -7< UniTE-huCD3-H1L1-S110A-LT41K166014110163.2x10 -7< UniTE-huCD3-H1L1-H101K-LT41K1321893765UniTE-huCD3-H1 L1-S110G-LT41K9493858736.1x10 -8< UniTE-huCD3-H1L1-H101N-LT41K84810602282.0x10 -7< UniTE-huCD3-H1L1-Y32A-LT41K719541497UniTE-huCD3-H1L1-H101F-LT41K641UniTE-huCD3-H1L1-H101I-LT41K5116036832.2x10 -7< UniTE-huCD3-H1L1-H101L-LT41K509812768UniTE-huCD3-H1L1-H101G-LT41K4921397129.7x10 -7<

[0366] For all binding assays below a selected panel of preferred heavy chain variants was tested (see Table 5)Octet binding affinity determination of IgG1-huCD3-H1L1-FEAL affinity mutants

[0367] Affinities of selected CD3 affinity variants in an IgG1-huCD3-H1L1-FEAL format were determined using Bio-Layer Interferometry on a ForteBio Octet HTX (ForteBio, UK) (Table 6). Anti-human Fc capture biosensors (cat: 18-5060, ForteBio, UK) were loaded for 600 s with hIgG (1 µg / mL). After a baseline (200 s) the association (1000 s) and dissociation (2000 s) of CD3E27-GSKa was determined, using a CD3E27-GSKa concentration range of 27.11 µg / mL - 0.04 µg mL (1000 nM - 1.4 nM) with three-fold dilution steps (sample diluent, cat: 18-5028, ForteBio, UK). For calculations, the theoretical molecular mass of CD3E27-GSKa based on the amino acid sequence was used, i.e. 27.11 kDa. Experiments were carried out while shaking at 1000 rpm and at 30 °C. Each antibody was tested in at at least two independent experiments (Table 6).

[0368] Data was analyzed with ForteBio Data Analysis Software v8.1, using the 1:1 model and a global full fit with 1000 s association time and 100 s dissociation time. Data traces were corrected by subtraction of a reference curve (antibody without CD3E27-GSKa), the Y-axis was aligned to the last 10 s of the baseline, and interstep correction as well as Savitzky-Golay filtering was applied. Data traces with a response <0.05 nm were excluded from analysis. Table 6 Average < KD> (nM)SDEVSEMCV<Kon>SDEVSEMCV<Kdis>SDEVSEMCVpKDnIgG1-huCD3-G105P-FEAL521454.7E+059.7E+045.6E+04212.5E-031.0E-035.8E-04408.33IgG1-huCD3-FEAL1563372.7E+055.1E+042.9E+04194.0E-031.6E-039.1E-04397.83IgG1-huCD3-Y114V-FEAL2984262.2E+053.3E+041.9E+04156.3E-039.7E-045.6E-04157.53IgG1-huCD3-T31P-FEAL4294211.9E+053.8E+041.6E+04207.8E-031.3E-035.3E-04177.46IgG1-huCD3-Y114M-FEAL42148332.6E+056.2E+043.6E+04241.0E-021.5E-038.7E-04157.43IgG1-huCD3-H101N-FEAL45137294.8E+052.2E+051.2E+05452.0E-023.1E-031.8E-03167.33IgG1-huCD3-Y114R-FEAL46106221.5E+054.1E+042.4E+04276.8E-034.1E-042.4E-0467.33IgG1-huCD3-S110A-FEAL72156211.8E+052.5E+041.0E+04141.3E-021.6E-036.4E-04127.16IgG1-huCD3-N57E-FEAL913017332.1E+052.8E+041.6E+04131.9E-024.0E-032.3E-03217.03IgG1-huCD3-T31 M-FEAL992313231.9E+052.5E+041.5E+04141.8E-022.6E-031.5E-03147.03IgG1-huCD3-Y32A-FEAL1053122292.2E+051.1E+057.5E+04482.2E-024.4E-033.1E-03207.02IgG1-huCD3-H101L-FEAL1073923372.7E+054.3E+042.5E+04162.8E-027.5E-034.4E-03277.03IgG1-huCD3-H101K-FEAL1209455792.2E+051.9E+051.1E+05841.7E-029.8E-035.7E-03586.93IgG1-huCD3-S110G-FEAL15312070793.8E+054.2E+052.4E+051122.6E-028.3E-034.8E-03326.83IgG1-huCD3-H101G-FEAL68316997253.0E+049.2E+035.3E+03302.0E-028.5E-044.9E-0446.23IgG1-huCD3-H101I-FEALnd0IgG1-huCD3-H101F-FEALnd0 T cell binding affinity determination of IgG1-huCD3-H1L1-FEAL affinity mutants

[0369] T cells from donor buffy coats (Sanquin, Amsterdam, The Netherlands) were isolated by using RosetteSep human T cell enrichment cocktail (Cat: 15021C.1, Stemcell Technologies, France) according to manufacturer's instructions. Briefly, 50 µL of T cell isolation cocktail was added to 1 mL of buffy coat and incubate at RT for 20 min. Next, the buffy coat was diluted (1:3, v / v) with PBS (cat: 3623140, B.Braun, Germany) and gently transferred to 50 mL falcon tubes (cat: 227261, Greiner bio-one, The Netherlands) filled with 15 mL lymphocyte separation medium (cat: 17-829E, Lonza, Switzerland). Tubes were centrifuged for 20 min at RT 1200xg without brakes. Collect the T cells from the density medium and wash with PBS twice.

[0370] 2x10E6 T cells / mL were resuspended in FACS buffer and transferred to 50 µL into round bottom 96 well plates (cat: 650101, Greiner bio-one, The Netherlands). 50 µL of the antibody solutions in five-fold dilutions was added starting with 5 µg / mL and incubated for 30 min at 4 °C. The 96 plates were centrifuged at 300xg for 5 min at 4 °C and the supernatant discarded. Cells were washed twice with ice cold FACS buffer on ice and the 1:200 diluted secondary antibody (anti IgG Fcy-PE (fab)'2, cat: 109-116-098, Jackson Immuno Research, UK) added to 100 µL / well and incubated for 30 min and washed twice with FACS buffer. Fluorescence intensity was measured on FACS Canto and geometric mean calculated by FlowJo V10 software. Graphs were made by GraphPad (V6.04). See Figure 5.Example 9: In vitro cytotoxicity screening of CD3 affinity mutants Cytotoxicity of CD3 affinity mutants on solid tumor cell lines (alamar blue assay)

[0371] T cells from donor buffy coats (Sanquin, Amsterdam, The Netherlands) were isolated by using RosetteSep human T cell enrichment cocktail (Cat: 15021C.1, Stemcell Technologies, France) according to manufacturer's instructions. NCI-N87 (25.000 cells / well) (Figure 6A), SKOV3 (16.000 cells / well) (Figure 6B) and MDA-MB-231 (16.000 cells / well) (Figure6C) cells were seeded into flat bottom 96 well plates (cat: 655180, Greiner-bio-one, The Netherlands) and adhered for 3-5h at 37 °C. T cells were added to tumor cells in the following ratios: NCI-N87 cell: T cells, 1:3; SKOV3 cell:T cell, 1:4; MDA-MB-231 cell: T cell, 1:8. Subsequently antibody solutions were added in ten-fold dilutions and plates were incubated for 2 days at 37 °C. Next, supernatants were discarded and adhered cells were washed twice with PBS. 150 µL of 10% alamar blue (cat: DAL1100, Life Technologies, The Netherlands) solution prepared in RPMI-1640 (cat: BE12-115F, Lonza, Switzerland) medium containing 10% donor bovine serum with iron (cat: 10371-029, Life Technologies, The Netherlands) was added to wells and incubated for 3-5h at 37 °C. The absorbance was measured with Envision multilabel plate reader (PerkinElmer, US). Staurosporine (cat: S6942, Sigma-Aldrich, US) treated cells were set as 100% kill and untreated cells were set as 0% kill. Viable cells were calculated by subtracting staurosporine treated cells from all groups and the percentage was plotted against the untreated group. Graphs were made by GraphPad (V6.04). See Figure 6.Cytotoxicity of CD3 affinity mutants on a hematological cell line (chromium release assay)

[0372] 5x10E6 Daudi cells / mL were incubated in complete culture medium with 100 µCi chromium for 1h under shaking conditions at 37°C. Next, cells were washed twice in PBS and resuspended in 5 mL complete cell culture medium (10% donor bovine serum with iron in RPMI 1640). 5.000 Daudi cells were seeded into round bottom 96 well plates. T cells from donor buffy coats (purchased from Sanquin, Amsterdam, The Netherlands) were isolated by using RosetteSep human T cell enrichment cocktail (Cat: 15021C.1, Stemcell Technologies, France) according to manufacturer's instructions. T cells are added in (tumor cell: T cell) 1:10 ratio to Daudi cells followed by the addition of the antibody solutions in two-fold dilutions. Plates were in incubated 24h at 37 °C. After 24h, plates were centrifuges at 300xg for 3 min, supernatant was harvested and measured for radioactivity. See Figure 7. Tabel 7% of killHERZ copy#CellsNo killkill ≤ 50%kill ≤ wtkill = wtHER2 ≥ 1.000.000NCI-N87H101I, H101L, H101K, Y32AH101F S110G, H101NN57E, H101GS110A, G105P, S110A, T31M, T31P, Y114M, Y114R, Y114V50.000 ≤ HER2 ≤ 180.000SKOV3H101I, H101F, H101L, Y32AH101KH101G, S110G, H101N, N57ES110A, G105P, S110A, T31M, T31P, Y114M, Y114R, Y114VHER2 ≤ 30.000MDA-MB-231H101F, H101I, S110G, Y32A, H101L, H101K, H101N, N57EH101GS110A, G105P, S110A, T31M, T31P, Y114M, Y114R, Y114V Example 10: Tumor efficacy of CD3xHER2 bispecific antibodies in a , (human PBMC + NCI-N87 cells) co-engraftment model in NOD-SCID mice

[0373] The in vivo anti-tumor efficacy of several CD3xHER2 bispecific antibodies was evaluated in a subcutaneous NCI-N87 co-engraftment model (Figure 8). As a CD3-arm of the bispecific antibody, a humanized WT CD3 (huCD3-FEAL) and 4 different CD3 affinity variants (N57E, H101K, S110A, Y114M) were used. The HER2-targeting arm in all cases was the same (Herceptin-FEAR) BisG1-huCD3-FEALx1014-Herceptin-FEAR BisG1-huCD3-N57E-FEALx1014-Herceptin-FEAR BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR BisG1-huCD3-S11OA-FEALx1014-Herceptin-FEAR BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR

[0374] In this model, HLA-A-matched human unstimulated PBMCs, as a source of human T cells, were co-inoculated with NCI-N87 tumor cells at two different dose levels (0.5 and 0.05 mg / kg).

[0375] Mice were sorted into treatment groups (n=4 per treatment group. At day 0, a mixture containing HLA-A matched hPBMC (5x10E6, Sanquin) and NCI-N87 (5x10E6) cells in 200 µL PBS / 0.1% BSA were inoculated subcutaneously (s.c.) in the right flank of each female NOD-SCID mice (NOD.C.B-17-Prkdc scid< / J), age 6-11 weeks old (Charles-River)). Directly after tumor cell injection, single intravenous dosing (150 µL) of five different CD3xHER2 antibodies was performed at two different concentrations (0.5 and 0.05 mg / kg) for all bispecific antibodies. Tumor volumes were determined at least two times per week. Tumor volumes (mm 3< ) were calculated from caliper (PLEXX) measurements as: 0.52 x (length) x (width) 2< .

[0376] NCI-N87 cells (ATCC# CRL-5822, gastric carcinoma arising from stomach) were thawed, cultured in RPMI 1640 (Lonza, BE12-115F) supplemented with 10% donor bovine serum with iron (Gibco, cat. no. 10371-029), penicillin / streptomycin and 0.45% glucose (Sigma, G8769), sodium pyruvate (Cambrex, BE13-115E) and 0.075% sodium bicarbonate (Cambrex, BE17-613E). Cells were grown in CellSTACK culture chambers and harvested in log-phase and counted by trypan blue exclusion.

[0377] For each study, hPBMCs were isolated from human HLA-A matched donors for NCI-N87 (HLA-A-01,23) from a buffy coat (Sanquin) by Ficoll density centrifugation. Isolated cells were frozen in nitrogen and thawed before use. All cells were washed in PBS / 0.1% BSA, filtered through a cell strainer and resuspended to a concentration of 50 x 10E6 cells / mL in PBS / 0.1% BSA.

[0378] The results are shown in Figure 8.Figure 6A-B show average tumor volumes after treatment over time. Figure 6C-D show dot plot representations of average NCI-N87 tumor volume at day 44. Statistical analysis (Mann-Whitney) on the tumor volumes at day 44, last day that all groups were still intact, revealed at a dose of 0.05 mg / kg a significant tumor growth inhibition (p<0.05) of BisG1-huCD3-FEALx1014-Herceptin-FEAR, BisG1-huCD3-S110A-FEALx1014-Herceptin-FEAR and BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR compared to the control (PBMCs) and not of BisG1-huCD3- N57E-FEALx1014-Herceptin-FEAR and BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR. At a dose of 0.5 mg / kg BisG1-huCD3-FEALx1014-Herceptin-FEAR and all CD3-arm affinity variants, showed significant tumor growth (p<0.05) inhibition compared to the PBS (PBMCs) control group, except BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR.

[0379] BisG1-huCD3-FEALx1014-Herceptin-FEAR, BisG1-huCD3-S110A-FEALx1014-Herceptin-FEAR, and BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR significantly (p<0.05) reduced NCI-N87 tumor volume at dosages of 0.05 and 0.5 mg / kg. BisG1-huCD3-N57E-FEALx1014-Herceptin-FEAR reduced significantly (p<0.05) NCI-N87 tumor volume only at a dosage of 0.5 mg / kg. BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR did not affect NCI-N87 tumor growth at both tested dosages.List of References

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Claims

1. A humanized antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region and a variable light chain (VL) region, wherein said VL region comprises CDR1, CDR2, and CDR3 regions having the CDR sequences as set forth in SEQ ID NO:6, GTN and 7, wherein said VH region comprises the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 176, wherein the antibody is of an IgG1 isotype, and wherein said VH region has a VH sequence set forth in SEQ ID NO: 177, and wherein said VL region comprises a VL sequence selected from the group consisting of; a) a VL sequence as set forth in SEQ ID NO:8; b) a VL sequence as set forth in SEQ ID NO:10.

2. The antibody according to claim 1, wherein the VL region comprises the VL sequence set forth in SEQ ID NO:8.

3. The antibody according to claim 1, wherein the VL region comprises the VL sequence set forth in SEQ ID NO:10.

4. The antibody according to any one of the preceding claims, wherein human CD3 is human CD3 epsilon as specified in SEQ ID NO: 399.

5. The antibody according to any one of the preceding claims, wherein the antibody is a full-length antibody.

6. The antibody according to any one of the preceding claims, wherein the antibody is monovalent, bivalent or multivalent.

7. The antibody according to any one of the preceding claims, wherein said antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

8. The antibody according to any one of the preceding claims, wherein said antibody comprises a first and a second immunoglobulin heavy chain, wherein in at least one of said first and second immunoglobulin heavy chains one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering, are not L, L, D, N, and P, respectively.

9. The antibody according to claim 8, wherein in at least one of said first and second immunoglobulin heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain, is not D.

10. The antibody according to claim 8, wherein in at least one of said first and second heavy chains the amino acid in the position corresponding to position N297 in a human IgG1 heavy chain, is not N.

11. The antibody according to claim 8, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are not L and L, respectively.

12. The antibody according to any of claims 8 and 11, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E; or A and A, respectively.

13. The antibody according to claim 12, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are F and E, respectively.

14. The antibody according to claim 13, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain, are A and A, respectively.

15. The antibody according to claim 7, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively.

16. The antibody according to claim 15, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively.

17. The antibody according to any one of claims 15 to 16, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

18. The antibody according to any one of claims 15 to 16, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are A, A, and A, respectively.

19. The antibody according to claim 8, wherein in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are F, E, A, Q, and S, respectively.

20. A bispecific antibody comprising a first binding region of an antibody according to any one of claims 1 to 19, and a second binding region which binds a different target than said first antigen binding region.

21. The bispecific antibody according to claim 20 wherein said antibody comprises a first and a second heavy chain.

22. The bispecific antibody according to any one of claims 20 to 21, wherein each of said first and second heavy chain comprises at least a hinge region, a CH2 and CH3 region, wherein in said first heavy chain at least one of the amino acids in the positions corresponding to a positions selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and in said second heavy chain at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and wherein said first and said second heavy chains are not substituted in the same positions.

23. The bispecific antibody according to claim 22, wherein the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in said first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in said second heavy chain, or vice versa.

24. The bispecific antibody according to any one of claims 20 to 23, wherein said first binding region is according to any one of claims 1 to 7, and said second binding region binds a different target than said first binding region.

25. An expression vector comprising i. a nucleic acid sequence encoding a heavy chain sequence of a humanized antibody according to any one of claims 1 to 24; and ii. a nucleic acid sequence encoding a light chain sequence of a humanized antibody according to any one of claims 1 to 24.

26. A host cell comprising an expression vector of claim 25.

27. The host cell according to claim 26, wherein said host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell.

28. A composition comprising an antibody according to any one of claims 1 to 7 or a bispecific antibody according to claims 8 to 24.

29. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 7, or the bispecific antibody according to any one of claims 8 to 24 and a pharmaceutical acceptable carrier.

30. The antibody according to any one of claims 1 to 24, the composition according to claim 29, or the pharmaceutical composition according to claim 29 for use as a medicament.

31. The antibody according to any one of claims 1 to 24, the composition according to claim 28, or the pharmaceutical composition according to claim 29 for use as a medicament in treatment of cancer, an infectious disease, or autoimmune diseases.

32. The antibody according to any one of claims 1 to 7, the bispecific antibody according to claims 8 to 24, the composition according to claim 28, or the pharmaceutical composition according to claim 29, for use in the treatment of a disease.

33. A method for producing an antibody according to any one of claims 1 to 7 comprising the steps of a. culturing a host cell according to any one of claims 26 to 27; and b. purifying said antibody from the culture media.

34. A diagnostic composition comprising an antibody according to any one of claims 1 to 7 or a bispecific antibody according to any one of claims 8 to 24.

35. A method for detecting the presence of CD3 antigen, or a cell expressing CD3, in a sample comprising the steps of; a. contacting the sample with an antibody according to any one of claims 1 to 7 or a bispecific antibody according to any one of claims 8 to 24, under conditions that allow for formation of a complex between said antibody or bispecific antibody and CD3; and b. analyzing whether a complex has been formed.

36. A kit for detecting the presence of CD3 antigen, or a cell expressing CD3, in a sample comprising i) an antibody according to any one of claims 1 to 6 or a bispecific antibody according to any one of claims 8 to 24 ; and ii) instructions for use of said kit.

37. An anti-idiotypic antibody which binds to an antibody of any one of claims 1-7.