Humanized or chimeric CD3 antibodies

Abstract

The present invention relates to a humanized or chimeric antibody that binds to CD3. It further relates to bispecific antibodies, compositions, pharmaceutical compositions, the use of said antibodies in the treatment of diseases, and methods of treatment.

Description

[0001] This application is a divisional application of the following application: Application date: July 4, 2014; Application number: 201480048350.3 (PCT / EP2014 / 064326); Invention title: same as above. Field of the Invention

[0002] The present invention relates to humanized or chimeric antibodies that bind to human CD3, compositions comprising the humanized or chimeric antibodies, and the use of the humanized or chimeric antibodies in the treatment of diseases. Background Art

[0003] Cluster of differentiation 3 (CD3) has been known for many years and has thus been a subject of interest in many respects. Specific antibodies against CD3 or the T cell receptor complex of which CD3 is a part are known. The in vitro characterization of five humanized OKT3 effector function variant antibodies has been described [1].

[0004] Treatment with the anti-CD3 monoclonal antibody hOKT3γ1(Ala-Ala) resulted in improved C-peptide responses and clinical parameters for at least 2 years after the onset of type 1 diabetes in the absence of continuous immunosuppressive agents [2].

[0005] A promising approach to improving targeted antibody therapy is to specifically deliver cytotoxic cells to antigen-expressing cancer cells. This concept of using T cells for the efficient killing of tumor cells has been described [3]. However, initial clinical studies have been rather disappointing, mainly due to low efficacy, severe adverse effects (cytokine storm), and the immunogenicity of bispecific antibodies [4]. Advances in the design and application of bispecific antibodies have partially overcome the initial hurdle of cytokine storm and improved clinical efficacy without dose-limiting toxicity [5].

[0006] For example, certain bispecific antibodies with one arm targeting an antigen on tumor cells and the other arm targeting, for example, CD3 on T cells provide Fc receptor binding via the Fc region. After binding, a complex of T cells, tumor cells, and effector cells that bind to the Fc region of the antibody is formed, resulting in the killing of tumor cells [4]. Catumaxomab consists of a murine IgG2a / rat IgG2b heterodimer and has been found to be successfully used for the treatment of cancer-related ascites after intraperitoneal application [6]. However, the mouse / rat hybrid is immunogenic [7] and cannot be used for long-term intravenous treatment in humans. Frequent treatment-related adverse events attributed to catumaxomab include cytokine release-related symptoms (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) [8]-[9], which involve the effector function of the Fc region of catumaxomab. Another antibody is ertumaxomab (HER2xCD3), which induces cytotoxicity in cell lines with low HER2 expression. Ertumaxomab has been in phase II clinical development for metastatic breast cancer

[10] -

[11] .

[0007] CD3 antibodies that cross-react with cynomolgus and / or rhesus macaque CD3 have been described

[12] -

[13] , however, further improvement of such cross-reactive antibodies is needed. SUMMARY OF THE INVENTION

[0008] An object of the present invention is to provide humanized or chimeric CD3 antibodies. Thus, in one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region that comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 4, the sequence GTN, and the sequence shown in SEQ ID NO: 5, respectively.

[0009] In another aspect, the present invention relates to a bispecific antibody that comprises a first binding region of an antibody according to the present invention and a second binding region that binds to a target different from the first antigen-binding region.

[0010] In another aspect, the present invention relates to a nucleic acid construct encoding one or more amino acid sequences according to the present invention.

[0011] In another aspect, the present invention relates to an expression vector comprising: (i) a nucleic acid sequence encoding the heavy chain sequence of a humanized or chimeric antibody according to the present invention, (ii) a nucleic acid sequence encoding the light chain sequence of a humanized or chimeric antibody according to the present invention, or (iii) both (i) and (ii).

[0012] In another aspect, the invention relates to a host cell comprising the expression vector according to the invention.

[0013] In another aspect, the invention relates to a composition comprising an antibody or bispecific antibody according to the invention.

[0014] In another aspect, the invention relates to a pharmaceutical composition comprising an antibody or bispecific antibody according to the invention and a pharmaceutically acceptable carrier.

[0015] In another aspect, the invention relates to an antibody or bispecific antibody, composition or pharmaceutical composition according to the invention for use as a medicament.

[0016] In another aspect, the invention relates to an antibody or bispecific antibody, composition or pharmaceutical composition according to the invention for the treatment of a disease.

[0017] In another aspect, the invention relates to a method for treating a disease, which comprises administering to a subject in need thereof an antibody or bispecific antibody, composition or pharmaceutical composition according to the invention.

[0018] In one aspect, the invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, which comprises administering to a subject a humanized or chimeric antibody, composition or pharmaceutical composition according to the invention, optionally, wherein the humanized or chimeric antibody is labeled with a detectable reagent.

[0019] In another aspect, the invention relates to a method for producing an antibody or bispecific antibody according to the invention, which comprises the steps of a) culturing a host cell according to the invention, and b) purifying the antibody from the culture medium.

[0020] In another aspect, the invention relates to a diagnostic composition comprising an antibody or bispecific antibody according to the invention.

[0021] In another aspect, the invention relates to a method for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, which comprises the steps of a) contacting the sample with an antibody or bispecific antibody according to the invention under conditions allowing the formation of a complex between the antibody or bispecific antibody and CD3, and b) analyzing whether a complex has been formed.

[0022] In another aspect, the invention relates to a kit for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, which comprises i) an antibody or bispecific antibody according to the invention, and ii) instructions for use of the kit.

[0023] In another aspect, the invention relates to an anti-idiotypic antibody that binds to an antibody according to the invention. Brief Description of the Drawings

[0024] Figure 1 : shows ([[]] Figure 1 A) the binding curves of the monospecific antibody variants of IgG1-huCD3 and ([[]] Figure 1 B) the bispecific antibody variant bsIgG1 huCD3xHER2 to the human T cell line Jurkat. The data shown are the mean fluorescence intensities (MFI) of a representative experiment, as described in Example 2. The table shows the antibody concentrations (µg / mL) that result in half-maximal binding (EC50).

[0025] Figure 2 : shows ([[]] Figure 2 A) the binding curves of the monospecific antibody variants of IgG1-huCD3 and ([[]] Figure 2 B) the bispecific antibody variant bsIgG1 huCD3xHER2 to the cynomolgus monkey T cell line HSC-F. The data shown are the mean fluorescence intensities (MFI) of a representative experiment, as described in Example 2.

[0026] Figure 3 : T cell activation by IgG1-huCD3 antibody variants. CD69 expression on T cells from human ([[]] Figure 3 A) and cynomolgus monkey ([[]] Figure 3 B) origin in PBMC cultures was measured by FACS analysis, as described in Example 3. These experiments were performed twice, and representative results from one experiment are shown.

[0027] Figure 4 : T cell proliferation induced by IgG1-huCD3 antibody variants. Human ([[]] Figure 4 A) or cynomolgus monkey ([[]] Figure 4 B) PBMCs were incubated with IgG1-huCD3 antibody variants for 3 days, after which proliferation was measured by cell proliferation ELISA, as described in Example 4. Representative results from two independent experiments are shown.

[0028] Figure 5 : As described in Example 5, the human ([[]] Figure 5 A) and cynomolgus monkey ([[]] Figure 5 B) T cell-mediated cytotoxicity induced by huCD3 antibody variants with inactivated LFLEDA mutations was determined. Representative results from two independent experiments performed in duplicate are shown.

[0029] Figure 6 : shows the inactivated, monospecific antibody variants of IgG1-huCD3 Figure 6(A) and the inactivated, bispecific antibody variant bsIgG1-huCD3xHER2 ( Figure 6 (B) Binding curves to the human T cell line Jurkat. The data shown are the mean fluorescence intensity (MFI) of a representative experiment, as described in Example 2. The table shows the antibody concentration (µg / mL) that results in half-maximal binding (EC50).

[0030] Figure 7 : Shows the inactivated, monospecific antibody variant of IgG1-huCD3 ( Figure 7 (A) and the inactivated, bispecific antibody variant bsIgG1-huCD3xHER2 ( Figure 7 (B) Binding curves to the cynomolgus monkey T cell line HSC-F. The data shown are the mean fluorescence intensity (MFI) of a representative experiment, as described in Example 2. The table shows the antibody concentration (µg / mL) that results in half-maximal binding (EC50).

[0031] Figure 8 : T cell activation by inactivated monospecific IgG1-huCD3 ( Figure 8 (A and B) or inactivated bispecific bsIgG1-huCD3xHER2 antibody variants ( Figure 8 (C and D). CD69 expression on T cells from human ( Figure 8 (A and C) and cynomolgus monkey ( Figure 8 (B and D) origin in PBMC cultures was measured by FACS analysis, as described in Example 3. These experiments were performed twice, and representative results from one experiment are shown.

[0032] Figure 9 : T cell proliferation induced by inactivated monospecific IgG1-huCD3 ( Figure 9 (A and B) or inactivated bispecific bsIgG1-huCD3xHER2 antibody variants ( Figure 9 (C and D). T cell proliferation was measured in human ( Figure 9 (A and C) or cynomolgus monkey ( Figure 9 (B and D) PBMCs, which were incubated with the various antibody variants for 3 days, after which proliferation was measured by cell proliferation ELISA, as described in Example 4. Representative results from two independent experiments are shown.

[0033] Figure 10 : As described in Example 5, the induction of human ( Figure 10 (A) and cynomolgus monkey (( Figure 10B) T cell-mediated cytotoxicity. Representative results from two independent experiments performed in duplicate are shown.

[0034] Figure 11 : Activation of rhesus T cells by IgG1-huCD3 antibody variants. CD69 expression on T cells of rhesus origin in PBMC cultures was measured by FACS analysis as described in Example 6.

[0035] Figure 12 : Activation of T cells by non-activated variants of huCLB-T3 / 4 antibody. IgG1-huCLB-T3 / 4 variants were titrated on PBMCs. CD69 expression on T cells in PBMC cultures was measured by FACS analysis as described in Example 7. Representative examples of 3 experiments are shown.

[0036] Figure 13 : Proliferation of T cells by non-activated variants of huCLB-T3 / 4 antibody. PBMCs were incubated with the antibody for three days, after which proliferation was measured by cell proliferation ELISA as described in Example 8. Representative results from two independent experiments are shown.

[0037] Figure 14 : In vitro T cell-mediated cytotoxicity induced by non-activated antibody variants of CD3 antibody. As described in Example 9, T cell-mediated cytotoxicity induced by antibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS Figure 14 A-G]) was determined. The mean values from two experiments performed in duplicate are shown.

[0038] Figure 15 : In vitro T cell-mediated cytotoxicity induced by non-activated huCLB-T3 / 4 variants. As described in Example 9, T cell-mediated cytotoxicity induced by antibody variants (LFLEDA LAL Figure 15 A-C] was determined. The mean value from one experiment performed in duplicate is shown.

[0039] Figure 16 : Evaluation of the binding of C1q to non-activated huCLB-T3 / 4 antibody variants. As described in Example 10, the binding of C1q to monospecific IgG1 huCLB-T3 / 4 ( Figure 16 A-C) and bsIgG1-huCLB-T3 / 4xHER2 (Figure B-D) and their non-activated antibody variants was evaluated by ELISA. The results in the graph represent n = 2 experiments.

[0040] Figure 17: As described in Example 11, the pharmacokinetic (PK) analysis of the inactivated huCLB-T3 / 4 antibody variant was compared to that of the wild-type IgG1-huCLB-T3 / 4 antibody. The plasma concentration of human IgG1 was plotted against time ( Figure 17 A). As described in Example 11, the plasma clearance was calculated ( Figure 17 B). The horizontal dashed line represents the mean clearance rate of human IgG1 antibody in SCID mice (10 mL / day / kg).

[0041] Figure 18 : Frequency of positive T cell responses in healthy HLA-typed donors. An SI index of ≥1.9 in the proliferation and IL-2 secretion assays was considered a positive response. Humanized A33 was used as a clinical benchmark control antibody, which showed a high level of immunogenicity in clinical settings and routinely induced 20 - 30% T cell responses in the EpiScreen assay. KLH responses were included to check PBMC quality (after thawing).

[0042] Figure 19 : Sequence alignment of the heavy chain (VH) and light chain (VL) variable regions of the humanized CD3 antibody according to the present invention. Detailed Description

[0043] In one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region that comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 4, the sequence GTN, and the sequence shown in SEQ ID NO: 5, respectively.

[0044] As used herein, the term "antibody" means an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, which has the ability to specifically bind to an antigen under normal physiological conditions and has a half-life of a significant period 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 (e.g., a time sufficient to induce, promote, enhance, and / or regulate a physiological response associated with antibody and antigen binding, and / or a time sufficient for antibody recruitment of effector activity). The binding region that interacts with the antigen (or the binding domain, which may also be used herein, and the two terms have the same meaning) comprises the variable regions of both the heavy and light chains of the immunoglobulin molecule. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., 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 shown above, unless the context otherwise indicates or is clearly contradictory, the term antibody as used herein includes antibody fragments that retain the ability to selectively interact (e.g., bind) with an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antibody" include (i) Fab' or Fab fragments, monovalent fragments consisting of the V L 、V H 、C L and C H 1 domains, or monovalent antibodies as described in WO2007059782 (Genmab A / S); (ii) F(ab')2 fragments, divalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragments consisting essentially of V H and C H 1 domains; and (iv) Fv fragments consisting essentially of the V L and V H domains of a single arm of an antibody. In addition, although the two domains V L and V H of the Fv fragment are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be prepared as a single protein chain, in which V L and V HThe regions pair to form monovalent molecules (referred to as single-chain antibodies or single-chain Fvs (scFvs); see, e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are also intended to be encompassed within the term antibody unless the context clearly indicates otherwise. Although such fragments are generally included within the meaning of antibody, they are both collectively and individually unique features of the present invention, exhibiting different biological properties and utilities. These and other useful antibody fragments in the context of the present invention are discussed further herein. It should also be understood that unless otherwise indicated, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen, which are provided by any known technique such as enzymatic cleavage, peptide synthesis and recombinant techniques. Antibodies so generated can have any isotype.

[0045] As used herein, the term "immunoglobulin heavy chain", "heavy chain of an immunoglobulin" or "heavy chain" means one of the immunoglobulin chains. A heavy chain typically comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which define the isotype of the immunoglobulin. The heavy chain constant region typically comprises three domains: CH1, CH2 and CH3. The heavy chain constant region may further comprise a hinge region. As used herein, the term "immunoglobulin" means a class of structurally related glycoproteins consisting of two pairs of polypeptide chains: a pair of low molecular weight light (L) chains and a pair of heavy (H) chains, all four chains potentially interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, e.g.,

[14] ). Within the structure of an immunoglobulin, the two heavy chains are interconnected via disulfide bonds in the so-called "hinge region". Similarly to the heavy chains, each light chain typically comprises 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 comprises one domain: CL. In addition, the VH and VL regions may be further subdivided into hypervariable regions (or regions that may be hypervariable in sequence and / or in the form of structurally defined loops) also known as complementarity determining regions (CDRs), separated by more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see

[15] ). CDR sequences can be determined by using the methods provided by IMGT

[16] -

[17] .

[0046] As used herein, the term "isotype" refers to an immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof such as IgG1m(za) and IgG1m(f) [SEQ ID NO:15], which is encoded by a heavy chain constant region gene. Thus, in one embodiment, the antibody comprises an immunoglobulin of the IgG1 class or a heavy chain of any allotype thereof. Further, each heavy chain isotype can be combined with a kappa (κ) or lambda (λ) light chain.

[0047] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., derived from a rodent), and the constant region is derived from a different species such as human. Chimeric antibodies can be generated by antibody engineering. "Antibody engineering" is a term generally used for the modification of antibodies of different species and is a process well known to those skilled in the art. In particular, chimeric antibodies can be generated by using standard DNA techniques as described in

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

[0048] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody that contains human antibody constant domains and non-human variable domains that have been modified to contain a high level of sequence homology with human variable domains. This can be achieved by transplantation of six non-human antibody complementarity determining regions (CDRs), which together form the antigen-binding site on the homologous human receptor framework region (FR) (see

[19] -

[20] ). In order to fully reconstruct the binding affinity and specificity of the parental antibody, it may be necessary to substitute framework residues from the parental antibody (i.e., the non-human antibody) into the human framework region (backmutation). Structural homology modeling can assist in identifying amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody can contain non-human CDR sequences, predominantly human framework regions, optionally containing one or more amino acid backmutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications that are not necessarily backmutations can be applied to obtain a humanized antibody with preferred characteristics such as affinity and biochemical properties.

[0049] A humanized or chimeric antibody according to any aspect or embodiment of the present invention may be referred to as a "humanized or chimeric CD3 antibody", "the humanized or chimeric antibody of the present invention", "CD3 antibody", or "the CD3 antibody of the present invention", all of which have the same meaning and use, unless the context is otherwise contradictory.

[0050] The amino acid sequence of an antibody of non-human origin is different from that of an antibody of human origin, and thus when administered to a human patient, a non-human antibody is potentially immunogenic. However, despite the non-human origin of the antibody, its CDR regions are responsible for the ability of the antibody to bind to its target antigen, and humanization is aimed at maintaining the specificity and binding affinity of the antibody. Thus, a non-human therapeutic antibody is humanized to minimize its immunogenicity in humans while such humanized antibody maintains the specificity and binding affinity of the antibody of non-human origin.

[0051] As used herein, the term "binding region" refers to an antibody region capable of binding to any molecule, such as a polypeptide, present on, for example, a cell, bacterium, or virus particle.

[0052] As used herein, the term "binding" refers to the binding of an antibody to a predetermined antigen or target, which, when using the antigen as a ligand and the antibody as an analyte, as determined by surface plasmon resonance (SPR) technology in, for example, a BIAcore 3000 instrument, typically has a K corresponding to the following D affinity: about 10 -6 M or less, such as 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, and compared to the affinity for binding non-specific antigens (such as BSA, casein) other than the predetermined antigen or closely related antigens, binds to the predetermined antigen with an affinity corresponding to the following K D affinity: at least ten-fold lower, such as at least 100-fold lower, such as at least 1,000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower. The degree of reduction in affinity depends on the K of the antibody D such that when the K of the antibody D is extremely low (i.e., the antibody is highly specific), the degree to which the affinity for the antigen is lower than that for non-specific antigens can be at least 10,000-fold. As used herein, the term "K D "(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.

[0053] As used herein, the term "human CD3" refers to the human Cluster of Differentiation 3 protein, which is part of the T cell co-receptor protein complex and consists of four different chains. CD3 has also been found in other species, and thus, the term "CD3" may be used herein and is not limited to human CD3, unless contradicted by the context. In mammals, the complex contains the CD3γ (gamma) chain (human CD3γ chain Swissprot P09693, or cynomolgus monkey CD3γ Swissprot Q95LI7), the CD3δ (delta) chain (human CD3δ Swissprot P04234, or cynomolgus monkey CD3δ Swissprot Q95LI8), two CD3ε (epsilon) chains (human CD3ε Swissprot P07766; or cynomolgus monkey CD3ε Swissprot Q95LI5), rhesus monkey CD3ε (Swissprot G7NCB9), and the CD3ζ - chain (zeta) chain (human CD3ζ Swissprot P20963, cynomolgus monkey CD3ζ Swissprot Q09TK0). These chains bind to a molecule called the T cell receptor (TCR) and generate an activation signal in T lymphocytes. The TCR and CD3 molecules together comprise the TCR complex.

[0054] The amino acid sequences mentioned as Swissprot numbers include the signal peptides removed after protein translation, which is within the knowledge of a person skilled in the art. Thus, proteins present on the cell surface such as CD3 do not include the signal peptides. In particular, the amino acid sequences listed in Table 1 do not contain such signal peptides. Such proteins listed in Table 1 may be referred to as "mature proteins". Thus, SEQ ID NO:14 shows the amino acid sequence of mature human CD3δ (delta), SEQ ID NO:13 shows the amino acid sequence of mature human CD3ε (epsilon), SEQ ID NO:21 shows the amino acid sequence of mature cynomolgus monkey CD3ε, and SEQ ID NO:23 shows the amino acid sequence of mature rhesus monkey CD3ε. Thus, as used herein, the term "mature" refers to a protein that does not contain any signal or leader sequence.

[0055] It is well known that signal peptide sequence homology, length, and cleavage site location vary significantly between different proteins. Signal peptides can be determined by different methods, for example, SEQ ID NO:13 of the present invention has been determined according to the SignalP application (available at http: / / www.cbs.dtu.dk / services / SignalP / ).

[0056] In a particular embodiment, the humanized or chimeric antibody of the invention binds to the epsilon chain of CD3, such as the epsilon chain of human CD3 (SEQ ID NO:13). In yet another particular embodiment, the humanized or chimeric antibody binds to an epitope within amino acids 1-27 of the N-terminal portion of human CD3ε (epsilon) (SEQ ID NO:13). In such particular embodiments, the antibody can even further cross-react with other non-human primate species such as cynomolgus monkeys (cynomolgus monkey CD3ε SEQ ID NO:21) and / or rhesus monkeys (rhesus monkey CD3ε SEQ ID NO:23).

[0057] As used herein, the term "cross-react" refers to the ability of an antibody, such as a humanized or chimeric antibody according to the invention, to bind its target on different species. In particular, the humanized CD3 antibody exemplified in the examples described herein has the ability to bind human (Example 2), cynomolgus monkey (Example 2), and rhesus monkey CD3.

[0058] An antibody according to the invention comprising CDR sequences as defined herein and further comprising framework regions can differ in the sequences outside the CDR sequences but still retain the full binding ability as compared to the original antibody. Accordingly, the invention also relates to antibodies comprising an amino acid sequence of a variable region having a certain sequence identity with any of the sequences described herein.

[0059] As used in the context herein, the term "sequence identity" refers to the percentage of identity between two sequences (i.e., % homology = # of identical positions / # of total positions x 100) based on the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for the best alignment of the two sequences and the length of each gap. The percentage of identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller

[21] . Additionally, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm

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

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

[0060] Accordingly, in one embodiment, the VH region has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with at least one amino acid sequence as shown in a VH sequence selected from:

[0061] a) the VH sequence as shown in SEQ ID NO:6;

[0062] b) a VH sequence as shown in SEQ ID NO:8;

[0063] c) a VH sequence as shown in SEQ ID NO:7; and

[0064] d) a VH sequence as shown in SEQ ID NO:9.

[0065] In a particular embodiment, the VH region has at least 96% amino acid sequence identity with at least one amino acid sequence as shown in a VH sequence selected from:

[0066] a) a VH sequence as shown in SEQ ID NO:6;

[0067] b) a VH sequence as shown in SEQ ID NO:8;

[0068] c) a VH sequence as shown in SEQ ID NO:7; and

[0069] d) a VH sequence as shown in SEQ ID NO:9.

[0070] In one embodiment, the VL region has at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity with at least one amino acid sequence as shown in a VL sequence selected from:

[0071] a) a VL sequence as shown in SEQ ID NO:10;

[0072] b) a VL sequence as shown in SEQ ID NO:11; and

[0073] c) a VL sequence as shown in SEQ ID NO:12.

[0074] In a particular embodiment, the VL region has at least 95% amino acid sequence identity with at least one amino acid sequence as shown in a VL sequence selected from:

[0075] a) a VL sequence as shown in SEQ ID NO:10;

[0076] b) a VL sequence as shown in SEQ ID NO:11; and

[0077] c) a VL sequence as shown in SEQ ID NO:12.

[0078] In one embodiment, the VH region is selected from:

[0079] a) a VH sequence as shown in SEQ ID NO:6;

[0080] b) a VH sequence as shown in SEQ ID NO:8;

[0081] c) a VH sequence as shown in SEQ ID NO:7; and

[0082] d) a VH sequence as shown in SEQ ID NO:9.

[0083] In one embodiment, the VL region is selected from:

[0084] a) a VL sequence as shown in SEQ ID NO:10;

[0085] b) a VL sequence as shown in SEQ ID NO:11; and

[0086] c) a VL sequence as shown in SEQ ID NO:12.

[0087] In one embodiment, only one of the VH or VL sequences is 100% identical to one of the sequences disclosed herein, and the other may have a sequence identity of at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to one of the sequences disclosed herein.

[0088] In a particular embodiment, the VH region has at least 97% amino acid sequence identity to at least one amino acid sequence as shown in a VH sequence selected from:

[0089] a) a VH sequence as shown in SEQ ID NO:6;

[0090] b) a VH sequence as shown in SEQ ID NO:7;

[0091] c) a VH sequence as shown in SEQ ID NO:8; and

[0092] d) a VH sequence as shown in SEQ ID NO:9;

[0093] and the VL sequence has at least 95% amino acid sequence identity to at least one amino acid sequence as shown in a VL sequence selected from:

[0094] i. a VL sequence as shown in SEQ ID NO:10;

[0095] ii. a VL sequence as shown in SEQ ID NO:11; and

[0096] iii. a VL sequence as shown in SEQ ID NO:12.

[0097] In one embodiment, the VH and VL sequences are selected from the following:

[0098] a) VH and VL sequences having at least 90% identity with the sequences shown in SEQ ID NO:6 and 10; 7 and 10; 8 and 10; 9 and 10; 6 and 11; 7 and 11; 8 and 11; 9 and 11; 6 and 12; 7 and 12; 8 and 12; and 9 and 12, respectively;

[0099] b) VH and VL sequences having at least 95% identity with the sequences shown in SEQ ID NO:6 and 10; 7 and 10; 8 and 10; 9 and 10; 6 and 11; 7 and 11; 8 and 11; 9 and 11; 6 and 12; 7 and 12; 8 and 12; and 9 and 12, respectively;

[0100] c) VH and VL sequences having at least 97% identity with the sequences shown in SEQ ID NO:6 and 10; 7 and 10; 8 and 10; 9 and 10; 6 and 11; 7 and 11; 8 and 11; 9 and 11; 6 and 12; 7 and 12; 8 and 12; and 9 and 12, respectively;

[0101] d) VH and VL sequences having at least 99% identity with the sequences shown in SEQ ID NO:6 and 10; 7 and 10; 8 and 10; 9 and 10; 6 and 11; 7 and 11; 8 and 11; 9 and 11; 6 and 12; 7 and 12; 8 and 12; and 9 and 12, respectively;

[0102] e) VH and VL sequences having at least 100% identity with the sequences shown in SEQ ID NO:6 and 10; 7 and 10; 8 and 10; 9 and 10; 6 and 11; 7 and 11; 8 and 11; 9 and 11; 6 and 12; 7 and 12; 8 and 12; and 9 and 12, respectively;

[0103] f) A VH sequence having at least 90% identity with the sequence shown in SEQ ID NO:6, and a VL sequence having at least 95% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0104] g) A VH sequence having at least 90% identity with the sequence shown in SEQ ID NO:6, and a VL sequence having at least 97% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0105] h) A VH sequence having at least 90% identity with the sequence shown in SEQ ID NO:6, and a VL sequence having at least 99% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0106] i) A VH sequence having at least 90% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0107] j) A VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0108] k) A VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0109] l) A VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 99% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0110] m) A VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0111] n) A VH sequence having at least 97% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0112] o) A VH sequence having at least 97% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 95% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0113] p) A VH sequence having at least 97% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 99% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0114] q) A VH sequence having at least 97% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0115] r) A VH sequence having at least 99% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0116] s) A VH sequence having at least 99% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 95% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0117] t) A VH sequence having at least 99% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0118] u) A VH sequence having at least 99% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0119] v) A VH sequence having at least 100% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0120] x) A VH sequence having at least 100% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 95% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0121] y) A VH sequence having at least 100% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0122] z) A VH sequence having at least 100% identity to the sequence shown in SEQ ID NO:6, and a VL sequence having at least 99% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0123] aa) A VH sequence having at least 90% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 95% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0124] ab) A VH sequence having at least 90% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11, or 12;

[0125] ac) A VH sequence having at least 90% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 99% identity to the sequence shown in SEQ ID NO:10, 11, or 12;

[0126] ad) A VH sequence having at least 90% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11, or 12;

[0127] ae) A VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11, or 12;

[0128] af) A VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11, or 12;

[0129] ag) A VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 99% identity to the sequence shown in SEQ ID NO:10, 11, or 12;

[0130] ah) A VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11, or 12;

[0131] ai) A VH sequence having at least 97% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11, or 12;

[0132] aj) A VH sequence having at least 97% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 95% identity to the sequence shown in SEQ ID NO:10, 11, or 12;

[0133] ak) A VH sequence having at least 97% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 99% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0134] al) A VH sequence having at least 97% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0135] am) A VH sequence having at least 99% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0136] an) A VH sequence having at least 99% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 95% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0137] ao) A VH sequence having at least 99% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0138] ap) A VH sequence having at least 99% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0139] aq) A VH sequence having at least 100% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0140] ar) A VH sequence having at least 100% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 95% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0141] as) A VH sequence having at least 100% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0142] at) a VH sequence having at least 100% identity to the sequence shown in SEQ ID NO:7, and a VL sequence having at least 99% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0143] ba) a VH sequence having at least 90% identity to the sequence shown in SEQ ID NO:8, and a VL sequence having at least 95% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0144] bb) a VH sequence having at least 90% identity to the sequence shown in SEQ ID NO:8, and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0145] bc) a VH sequence having at least 90% identity to the sequence shown in SEQ ID NO:8, and a VL sequence having at least 99% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0146] bd) a VH sequence having at least 90% identity to the sequence shown in SEQ ID NO:8, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0147] be) a VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:8, and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0148] bf) a VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:8, and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0149] bg) a VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:8, and a VL sequence having at least 99% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0150] bh) a VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:8, and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0151] bi) a VH sequence having at least 97% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 90% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0152] bj) a VH sequence having at least 97% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 95% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0153] bk) a VH sequence having at least 97% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 99% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0154] bl) a VH sequence having at least 97% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 100% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0155] bm) a VH sequence having at least 99% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 90% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0156] bn) a VH sequence having at least 99% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 95% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0157] bo) a VH sequence having at least 99% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 97% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0158] bp) a VH sequence having at least 99% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 100% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0159] bq) a VH sequence having at least 100% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 90% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0160] br) A VH sequence having at least 100% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 95% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0161] bs) A VH sequence having at least 100% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 97% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0162] bt) A VH sequence having at least 100% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 99% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0163] ca) A VH sequence having at least 90% identity with the sequence shown in SEQ ID NO:8, and a VL sequence having at least 95% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0164] cb) A VH sequence having at least 90% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 97% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0165] cc) A VH sequence having at least 90% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 99% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0166] cd) A VH sequence having at least 90% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 100% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0167] ce) A VH sequence having at least 95% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 90% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0168] cf) A VH sequence having at least 95% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 97% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0169] cg) A VH sequence having at least 95% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 99% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0170] ch) A VH sequence having at least 95% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 100% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0171] ci) A VH sequence having at least 97% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 90% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0172] cj) A VH sequence having at least 97% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 95% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0173] ck) A VH sequence having at least 97% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 99% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0174] cl) A VH sequence having at least 97% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 100% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0175] cm) A VH sequence having at least 99% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 90% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0176] cn) A VH sequence having at least 99% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 95% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0177] co) A VH sequence having at least 99% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 97% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0178] cp) A VH sequence having at least 99% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 100% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0179] cq) A VH sequence having at least 100% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 90% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0180] cr) A VH sequence having at least 100% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 95% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0181] cs) A VH sequence having at least 100% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 97% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0182] ct) A VH sequence having at least 100% identity with the sequence shown in SEQ ID NO:9, and a VL sequence having at least 99% identity with the sequence shown in SEQ ID NO:10, 11 or 12;

[0183] In one embodiment, the binding region comprises a VH and a VL selected from:

[0184] a) A VH sequence as shown in SEQ ID NO:6, and a VL sequence as shown in SEQ ID NO:10;

[0185] b) A VH sequence as shown in SEQ ID NO:8, and a VL sequence as shown in SEQ ID NO:10;

[0186] c) A VH sequence as shown in SEQ ID NO:9, and a VL sequence as shown in SEQ ID NO:10;

[0187] d) A VH sequence as shown in SEQ ID NO:6, and a VL sequence as shown in SEQ ID NO:11;

[0188] e) A VH sequence as shown in SEQ ID NO:6, and a VL sequence as shown in SEQ ID NO:12;

[0189] f) The VH sequence as shown in SEQ ID NO:7, and the VL sequence as shown in SEQ ID NO:10;

[0190] g) The VH sequence as shown in SEQ ID NO:7, and the VL sequence as shown in SEQ ID NO:11;

[0191] h) The VH sequence as shown in SEQ ID NO:7, and the VL sequence as shown in SEQ ID NO:12;

[0192] i) The VH sequence as shown in SEQ ID NO:8, and the VL sequence as shown in SEQ ID NO:11;

[0193] j) The VH sequence as shown in SEQ ID NO:8, and the VL sequence as shown in SEQ ID NO:12;

[0194] k) The VH sequence as shown in SEQ ID NO:9, and the VL sequence as shown in SEQ ID NO:11; and

[0195] l) The VH sequence as shown in SEQ ID NO:9, and the VL sequence as shown in SEQ ID NO:12.

[0196] In a particular embodiment, the binding region comprises a VH sequence and a VL sequence selected from the following:

[0197] a) The VH sequence as shown in SEQ ID NO:6, and the VL sequence as shown in SEQ ID NO:10;

[0198] b) The VH sequence as shown in SEQ ID NO:8, and the VL sequence as shown in SEQ ID NO:10; and

[0199] c) The VH sequence as shown in SEQ ID NO:9, and the VL sequence as shown in SEQ ID NO:10.

[0200] Humanized antibodies according to the present invention can be generated by comparing heavy and light chain variable region amino acid sequences against a database of human germline variable region sequences in order to identify human heavy and light chain sequences with an appropriate degree of homology to be used as the human variable framework regions. A series of humanized heavy and light chain variable regions can be designed by, for example, transplanting murine CDRs into the framework regions (identified as above) and, if necessary, by backmutating (mutating one or more of the human amino acid residues in the framework region back to the non-human amino acid at a particular position) to the specific murine sequence of the residue(s) identified, which may be critical for the restoration of antibody binding efficiency. As by applying techniques on computer chips; iTope TM and TCED TM (

[24] ,

[25] , and

[26] ), variant sequences with the lowest incidence of potential T cell epitopes can then be selected.

[0201] In addition, humanized antibodies according to the present invention can also be "deimmunized". Deimmunization may be desirable because within the protein sequence, for example, in a humanized antibody according to the present invention, the presence of human T cell epitopes can increase the immunogenic risk profile as they have the potential to activate helper T cells. Such activation of helper T cells can be avoided by deimmunization. Deimmunization can be carried out by introducing mutations into the amino acid sequence of the humanized antibody to remove T cell epitopes without significantly reducing the antibody affinity of the antibody.

[0202] Thus, in one embodiment of the present invention, a humanized antibody can be produced by a method comprising the steps of: (i) comparing a non-human full variable heavy chain sequence and / or full variable light chain sequence against a database of human germline sequences, (ii) selecting the human germline sequence with the highest homology to the non-human sequence to obtain a humanized sequence, (iii) optimizing the humanized sequence by backmutation if necessary, and (iv) expressing the sequence in a suitable expression system.

[0203] Thus, a full-length antibody according to the present invention can be produced by a method comprising the steps of: (i) comparing a non-human variable heavy chain sequence and variable light chain sequence against a database of human germline sequences, (ii) selecting the human germline sequence with the highest homology to the non-human sequence, (iii) transplanting non-human CDRs into the selected human germline to obtain a humanized sequence, (iv) optimizing the humanized sequence by backmutation if necessary, (v) identifying the constant heavy and light chain sequences, and (vi) expressing the full heavy chain sequence and full light chain sequence in a suitable expression system. A full-length antibody according to the present invention can thus be produced as described in Example 1. Generating a full-length antibody starting from the CDR sequences or full variable region sequences is within the knowledge of a person skilled in the art. Thus, a person skilled in the art will know how to generate a full-length antibody according to the present invention.

[0204] As used herein, the term "complete heavy chain sequence" refers to a sequence consisting of a variable heavy chain and a constant heavy chain sequence.

[0205] As used herein, the term "complete light chain sequence" refers to a sequence consisting of a variable light chain and a constant light chain sequence.

[0206] Back mutations can be introduced by standard DNA mutagenesis. Such standard techniques for DNA mutagenesis are described in

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

[0207] Thus, in one embodiment, the antibody is a humanized antibody.

[0208] Chimeric antibodies can be generated by replacing all constant region sequences of a non-human (e.g., murine) antibody with constant region sequences of human origin. Thus, the complete non-human variable region sequences are maintained in the chimeric antibody. Thus, a chimeric antibody according to the present invention can be produced by a method comprising the steps of expressing a non-human variable heavy chain (SEQ ID NO: 27), a non-human variable light chain sequence (SEQ ID NO: 28), a human constant heavy chain, and a human constant light chain sequence in a suitable expression system, and thereby generating a full-length chimeric antibody. Alternative methods can be used. Such methods for producing chimeric antibodies are within the knowledge of the person skilled in the art, and thus the person skilled in the art will know how to produce a chimeric antibody according to the present invention.

[0209] Thus, in one embodiment, the antibody is a chimeric antibody.

[0210] In one embodiment, the antibody is a full-length antibody. As used herein, the term "full-length antibody" refers to an antibody (e.g., a parental or variant antibody) that contains all heavy and light chain constant and variable domains corresponding to those typically found in the wild-type antibody of that isotype.

[0211] In one embodiment, the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

[0212] As used herein, the term "Fc region" refers to a region that comprises at least a hinge region, a CH2 region, and a CH3 region in the direction from the N-terminus to the C-terminus. The Fc region can further comprise a CH1 region at the N-terminus of the hinge region.

[0213] As used herein, the term "hinge region" 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 shown in Kabat.

[0214] Unless otherwise indicated or inconsistent with the context, the amino acids of the constant region sequences are numbered herein according to the Eu numbering index (described in

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

[0215] As used herein, the term "CH1 region" or "CH1 domain" 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 can also have any of the other subtypes described herein.

[0216] As used herein, the term "CH2 region" or "CH2 domain" 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 can also have any of the other subtypes described herein.

[0217] As used herein, the term "CH3 region" or "CH3 domain" 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 can also have any of the other subtypes described herein.

[0218] In one embodiment, the isotype of the immunoglobulin heavy chain is selected from IgG1, IgG2, IgG3, and IgG4. The immunoglobulin heavy chain can be any allotype within each immunoglobulin class, such as IgG1m(f) (SEQ ID NO:15). Thus, in a particular embodiment, the isotype of the immunoglobulin heavy chain is IgG1 or any of its allotypes, such as IgG1m(f) (SEQ ID NO:15).

[0219] When targeting the antigen CD3, which is part of the T cell receptor (TCR), the T cell - specific mechanism of cell killing is desired. Other effector functions such as complement activation can be undesirable, and thus a reduction in effector function is desired. C1q binding is the first step in the complement cascade and thus serves as an indicator of the complement - dependent cytotoxicity (CDC) ability of an antibody. If the binding of C1q to the antibody can be avoided, then the activation of the complement cascade can also be avoided.

[0220] Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that, compared to a wild-type antibody, binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% or 100%, wherein C1q binding is determined by ELISA.

[0221] As used herein, the term "modified" means that the amino acid sequence of the Fc region is not identical to the amino acid sequence of the wild-type Fc region. That is, an amino acid residue at a specific position in the wild-type Fc region has been substituted, deleted or inserted in order to alter, for example, the binding site for C1q, the binding site for other effector molecules or the binding to Fc receptors (FcR). Such modifications of the amino acid sequence can be prepared by substituting one or more amino acids with conservative amino acids, or can be prepared by substituting one or more amino acids with alternative amino acids that are physically and / or functionally similar to the amino acids present in the wild-type. Substitutions can also be prepared by substituting with non-conservative amino acids.

[0222] In the context of the present invention, amino acids can be described as conservative or non-conservative amino acids and can thus be classified accordingly. Amino acid residues can also be divided into classes defined by alternative physical and functional properties. Thus, amino acid classes can be reflected in one or both of the following tables:

[0223] Conservative classes of amino acid residues

[0224] Acidic residue D and E Basic residue K, R and H Hydrophilic uncharged residue S, T, N and Q Aliphatic uncharged residue G, A, V, L and I Nonpolar uncharged residue C, M and P Aromatic residue F, Y and W

[0225] Alternative physical and functional classifications of amino acid residues

[0226] Alcohol group-containing residue S and T Aliphatic residue I, L, V and M Cycloalkenyl-related residue F, H, W and Y Hydrophobic residue A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively charged residue D and E Polar residue C, D, E, H, K, N, Q, R, S and T Positively charged residue H, K and R Small residue A, C, D, G, N, P, S, T and V Minimal residue A, G and S Residue involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible residue Q, T, K, S, G, P, D, E and R

[0227] In the context of the present invention, substitutions in antibodies such as humanized or chimeric antibodies are as follows:

[0228] Original amino acid - position - substituted amino acid;

[0229] Referring to the recognized amino acid nomenclature, the three-letter code or single-letter code is used, including the codes Xaa and X to indicate any amino acid residue. Thus, the notation "L234F" or "Leu234Phe" means that the antibody contains a substitution of leucine at amino acid position 234 by phenylalanine.

[0230] An amino acid substitution at a given position by any other amino acid is referred to as:

[0231] Original amino acid - position; or for example "L234".

[0232] For modifications in which the original amino acid and / or substituted amino acid may contain more than one, but not all, amino acids, more than one amino acid may be separated by "," or " / ". For example, the substitution of leucine at position 234 with phenylalanine, arginine, leucine, or tryptophan is:

[0233] "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".

[0234] Such designations may be used interchangeably in the context of the present invention and have the same meaning and purpose.

[0235] In addition, the term "substitution" includes substitution with any one of the other nineteen natural amino acids, or other amino acids such as non-natural amino acids. For example, the substitution of amino acid L at 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, for example, equivalent to specifying 234X, where X specifies any amino acid other than the original amino acid. These substitutions may also be designated as L234A, L234C, etc., or L234A,C, etc., or L234A / C / , etc. This also applies analogously to each and every position mentioned herein to specifically include any one of such substitutions herein.

[0236] Antibodies according to the present invention may also contain deletions of amino acid residues. Such deletions may be indicated as "del" and include, for example, written as L234del. Thus, in such an embodiment, the leucine at position 234 has been deleted from the amino acid sequence.

[0237] The terms "amino acid" and "amino acid residue" may be used interchangeably herein.

[0238] As used herein, when the antibody binds to its antigen, the term "C1q binding" refers to the binding of C1q to the antibody. As used herein, the term "binds to its antigen" refers to the binding of the antibody to its antigen in vivo and in vitro.

[0239] As used herein, when referring to C1q binding, the term "reduced" means that the antibody according to the present invention reduces, minimizes, or even completely inhibits the ability of C1q to bind to the antibody when compared to the binding of C1q to the wild-type antibody.

[0240] As used herein, in the context of the use of an antibody according to the invention in a comparative assay, the term "wild-type antibody" refers to an antibody that is equivalent to the antibody to be tested, except that it is not inert. In this context, the term "inert" refers to a modified Fc region having reduced or no C1q binding, as determined in Example 10, i.e., where C1q binding is determined by ELISA; reduced or no Fc-mediated T cell proliferation (i.e., T cell proliferation) as determined as described in Example 4, measured in a peripheral blood mononuclear cell (PBMC)-based functional assay; and / or reduced or no Fc-mediated CD69 expression (i.e., Fc-mediated CD69 expression) as determined as described in Example 3, determined in a PBMC-based functional assay. Thus, a wild-type antibody contains the naturally occurring amino acids in the immunoglobulin heavy chain, i.e., an antibody without any amino acid modifications that might alter or reduce the ability of the antibody to interact with, for example, C1q, Fc receptors, and the like. Thus, such wild-type antibodies remain as activating antibodies that are capable of binding, for example, C1q. Wild-type antibodies and the antibodies of the invention may contain other amino acid modifications other than those that affect the ability of the antibody to induce effector functions, so as to render the antibody a bispecific antibody, etc.

[0241] As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay, which is a test that uses antibodies and color change to identify substances. A first specific antibody is attached to the plate surface. Thereby, proteins from a sample are added, and the binding to the first specific antibody is tested. A second antibody that binds the antibody from the sample is added. The second antibody is linked to an enzyme, and in a final step, a substance containing the substrate for the enzyme is added. A subsequent reaction produces a detectable signal, most commonly a color change in the substrate. The concept of the ELISA method is well known in the art, and the various methods of performing an ELISA are considered part of the methods for evaluating the antibodies according to the invention. Thus, this explanation should not be construed as limiting, as various forms of ELISA can be performed as described, for example, in Example 4.

[0242] Specifically, the ability of an antibody according to the invention to bind C1q can be determined by an ELISA comprising the following steps: (i) coating the antibody onto a 96-well plate, (ii) adding 3% serum, (iii) adding an anti-human C1q antibody, (iv) developing the plate, and (v) measuring the OD 405nm. Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that, compared to a wild-type antibody, binding of C1q to the antibody is reduced 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, the ELISA comprising the steps of: (i) coating the antibody onto a 96-well plate, (ii) adding 3% serum, (iii) adding anti-human C1q, (iv) developing the plate, and (v) measuring the OD 405 nm. Thus, in a particular embodiment, binding of C1q is evaluated as described in Example 10.

[0243] As used herein, the term "Fc receptor" or "FcR" refers to a protein found on the surface of certain cells. FcRs bind to the Fc region of an antibody. There are several different types of FcRs, which are classified based on the type of antibody they recognize. For example, Fcγ (gamma) receptors bind to antibodies of the IgG class.

[0244] As used herein, the term "Fcγ receptor", "Fc gamma receptor" or "FcγR" refers to a group of Fc receptors belonging to the immunoglobulin superfamily and is the most important Fc receptor for inducing phagocytosis of opsonized (coated) microorganisms. This family includes several members: FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), FcγRIIIb (CD16b), which differ in their antibody affinities due to their different molecular structures.

[0245] Fc-mediated effector functions form part of the biological activity of human immunoglobulin G (IgG) molecules. Examples of such effector functions include, for example, 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 the antibody Fc region to an FcR" refer to the binding of the Fc region to an Fc receptor (FcR) or an effector molecule. The terms "FcγR binding" and "FcγRI binding" refer to the binding of the Fc region to an Fc gamma receptor and an Fc gamma receptor I, respectively. When a CD3 antibody binds to a T cell, the wild-type Fc region of the CD3 antibody binds to FcRs present on other cells such as monocytes, which results in non-specific, Fc-mediated T cell activation. Such non-specific, Fc-mediated T cell activation may be undesirable. T cells can also be activated by targeted or target-specific T cell activation. Such targeted T cell activation may be highly desirable for a range of indications such as the treatment of cancer. As used herein, the term "targeted T cell activation" refers to the directed targeting of T cells to specific cells such as tumour cells by using a bispecific antibody that comprises a first binding region that binds to a specific target such as a tumour target on a tumour cell and a second binding region that binds to a T cell-specific target such as CD3. Thus, the targeting of T cells to specific cells such as tumour cells can be facilitated by using a bispecific antibody in which one of the binding regions binds to CD3 present on the T cell and the other binding region binds to a target-specific antigen such as on a tumour cell. Although non-specific Fc-mediated T cell activation may still be possible and thus such undesirable non-specific Fc-mediated T cell activation via Fc-mediated cross-linking should be avoided and can be abrogated by rendering the Fc region inert to such activity. Thereby, the interaction between the inert Fc region and the Fc receptor is prevented. When tested in several different assays, i.e. see Examples 3 - 5, the humanized antibodies of the present invention have proven to be inert. When tested in different assays, i.e. see Examples 7 - 10, another tested CD3 antibody huCLB-T3 / 4 comprising amino acid modifications in the Fc region has also proven to be inert. As described in the Examples, the humanized CD3 antibody according to the present invention comprising the amino acid substitutions L234F, L235E and D265A, shows low levels of CD69 expression on T cells (Example 3), abrogation of Fc-mediated T cell proliferation (Example 4), and non-specific target killing when in the form of a bispecific antibody (Example 5). Thus, the humanized antibodies of the present invention show excellent results in several assays when compared to wild-type antibodies.

[0246] Antibodies 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-activated antibody. As used herein, the terms "inert", "inertia" or "non-activated" refer to an Fc region that is at least unable to bind any Fcγ receptor, induce Fc-mediated FcR cross-linking, or induce FcR-mediated cross-linking of the target antigen via the two Fc regions of an individual antibody, or is unable to bind C1q. Using antibodies in a monospecific form, the inertia of the Fc region of a humanized or chimeric CD3 antibody is advantageously tested, although the inertia Fc regions so identified can be used in bispecific or other humanized or chimeric multispecific CD3 antibodies.

[0247] Several variants can be constructed to render the Fc region of an antibody inactive for interaction with Fc gamma receptors and C1q for therapeutic antibody development. Examples of such variants are described herein.

[0248] Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that, compared to the wild-type antibody, the antibody-mediated reduced Fc-mediated T cell proliferation is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein the T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMC).

[0249] As used herein, the term "reduction" refers to a reduction in activity or expression when compared to a control protein such as an antibody. In particular, when referring to T cell proliferation, the term "reduction" refers to the ability of an antibody according to the present invention to reduce, minimize or even completely inhibit T cell proliferation when compared to the T cell proliferation bound by a wild-type antibody. The ability of an antibody to reduce T cell proliferation can be evaluated by a functional assay based on PBMC as described in Examples 4 and 8. In one embodiment, the assay is performed with human PBMC. In another embodiment, the assay is performed with cynomolgus monkey PBMC. In yet another embodiment, the assay is performed with rhesus monkey PBMC. Since the antibodies according to the present invention are cross-reactive, the PBMC-based assays described herein can be performed with PBMC of any species to show a reduction in T cell proliferation, provided that the species PBMC used is within the cross-reactivity profile of the antibody, such as human, cynomolgus monkey or rhesus monkey.

[0250] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay for evaluating the functional characteristics of the antibodies of the present invention, such as the ability of the antibodies to affect T cell proliferation or CD69 expression, wherein the only cells present are peripheral blood mononuclear cells. Thus, in one embodiment, T cell proliferation is measured by a method comprising the steps of: incubating PBMC with an antibody in the range of 1 - 1000 ng / mL for three days at 37°C in a 5% (v / v) CO2 humidified incubator, adding a chemical compound such as BrdU incorporated into the DNA of proliferating cells, incubating for five hours, pelleting the cells, drying the cells, optionally storing the cells at 4°C, coating the cells onto an ELISA plate, incubating with anti-BrdU peroxidase for 90 minutes at room temperature, developing with 1 mg / mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) for approximately 30 minutes, adding 100 μL of 2% oxalic acid to stop the reaction, and measuring the absorbance at 405 nm in a suitable microplate reader.

[0251] As used herein, the term "proliferation" refers to cell growth in the context of cell division.

[0252] As used herein, the term "BrdU" refers to 5-bromo-2'-deoxyuridine, which is an analogue of thymidine. When BrdU is added to a 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 can be performed in an ELISA using anti-BrdU peroxidase. BrdU incorporation is thus a measure of proliferation.

[0253] In one embodiment, the antibody comprises an Fc region that has been modified such that, when compared to a wild-type antibody, the 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%, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0254] As used herein, the term "reduced" refers to a decrease in activity or expression as compared to a control protein, such as an antibody. In particular, when referring to the expression level of the T cell activation marker CD69, the term "reduced" refers to a decrease in the CD69 expression level when compared to the CD69 expression level when the T cell is bound by a wild-type antibody, provided that both binding regions of the antibody bind CD3. The ability of an antibody to reduce CD69 expression can be evaluated by a PBMC-based functional assay as described in Examples 3 and 7. Thus, in one embodiment, the expression of CD69 is measured by a method comprising the steps of: incubating PBMC with an antibody in the range of 1 - 1000 ng / mL at 37°C in a 5% (v / v) CO2 humidified incubator for 16 - 24 hours, washing the cells, staining the cells with mouse anti-human CD28-PE and mouse anti-human CD69-APC antibodies at 4°C, and determining the CD69 expression on CD28-positive cells by flow cytometry.

[0255] As used herein, the term "CD69" refers to cluster of differentiation 69, which is a human transmembrane C-type lectin protein encoded by CD69 a gene. Activation of T lymphocytes and natural killer (NK) cells in vivo and in vitro induces the expression of CD69. CD69 acts as a signaling receptor involved in cell activation events including proliferation, as a signaling receptor in lymphocytes including natural killer cells and platelets, and the induction of specific genes.

[0256] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay used to evaluate the functional characteristics of the antibodies of the present invention, such as the ability of the antibody to affect T cell proliferation or CD69 expression, wherein the only cells present are peripheral blood mononuclear cells. As described in Examples 3, 4, 5, and 7, the PBMC-based functional assay comprises the steps of: (i) incubating PBMC with an antibody at 37°C in a 5% (v / v) CO2 humidified incubator for about 16 - 24 hours, (ii) washing the cells, (iii) staining the cells with mouse anti-human CD28-PE and mouse anti-human CD69-APC antibodies at 4°C, and (iv) when evaluating CD69 expression, determining the CD69 expression on CD28-positive cells by flow cytometry. Thus, in one embodiment, the CD69 expression can be determined as described in Examples 3, 4, 5, or 7.

[0257] Thus, the amino acids in the Fc region that play a significant role in the interaction with C1q and Fc Gamma receptors can be modified. Examples of amino acid positions that can be modified include L234, L235, and P331. Combinations thereof such as L234F / L235E / P331S can cause a significant reduction in the binding to human CD64, CD32A, CD16, and C1q.

[0258] Thus, in one embodiment, the amino acids at at least one position corresponding to L234, L235, and P331 can be A, A, and S respectively ([1],

[28] ). The L234F and L235E amino acid substitutions can also result in the abrogation of the interaction of the Fc region with Fc Gamma receptors and C1q (

[29] -

[30] ). Thus, in one embodiment, the amino acids at the positions corresponding to L234 and L235 can be F and E respectively. The D265A amino acid substitution can reduce the binding to all Fc gamma receptors and block ADCC (

[31] ). Thus, in one embodiment, the amino acid at the position corresponding to D265 can be A. The binding to C1q can be abrogated by mutating the positions D270, K322, P329, and P331. Mutating these positions to D270A or K322A or P329A or P331A can render the antibody defective in CDC activity (

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

[0259] An alternative method of minimizing the interaction of the Fc region with Fc gamma receptors and C1q is by removing the glycosylation sites of the antibody. Mutating the position N297 to, for example, Q, A, and E removes the glycosylation sites critical for the IgG-Fc gamma receptor interaction. Thus, in one embodiment, the amino acid at the position corresponding to N297 can be G, Q, A, or E (

[33] ). Another alternative method of minimizing the interaction of the Fc region with Fc gamma receptors can be obtained by the following mutations: P238A, A327Q, P329A, or E233P / L234V / L235A / G236del (

[31] ).

[0260] Optionally, although interactions with Fcγ receptors have been reported (

[34] -

[35] ), human IgG2 and IgG4 subclasses are considered to be naturally compromised in their interactions with C1q and Fcγ receptors. Mutations that abrogate these residual interactions can be made in both isotypes, resulting in a reduction of unwanted side effects associated with FcR binding. For IgG2, these include L234A and G237A, and for IgG4, these include L235E. Thus, in one embodiment, the amino acids at positions corresponding to L234 and G237 in the human IgG2 heavy chain can be A and A, respectively. In one embodiment, the amino acid at the position corresponding to L235 in the human IgG4 heavy chain can be E.

[0261] Other methods for further minimizing the interactions of IgG2 antibodies with Fcγ receptors and C1q include those described in

[36] and

[37] .

[0262] The hinge region of an antibody can also have importance with respect to interactions with Fcγ receptors and complement (

[38] -

[39] ). Thus, mutations or deletions in the hinge region can affect the effector functions of the antibody.

[0263] As used herein, the term "crosslinking" refers to the indirect bridging of the Fab arms (monovalent or divalent) of an antibody that binds to a target antigen by cells bearing FcRs via binding to the Fc region of the antibody. Thus, an antibody that binds to a target antigen on a cell bearing that target antigen can crosslink to another cell expressing an FcR.

[0264] As used herein, the term "non-specific killing" refers to the killing of cells by the cytotoxic function of T cells or other effector cells via tumor target antigen-independent activation of said cells. Thus, non-specific killing means that cells bearing a tumor target can be killed by, for example, cytotoxic T cells, rather than the antibody binding to the tumor target by, for example, inducing CDC.

[0265] The inventors have shown (see Examples 3-5, 7-10) that a non-activated Fc region can be obtained by modifying one or more of at least five specific amino acid positions in the Fc region.

[0266] Thus, in one embodiment, an antibody comprises first and second immunoglobulin heavy chains, wherein in at least one of the first and second immunoglobulin heavy chains, one or more of the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0267] In one embodiment, in both the first and second immunoglobulin heavy chains, one or more of the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0268] In another embodiment, in at least one of the first and second heavy chains, one or more of the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are not L, L, and D, respectively, and the amino acids at positions corresponding to N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0269] As used herein, the term "amino acid corresponding to a position" refers to the amino acid position number in the human IgG1 heavy chain. Unless the context otherwise indicates or is inconsistent, the amino acids of the constant region sequences are numbered herein according to the Eu numbering index (described in

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

[0270] In the context of the present invention, amino acids can be defined as described above.

[0271] When referring to an amino acid in a heavy chain, the term "amino acid is not" or similar wording should be understood to mean that the amino acid is any other amino acid other than the specific amino acid. For example, the amino acid at the position corresponding to L234 in the human IgG1 heavy chain is not L, meaning that the amino acid can be any one of the other natural or non-natural amino acids other than L.

[0272] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is not D.

[0273] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to D265 in the human IgG1 heavy chain is not D, and the amino acids at positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0274] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is a hydrophobic or polar amino acid.

[0275] As used herein, the term "hydrophobic" with respect to an amino acid residue refers to an amino acid residue selected from the following: A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from the following amino acids: A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0276] As used herein, the term "polar" with respect to an amino acid residue refers to any amino acid residue selected from the following: C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human heavy chain is selected from: C, E, H, K, N, Q, R, S, and T.

[0277] In another embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is an aliphatic uncharged, aromatic, or acidic amino acid.

[0278] As used herein, the term "aliphatic uncharged" with respect to an amino acid residue refers to any amino acid residue selected from the following: A, G, I, L, and V. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: A, G, I, L, and V.

[0279] As used herein, the term "aromatic" with respect to an amino acid residue refers to any amino acid residue selected from the following: F, T, and W. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: F, T, and W.

[0280] As used herein, the term "acidic" with respect to an amino acid residue refers to any amino acid residue selected from the following: D and E. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: D and E.

[0281] In a particular embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: A, E, F, G, I, L, T, V, and W.

[0282] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is not D.

[0283] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to D265 in the human IgG1 heavy chain is not D, and the amino acids at the positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0284] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is a hydrophobic or polar amino acid.

[0285] As used herein, the term "hydrophobic" with respect to an amino acid residue refers to an amino acid residue selected from the following: A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from the following amino acids: A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0286] As used herein, the term "polar" with respect to an amino acid residue refers to any amino acid residue selected from the following: C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human heavy chain is selected from: C, E, H, K, N, Q, R, S, and T. In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from the following amino acids: A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0287] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human heavy chain is selected from: C, E, H, K, N, Q, R, S, and T.

[0288] In another embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is an aliphatic uncharged, aromatic, or acidic amino acid.

[0289] As used herein, the term "aliphatic uncharged" with respect to an amino acid residue refers to any amino acid residue selected from the following: A, G, I, L, and V. Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: A, G, I, L, and V.

[0290] As used herein, the term "aromatic" with respect to an amino acid residue refers to any amino acid residue selected from: F, T, and W. Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: F, T, and W.

[0291] As used herein, the term "acidic" with respect to an amino acid residue refers to any amino acid residue selected from: D and E. Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: D and E.

[0292] In a particular embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: A, E, F, G, I, L, T, V, and W.

[0293] In a further embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position N297 in the human IgG1 heavy chain is not N.

[0294] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to N297 in the human IgG1 heavy chain is not N, and the amino acid at the position corresponding to position P331 in the human IgG1 heavy chain is P.

[0295] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position N297 in the human IgG1 heavy chain is not N.

[0296] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to N297 in the human IgG1 heavy chain is not N, and the amino acid at the position corresponding to position P331 in the human IgG1 heavy chain is P.

[0297] In a further embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are not L and L, respectively.

[0298] In one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are not L and L, respectively, and the amino acids at the positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0299] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 in the human IgG1 heavy chain are selected from: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V.

[0300] In one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are hydrophobic or polar amino acids.

[0301] As used herein, the term "hydrophobic" with respect to an amino acid residue refers to an amino acid residue selected from: A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each selected from: A, C, F, G, H, I, M, R, T, V, W, and Y.

[0302] As used herein, the term "polar" with respect to an amino acid residue refers to any amino acid residue selected from: C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each selected from the following amino acids: C, D, E, H, K, N, Q, R, S, and T.

[0303] In a particular embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each selected from: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0304] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are not L and L, respectively.

[0305] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to L234 and L235 in the human IgG1 heavy chain are not L and L, respectively, and the amino acids at the positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0306] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are hydrophobic or polar amino acids.

[0307] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from: A, C, F, G, H, I, M, R, T, V, W, and Y.

[0308] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from the following amino acids: C, D, E, H, K, N, Q, R, S, and T.

[0309] In a particular embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0310] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are aliphatic uncharged, aromatic, or acidic amino acids.

[0311] As used herein, the term "aliphatic uncharged" with respect to an amino acid residue refers to any amino acid residue selected from: A, G, I, L, and V. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from: A, G, I, and V.

[0312] As used herein, the term "aromatic" with respect to an amino acid residue refers to any amino acid residue selected from: F, T, and W. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from: F, T, and W.

[0313] As used herein, the term "acidic" with respect to an amino acid residue refers to any amino acid residue selected from: D and E. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from: D and E.

[0314] In a particular embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 are each independently selected from: A, D, E, F, G, I, T, V, and W.

[0315] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively; or A and A.

[0316] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in the human IgG1 heavy chain are F and E, respectively; or A and A, and the amino acids at positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0317] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively; or A and A.

[0318] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in the human IgG1 heavy chain are F and E, respectively; or A and A, and the amino acids at positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0319] In a particular embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively.

[0320] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively.

[0321] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to at least positions L234 and L235 in the human IgG1 heavy chain are A and A, respectively.

[0322] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to at least positions L234 and L235 in the human IgG1 heavy chain are A and A, respectively.

[0323] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are not L, L, and D, respectively.

[0324] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are not L, L, and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0325] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 in the human IgG1 heavy chain are selected from: 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: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, and W.

[0326] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are hydrophobic or polar amino acids.

[0327] As used herein, the term "hydrophobic" with respect to an amino acid residue refers to an amino acid residue selected from: A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from the following amino acids: A, C, F, G, H, I, L, M, R, T, V, W, and Y, and the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each selected from: A, C, F, G, H, I, M, R, T, V, W, and Y.

[0328] As used herein, the term "polar" with respect to an amino acid residue refers to any amino acid residue selected from: C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each selected from the following amino acids: C, D, E, H, K, N, Q, R, S, and T, and the amino acid at the position corresponding to position D265 in the human heavy chain is selected from: C, E, H, K, N, Q, R, S, and T.

[0329] In a particular embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0330] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are hydrophobic or polar amino acids.

[0331] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from the following amino acids: A, C, F, G, H, I, L, M, R, T, V, W, and Y, and the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from: A, C, F, G, H, I, M, R, T, V, W, and Y.

[0332] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from the following amino acids: C, D, E, H, K, N, Q, R, S, and T, and the amino acid at the position corresponding to position D265 in the human heavy chain is selected from: C, E, H, K, N, Q, R, S, and T.

[0333] In a particular embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each independently selected from: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0334] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are aliphatic uncharged, aromatic, or acidic amino acids.

[0335] As used herein, the term "aliphatic uncharged" with respect to an amino acid residue refers to any amino acid residue selected from the following: A, G, I, L, and V. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: A, G, I, L, and V, and the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each selected from: A, G, I, and V.

[0336] As used herein, the term "aromatic" with respect to an amino acid residue refers to any amino acid residue selected from the following: F, T, and W. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are each selected from: F, T, and W.

[0337] As used herein, the term "acidic" with respect to an amino acid residue refers to any amino acid residue selected from the following: D and E. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are each selected from: D and E.

[0338] In a particular embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from: A, E, F, G, I, L, T, V, and W, and the amino acids at the positions corresponding to L234 and L235 are each selected from: A, D, E, F, G, I, T, V, and W.

[0339] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are not L, L, and D, respectively.

[0340] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are not L, L, and D, respectively, and the amino acids at the positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0341] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are aliphatic uncharged, aromatic, or acidic amino acids.

[0342] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to position D265 in the human IgG1 heavy chain are selected from: A, G, I, L, and V, and the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each selected from: A, G, I, and V.

[0343] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are each selected from: D and E.

[0344] In a particular embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to position D265 in the human IgG1 heavy chain are selected from: A, E, F, G, I, L, T, V, and W, and the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are each selected from: A, D, E, F, G, I, T, V, and W.

[0345] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A.

[0346] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A, and the amino acids at positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0347] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A.

[0348] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A, and the amino acids at positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively.

[0349] In a particular embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0350] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0351] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are A, A, and A, respectively.

[0352] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are A, A, and A, respectively.

[0353] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are F, E, A, Q, and S, respectively.

[0354] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are F, E, A, Q, and S, respectively.

[0355] In a particular embodiment, the antibody according to the invention comprises a VH sequence as shown in SEQ ID NO:8, a VL sequence as shown in SEQ ID NO:10, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0356] In another embodiment, the antibody according to the invention comprises a VH sequence as shown in SEQ ID NO:8, a VL sequence as shown in SEQ ID NO:12, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0357] In another embodiment, the antibody according to the invention comprises a VH sequence as shown in SEQ ID NO:6, a VL sequence as shown in SEQ ID NO:10, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0358] In another embodiment, an antibody according to the invention comprises a VH sequence as shown in SEQ ID NO:6, a VL sequence as shown in SEQ ID NO:12, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0359] In another embodiment, an antibody according to the invention comprises a VH sequence as shown in SEQ ID NO:9, a VL sequence as shown in SEQ ID NO:10, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0360] In another embodiment, an antibody according to the invention comprises a VH sequence as shown in SEQ ID NO:9, a VL sequence as shown in SEQ ID NO:12, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0361] In one aspect, the invention relates to a multispecific antibody comprising at least a first binding region of an antibody according to any aspect or embodiment described herein, and one or more binding regions that bind to one or more targets different from the first binding region. Such multispecific antibodies can be bispecific antibodies.

[0362] Thus, in one aspect, the invention relates to a bispecific antibody comprising a first binding region of an antibody according to any aspect or embodiment described herein, and a second binding region that binds to a target different from the first binding region.

[0363] The term "multispecific antibody" refers to an antibody that is specific for at least two different, for example at least three, typically non-overlapping epitopes. Such epitopes can be on the same or different targets. If the epitopes are on different targets, such targets can be on the same cell or different cells or cell types.

[0364] The term "bispecific antibody" refers to an antibody that is specific for at least two different, typically non-overlapping epitopes. Such epitopes can be on the same or different targets. If the epitopes are on different targets, such targets can be on the same cell or different cells or cell types.

[0365] In one embodiment, the bispecific antibody comprises a first and a second heavy chain.

[0366] Embodiments involving modifications of the Fc region and embodiments involving specific amino acid substitutions are considered part of any bispecific antibody according to the invention. Thus, in one embodiment, at least one of the first and second heavy chains comprises one or more amino acids modified as defined in any of the embodiments described herein, such as those described with respect to providing an inert Fc region. In one embodiment, both the first and second heavy chains comprise one or more amino acids modified as defined in any of the embodiments described herein, such as those described with respect to providing an inert Fc region. Thus, the bispecific antibody comprises an Fc region modified according to any aspect or embodiment described herein; or at least one of the first and second heavy chains comprises one or more amino acids modified as defined in any aspect or embodiment described herein.

[0367] Thus, in one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:10; and wherein the Fc region has been modified such that, compared to the wild-type antibody, the binding of C1q to the antibody is reduced 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.

[0368] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:12; and wherein the Fc region has been modified such that, compared to the wild-type antibody, the binding of C1q to the antibody is reduced 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.

[0369] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:10; and wherein the Fc region has been modified such that, compared to the wild-type antibody, the binding of C1q to the antibody is reduced 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.

[0370] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:12; and wherein the Fc region has been modified such that, compared to the wild-type antibody, the binding of C1q to the antibody is reduced 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.

[0371] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:10; and wherein the Fc region has been modified such that, compared to the wild-type antibody, the binding of C1q to the antibody is reduced 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.

[0372] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:12; and wherein the Fc region has been modified such that, compared to the wild-type antibody, the binding of C1q to the antibody is reduced 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.

[0373] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:10; and wherein the Fc region has been modified such that, compared to the wild-type antibody, the reduced Fc-mediated T cell proliferation mediated by the antibody is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein the T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

[0374] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:12; and wherein the Fc region has been modified such that, compared to the wild-type antibody, the reduced Fc-mediated T cell proliferation mediated by the antibody is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein the T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

[0375] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:10; and wherein the Fc region has been modified such that, compared to a wild-type antibody, the antibody-mediated reduced Fc-mediated T cell proliferation is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein the T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

[0376] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:12; and wherein the Fc region has been modified such that, compared to a wild-type antibody, the antibody-mediated reduced Fc-mediated T cell proliferation is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein the T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

[0377] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:10; and wherein the Fc region has been modified such that, compared to a wild-type antibody, the antibody-mediated reduced Fc-mediated T cell proliferation is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein the T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

[0378] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:12; and wherein the Fc region has been modified such that, compared to a wild-type antibody, the antibody-mediated reduced Fc-mediated T cell proliferation is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein the T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

[0379] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:10; and wherein the Fc region has been modified such that when compared to a wild-type antibody, the 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%, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0380] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:12; and wherein the Fc region has been modified such that when compared to a wild-type antibody, the 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%, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0381] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:10; and wherein the Fc region has been modified such that when compared to a wild-type antibody, the 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%, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0382] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:12; and wherein the Fc region has been modified such that when compared to a wild-type antibody, the 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%, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0383] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:10; and wherein the Fc region has been modified such that, when compared to a wild-type antibody, the 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%, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0384] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:12; and wherein the Fc region has been modified such that, when compared to a wild-type antibody, the 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%, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0385] In a particular embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:10; and wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0386] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:12; and wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0387] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:10; and wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0388] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:12; and wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0389] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:10; and wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0390] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:12; and wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0391] Examples of bispecific antibody molecules that can be used in the present invention include (i) a single antibody having two arms comprising different antigen-binding regions, (ii) single-chain antibodies specific for two different epitopes, such as two scFvs tandemly linked via an additional peptide linker; (iii) dual variable domain antibodies (DVD-Ig TM ), wherein each light and heavy chain contains two variable domains tandemly linked by a short peptide (

[40] ); (iv) chemically linked bispecific (Fab’)2 fragments; (v) TandAb®, which is a fusion of two single-chain diabodies, resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) flexibody, which is a combination of an scFv and a diabody, resulting in a multivalent molecule; (vii) the so-called "dock and lock" molecule (Dock-and-Lock®), based on the "dimerization and docking domain" in protein kinase A, which can result in a trivalent bispecific binding protein composed of two identical Fab fragments linked to different Fab fragments when applied to Fab; (viii) the so-called Scorpion molecule, which comprises, for example, two scFvs fused to the two ends of a human Fab arm; and (ix) diabodies.

[0392] In one embodiment, the bispecific antibodies of the invention are diabodies, cross-bodies or bispecific antibodies obtained via controlled Fab-arm exchange, such as DuoBody® (e.g., as described in

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

[0393] 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 where each side of the molecule contains Fab fragments or portions of Fab fragments of at least two different antibodies; (iii) IgG fusion molecules where a full-length IgG antibody is fused to an additional Fab fragment or portion of a Fab fragment; (iv) Fc fusion molecules where a single-chain Fv molecule or a stable diabody is fused to a heavy-chain constant domain, Fc region or a portion thereof; (v) Fab fusion molecules where different Fab fragments are fused together and fused to a heavy-chain constant domain, Fc region or a portion thereof; and (vi) ScFv- and diabody-based antibodies and heavy-chain antibodies (e.g., domain antibodies, Nanobodies®), where different single-chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, Nanobodies®) are fused to each other or fused to another protein or carrier molecule, and the other protein or carrier molecule is fused to a heavy-chain constant domain, Fc region or a portion thereof.

[0394] Examples of IgG-like molecules with complementary CH3 domains include, but are not limited to, Triomab® (Trion Pharma / Fresenius Biotech,

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

[43] ), CrossMAbs (Roche,

[44] ) and electrostatically matched (Amgen,

[45] -

[46] ; Chugai,

[47] ; Oncomed,

[48] ), LUZ-Y (Genentech), DIG-bodies and PIG-bodies (Pharmabcine), (strand-exchange engineered domain bodies) (SEED bodies) (EMD Serono,

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

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

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

[52] ), mAb-Fv (Xencor,

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

[41] ).

[0395] Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to, dual-targeting (DT)-Ig (GSK / Domantis), two-in-one antibody (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star,

[54] ), Zybodies TM (Zyngenia), methods using a common light chain (Crucell / Merus,

[55] ), kappa lambda bodies (NovImmune), and CovX-body® (CovX / Pfizer).

[0396] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig TM (Abbott,

[56] ), dual domain diabody (Unilever; Sanofi Aventis,

[57] ), IgG-like bispecific (ImClone / Eli Lilly), Ts2Ab (MedImmune / AZ), and BsAb (Zymogenetics), HERCULES (Biogen Idec,

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

[59] ), and TvAb (Roche,

[59] ,

[60] ).

[0397] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), dual affinity retargeting technology (Fc-DART TM ) (MacroGenics,

[62] ,

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

[0398] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), dual action or dual Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0399] Examples of ScFv-, diabody-based antibodies, and domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE®) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Re-Targeting technology (DART TM ) (MacroGenics), single-chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Biotech), Dual Targeting Nanobody® (Ablynx), Dual Targeting Heavy Chain Only Domain Antibodies.

[0400] Any monospecific antibody that further meets the assay conditions described herein can form the basis of a bispecific antibody. That is, a bispecific antibody in which one of the binding regions binds CD3 can be derived from any monospecific CD3 antibody that is tested in a functional assay and meets the requirements described herein. Such bispecific antibodies can be provided by the methods described in

[41] , which reference is incorporated herein by reference.

[0401] Thus, in a particular embodiment, the first and second heavy chains each comprise at least a hinge region, CH2, and CH3 regions, wherein in the first heavy chain, at least one of the amino acids at positions corresponding to positions selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain has been replaced, and in the second heavy chain, at least one of the amino acids at positions corresponding to positions selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain has been replaced, and wherein the first and the second heavy chains are not replaced at the same positions. In this context, the term "replaced" means that the amino acid at a specific amino acid position has been replaced by another naturally or non-naturally occurring amino acid. Thus, a "replaced" amino acid at a position corresponding to a position in the human IgG1 heavy chain means that the amino acid at a specific position is different from the naturally occurring amino acid in the IgG1 heavy chain.

[0402] In one embodiment, in the first heavy chain, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is not K, L, or M, and optionally, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is F, and in the second heavy chain, at least one of the amino acids at positions corresponding to positions selected from T366, L368, K370, D399, F405, and Y407 in the human IgG1 heavy chain has been replaced.

[0403] In one embodiment, in the first heavy chain, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is not K, L, or M, and in the second heavy chain, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is not F, and optionally, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is K.

[0404] In one embodiment, in the first heavy chain, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is not F, R, or G, and in the second heavy chain, the amino acid at the position corresponding to a position selected from T366, L368, K370, D399, Y407, and K409 in the human IgG1 heavy chain has been substituted.

[0405] In one embodiment, in the first heavy chain, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is not K, L, or M, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is not F.

[0406] In a further embodiment, in the first heavy chain, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R, or vice versa.

[0407] Thus, in one embodiment, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the second heavy chain.

[0408] In a further embodiment, the humanized or chimeric CD3 antibody of the invention contains one or more of the inactivating substitutions disclosed in any of the above embodiments in at least one of the first and second heavy chains, such as L234F, L235E, and D265A; and the amino acid at the position corresponding to F405 is not F. In one embodiment, the humanized or chimeric CD3 antibody of the invention contains one or more of the inactivating substitutions disclosed in any of the above embodiments in at least one of the first and second heavy chains, such as L234F, L235E, and D265A; and a further substitution at the K409 position, such as K409R. In particular, in one embodiment, the humanized or chimeric CD3 antibody of the invention contains one or more of the inactivating substitutions disclosed in any of the above embodiments in both the first and second heavy chains, such as L234F, L235E, and D265A; and a substitution at the F405 position, such as F405L. In one embodiment, the humanized or chimeric CD3 antibody of the invention contains one or more of the inactivating substitutions disclosed in any of the above embodiments in both the first and second heavy chains, such as L234F, L235E, and D265A; and a further substitution at the K409 position, such as K409R. Such antibodies can be used to generate bispecific antibodies.

[0409] Thus, in a further embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A respectively, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain, and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain.

[0410] In one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are F, E, A, N, and P respectively, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain, and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain.

[0411] In an alternative embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A respectively, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the second heavy chain.

[0412] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are F, E, A, N, and P, respectively, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the second heavy chain.

[0413] In another embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain, and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain.

[0414] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are F, E, A, N, and P, respectively, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain, and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain.

[0415] In an alternative embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the second heavy chain.

[0416] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are F, E, A, N, and P, respectively, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the second heavy chain.

[0417] As described herein, the recruitment of T cells to specific target cells such as cancer or tumor cells provides a method for killing the target cells. The inventors have shown that, as described in Example 5, a bispecific CD3xHER2 antibody comprising specific amino acid substitutions L234F, L235E, and D265A in two heavy chains is capable of killing AU565 cells. T cell-mediated killing can be achieved with a bispecific antibody that targets CD3 with a first binding region and another target with a second binding region. Thus, in one embodiment, the first binding region is according to any of the embodiments described herein for a humanized or chimeric CD3 antibody, and the second binding region binds a target different from the first binding region. It should be understood that when the antibody is a bispecific antibody, at least half of the antibody, i.e., a pair of heavy and light chains of the antibody, is a humanized or chimeric antibody as described herein. Thus, one half of the bispecific antibody is a humanized or chimeric antibody that binds CD3 according to the present invention, and the other half can be humanized, chimeric, fully non-human, or fully human that binds a second target. Thus, in one embodiment, the antibody comprises a first and a second heavy chain, and a first and a second light chain, wherein the first heavy chain and the first light chain are humanized or chimeric and are linked via a disulfide bridge to form a first binding region; and the second heavy and light chains are fully human and are linked via a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds a different target. In one embodiment, the antibody comprises a first and a second heavy chain, and a first and a second light chain, wherein the first heavy chain and the first light chain are humanized or chimeric and are linked via a disulfide bridge to form a first binding region; and the second heavy and light chains are humanized or chimeric and are linked via a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds a CD3 epitope different from the first binding region.

[0418] Thus, in one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8, and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:19, and a VL sequence as shown in SEQ ID NO:20; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0419] In one embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0420] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0421] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0422] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:12; a second binding region comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0423] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:12; a second binding region comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0424] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:12; a second binding region comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0425] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:8 and a VL sequence as shown in SEQ ID NO:12; a second binding region comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0426] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0427] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0428] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0429] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0430] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:6 and a VL sequence as shown in SEQ ID NO:12; a second binding region comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0431] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as set forth in SEQ ID NO:6 and a VL sequence as set forth in SEQ ID NO:12; a second binding region comprising a VH sequence as set forth in SEQ ID NO:29 and a VL sequence as set forth in SEQ ID NO:30; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0432] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as set forth in SEQ ID NO:6 and a VL sequence as set forth in SEQ ID NO:12; a second binding region comprising a VH sequence as set forth in SEQ ID NO:19 and a VL sequence as set forth in SEQ ID NO:20; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0433] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as set forth in SEQ ID NO:6 and a VL sequence as set forth in SEQ ID NO:12; a second binding region comprising a VH sequence as set forth in SEQ ID NO:29 and a VL sequence as set forth in SEQ ID NO:30; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0434] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0435] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0436] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0437] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:10; a second binding region comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0438] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:12; a second binding region comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0439] In another embodiment, the bispecific antibody comprises a first binding region comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:12; a second binding region comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0440] In another embodiment, the bispecific antibody comprises a first binding domain comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:12; a second binding domain comprising a VH sequence as shown in SEQ ID NO:19 and a VL sequence as shown in SEQ ID NO:20; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0441] In another embodiment, the bispecific antibody comprises a first binding domain comprising a VH sequence as shown in SEQ ID NO:9 and a VL sequence as shown in SEQ ID NO:12; a second binding domain comprising a VH sequence as shown in SEQ ID NO:29 and a VL sequence as shown in SEQ ID NO:30; wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively; and wherein in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to position K409 in the human IgG1 heavy chain is R.

[0442] As used herein, the term "disulfide bridge" refers to a covalent bond between two cysteine residues, i.e., the interaction can also be designated as a Cys-Cys interaction.

[0443] As used herein, the term "target" refers to a molecule to which the binding domain of an antibody according to the invention binds, and when used in the context of antibody binding, the term includes any antigen against which the generated antibody is directed.

[0444] In a particular embodiment, the first heavy and light chains are humanized or chimeric and are linked via a disulfide bridge to form a first binding domain; and the second heavy and light chains are fully human and are linked via a disulfide bridge to form a second binding domain, wherein the first binding domain is according to any aspect or embodiment described herein and the second binding domain binds to a different target; and wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain are F, E and A, respectively.

[0445] In a particular embodiment, the first heavy and first light chains are humanized or chimeric and are linked via a disulfide bridge to form a first binding region; and the second heavy and light chains are fully human and are linked via a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds to a CD3 epitope different from the first binding region; and wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0446] In a particular embodiment, the first heavy and first light chains are humanized or chimeric and are linked via a disulfide bridge to form a first binding region; and the second heavy and light chains are fully human and are linked via a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds to a different target; and wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0447] In a particular embodiment, the first heavy and first light chains are humanized or chimeric and are linked via a disulfide bridge to form a first binding region; and the second heavy and light chains are fully human and are linked via a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds to a CD3 epitope different from the first binding region; and wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0448] Nucleic acid construct, expression vector and host cell

[0449] In one aspect, the invention relates to a nucleic acid construct encoding one or more of the sequences shown in Table 1. Accordingly, the invention relates to a nucleic acid construct encoding any one of the sequences shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26.

[0450] In a further aspect, the invention relates to a nucleic acid construct encoding a sequence of a humanized or chimeric CD3 antibody according to the invention, an expression vector comprising the nucleic acid construct according to the invention, a host cell comprising such an expression vector, and a method for producing such an antibody by culturing such a host cell under suitable conditions and optionally recovering the antibody. The humanized CD3 antibody may also be designated as "huCD3".

[0451] In one embodiment, the present invention provides an expression vector comprising: (i) a nucleic acid sequence encoding a heavy chain sequence of a humanized or chimeric antibody according to the present invention, (ii) a nucleic acid sequence encoding a light chain sequence of a humanized or chimeric antibody according to the present invention, and (iii) both (i) and (ii). Thus, the expression vector comprises one or more nucleic acid constructs or nucleic acid sequences according to any aspect or embodiment described herein.

[0452] In one embodiment, the expression vector of the present invention comprises a nucleic acid sequence encoding one or more of the heavy and light chain CDR sequences selected from the following: SEQ ID NO.: 1, 2, 3, 4, and 5; and the sequence GTN.

[0453] In one embodiment, the present invention provides an expression vector comprising a nucleic acid sequence encoding one or more amino acid sequences selected from SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 19, 20, 27, 28, 29, and 30, or any combination thereof. In another embodiment, the expression vector comprises a nucleic acid sequence encoding the VH CDR3 amino acid sequence as shown in SEQ ID NO: 3. In another embodiment, the expression vector comprises a nucleic acid sequence encoding a VH amino acid sequence selected from SEQ ID NO: 6, 7, 8, 9, 19, 27, and 29. In another embodiment, the expression vector comprises a nucleic acid sequence encoding a VL amino acid sequence selected from SEQ ID NO: 10, 11, 12, 20, 28, and 30. In another embodiment, the expression vector comprises a nucleic acid sequence encoding the constant region of a human antibody light chain, a human antibody heavy chain, or both. In another embodiment, the present invention provides an expression vector comprising a nucleic acid sequence encoding the amino acid sequences according to SEQ ID NO: 15, 16, 23, 24, 25, and 26.

[0454] In a particular embodiment, the expression vector comprises a nucleic acid sequence encoding a variant of one or more of the above amino acid sequences, said variant having up to 25 amino acid modifications, such as up to 20, such as up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modification, such as a deletion or insertion, preferably a substitution, such as a conservative or non-conservative substitution, or at least 80% identity with any one of said sequences, such as at least 85% identity or 90% identity or 95% identity with any one of the above amino acid sequences, such as 96% identity or 97% identity or 98% identity or 99% identity. The invention also relates to such nucleic acid sequences which differ from the above nucleic acid sequences, but due to the degeneracy of the genetic code, encode the same amino acid sequence as the antibodies of the invention. For example, the nucleic acid sequence may vary, but results in an amino acid sequence equivalent to any of the amino acid sequences described herein. It is well known to those skilled in the art how to identify such further nucleic acid sequences based on the genetic code.

[0455] In a further embodiment, the expression vector further comprises a nucleic acid sequence encoding the constant region of the light chain, the heavy chain or both the light and heavy chains of an antibody, such as a human antibody.

[0456] Such expression vectors as described above can be used for the recombinant production of the antibodies of the invention.

[0457] The expression vector in the context of the present invention can be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the humanized or chimeric CD3 antibody is contained in a naked DNA or RNA vector, which includes, for example, linear expression elements (as described, for example, in

[64] ), compact nucleic acid vectors (as described, for example, in

[65] and / or

[66] ), plasmid vectors such as pBR322, pUC 19 / 18 or pUC 118 / 119, "midge" minimal-sized nucleic acid vectors (as described, for example, in

[67] ), or nucleic acid vector constructs as precipitates, such as CaPO4 - precipitated constructs (as described, for example, in

[68] ,

[69] ,

[70] and

[71] ). Such nucleic acid vectors and their use are well known in the art (see, for example,

[72] and

[73] ).

[0458] In one embodiment, the vector is suitable for expressing a humanized or chimeric CD3 antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (

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

[0459] The 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 can be employed. Suitable vectors include, for example, vectors containing constitutive or inducible promoters, such as the α-factor, alcohol oxidase, and PGH (reviewed in

[75] and

[76] ).

[0460] The nucleic acid construct and / or vector may also contain 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 within the cell culture medium. Such sequences are known in the art and include secretion leaders or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization / anchoring sequences (e.g., stop-transfer sequences, GPI-anchoring sequences), and the like, which are well known in the art.

[0461] In the expression vectors of the present invention, the nucleic acid encoding the humanized or chimeric CD3 antibody may contain or be associated with any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include strong expression promoters (e.g., the human CMVIE promoter / enhancer and the RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effector poly(A) termination sequences, origins of replication for plasmid products in E. coli, antibiotic resistance genes as selectable markers, and / or convenient cloning sites (e.g., polylinkers). The nucleic acid construct and / or vector may also contain an inducible promoter as opposed to a constitutive promoter, such as CMV IE (those skilled in the art will recognize that such terms are actually descriptors of the degree of gene expression under certain conditions).

[0462] In one embodiment, the expression vector encoding the humanized or chimeric CD3 antibody is placed and / or delivered to a host cell or host animal via a viral vector.

[0463] Such expression vectors can be used for the recombinant production of humanized or chimeric CD3 antibodies.

[0464] In one aspect, the present invention provides a host cell comprising the expression vector according to the present invention.

[0465] In one aspect, the humanized or chimeric CD3 antibodies of any aspect or embodiment described herein are provided by using recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cells that produce the antibodies. Accordingly, the present invention provides recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cells that produce a humanized or chimeric CD3 antibody or immunoglobulin as defined herein. Examples of host cells include yeast, bacteria, and mammalian cells such as CHO or HEK-293 cells. For example, in one embodiment, the host cell contains a nucleic acid sequence stably integrated into the cell genome that contains a sequence encoding the expression of the humanized or chimeric CD3 antibody described herein. In another embodiment, the host cell contains a non-integrated nucleic acid sequence such as a plasmid, cosmid, phagemid, or linear expression element that contains a sequence encoding the expression of the humanized or chimeric CD3 antibody described herein.

[0466] As used herein, the term "recombinant host cell" (or simply "host cell") means a cell into which an expression vector or nucleic acid construct or sequence has been introduced. It should be understood that such terms refer not only to a particular subject cell but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not in fact be identical to the parental 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 lymphocytes, as well as prokaryotic cells such as Escherichia coli ( E. coli ) and other eukaryotic hosts such as plant cells and fungi.

[0467] In a further aspect, the present invention relates to a method for producing the humanized or chimeric CD3 antibodies of the present invention, the method comprising the steps of:

[0468] a) culturing the host cells of the present invention as described above herein, and

[0469] b) recovering and / or purifying the antibodies of the present invention from the culture medium.

[0470] In a further aspect, the nucleotide sequence encoding the humanized or chimeric CD3 antibody further encodes a second moiety, such as a therapeutic polypeptide. Exemplary therapeutic polypeptides are described elsewhere herein. In one embodiment, the present invention relates to a method for producing a humanized or chimeric CD3 antibody fusion protein, the method comprising the steps of:

[0471] a) culturing a host cell containing an expression vector that contains such a nucleotide sequence, and

[0472] b) recovering and / or purifying the humanized or chimeric CD3 antibody fusion protein from the culture medium.

[0473] Composition

[0474] In one aspect, the present invention provides a composition comprising an antibody or bispecific antibody according to any aspect and embodiment described herein.

[0475] In one aspect, the present invention provides a pharmaceutical composition comprising an antibody or bispecific antibody as defined in any one of the aspects and embodiments described herein, and a pharmaceutically acceptable carrier.

[0476] The pharmaceutical composition can be formulated with pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients according to conventional techniques such as those disclosed in

[77] .

[0477] The pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients should be suitable for the humanized or chimeric antibody of the present invention and the selected mode of administration. The suitability of the carrier and other components of the pharmaceutical composition is determined based on the lack of a significant negative impact on antigen binding (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.)) of the desired biological properties of the selected compound or pharmaceutical composition of the present invention.

[0478] The pharmaceutical composition of the present invention may also include diluents, fillers, salts, buffers, detergents (e.g., non-ionic detergents 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 the pharmaceutical composition.

[0479] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response with respect to a particular patient, composition, and mode of administration, and is non-toxic to the patient. The selected dosage level depends on various pharmacokinetic factors, including the activity of the particular composition or its amide of the present invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, the compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient to be treated, and similar factors well known in the medical arts.

[0480] The pharmaceutical composition can be administered by any route and mode. Suitable routes for administering the humanized or chimeric antibody of the present invention in vivo and in vitro are well known in the art and can be selected by one of ordinary skill in the art.

[0481] In one embodiment, the pharmaceutical composition of the present invention is administered parenterally.

[0482] As used herein, the phrases "parenteral administration" and "parenterally administered" mean modes of administration other than enteral and topical administration, typically by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.

[0483] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0484] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, absorption delaying agents and the like that are physiologically compatible with the humanized or chimeric antibodies of the present invention.

[0485] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffer solutions, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth and injectable organic esters such as ethyl oleate and / or different buffers. Other carriers are well known in the pharmaceutical art.

[0486] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically acceptable substances is known in the art. Unless any conventional media or reagent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated. When referring to "active compound", it is contemplated to also mean the humanized or chimeric antibodies according to the present invention.

[0487] Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions and by using surfactants.

[0488] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0489] The pharmaceutical composition of the present invention may also contain an isotonic agent in the composition, such as sugar, polyols such as mannitol, sorbitol, glycerol or sodium chloride.

[0490] The pharmaceutical composition of the present invention may also contain one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffering agents, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The humanized or chimeric antibody of the present invention can be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers alone or containing waxes such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art (see, for example,

[78] ).

[0491] In one embodiment, the humanized or chimeric antibody of the present invention can be formulated to ensure proper distribution in the body. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically acceptable substances is known in the art. Unless any conventional media or reagent is incompatible with the active compound, its use in the pharmaceutical composition of the present invention is contemplated. Other active or therapeutic compounds may also be incorporated into the composition.

[0492] A pharmaceutical composition for injection must generally be sterile and stable under the manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier can be an aqueous or non-aqueous solvent or dispersion medium, containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained by, for example, using coatings such as lecithin, in the case of dispersions by maintaining the required particle size and by using surfactants. In many cases, it will be preferred to include in the composition isotonic agents such as sugars, polyols such as glycerol, mannitol, sorbitol, or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, such as monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in an amount required, into a suitable solvent having one or a combination of the ingredients listed above, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of the preparation method are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient plus any additional required ingredients from its previously sterile-filtered solution.

[0493] Sterile injectable solutions can be prepared by incorporating the active compound in an amount required, into a suitable solvent having one or a combination of the ingredients listed above, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of the preparation method are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient plus any additional required ingredients from its previously sterile-filtered solution.

[0494] Therapeutic application

[0495] In another aspect, the present invention relates to a humanized or chimeric antibody of the present invention, or a pharmaceutical composition, as defined in any aspect or embodiment described herein, for use as a medicament.

[0496] In another aspect, the present invention relates to a humanized or chimeric antibody of the present invention, or a pharmaceutical composition, as defined in any aspect or embodiment described herein, for the treatment of diseases.

[0497] The humanized or chimeric antibodies or pharmaceutical compositions of the invention can be used in the treatment of any cancer in which the effector mechanism of cytotoxic T cells is required. For example, the humanized or chimeric antibodies can be administered, for example, ex vivo or ex vivo then in vivo to cells in culture, or, for example, in vivo to a human subject, to treat or prevent a disorder such as cancer, an inflammatory or autoimmune disorder. As used herein, the term "subject" is generally a human who responds to the humanized or chimeric antibodies or pharmaceutical compositions. A subject can include, for example, a human patient having a disorder that can be corrected or ameliorated by modulating a target function or by directly or indirectly causing cell killing.

[0498] In another aspect, the invention provides a method of treating or preventing a disorder such as cancer, wherein recruitment of T cells contributes to the treatment or prevention, the method comprising administering to a subject in need thereof a therapeutically effective amount of the humanized or chimeric antibody or pharmaceutical composition of the invention. The method generally involves administering to the subject a humanized or chimeric antibody in an amount effective to treat or prevent the disorder.

[0499] In a particular aspect, the invention relates to a method of treating cancer, which comprises administering to a subject in need thereof a humanized or chimeric antibody or pharmaceutical composition of the invention as defined in any aspect and embodiment described herein.

[0500] In another aspect, the invention relates to a use or method as defined in any aspect or embodiment described herein, wherein the humanized or chimeric antibody is a bispecific antibody that specifically binds to CD3 and a cancer-specific target, or a target that is overexpressed in cancer or is cancer-associated, such as HER2, CD19, EpCAM, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2 or MCSP (or HMW-MAA), 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 head and neck squamous cell carcinoma, cervical cancer, pancreatic cancer, testicular cancer, malignant melanoma, soft tissue cancer (such as synovial sarcoma), indolent or aggressive forms of B cell lymphoma, chronic lymphocytic leukemia or acute lymphoblastic leukemia.

[0501] The effective dose and dosage regimen of the humanized or chimeric antibody depend on the disease or condition to be treated and can be determined by those skilled in the art.

[0502] A physician having ordinary skill in the art can readily determine and prescribe an effective amount of the desired pharmaceutical composition. For example, the physician can start with a dose of the humanized or chimeric antibody employed in the pharmaceutical composition at a level lower than that desired to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, the appropriate dose of the compositions of the present invention will be that amount of the humanized or chimeric antibody which is the lowest dose effective to produce a therapeutic effect according to a particular dosing regimen. Such effective doses generally depend on the factors described above.

[0503] For example, an “effective amount” for therapeutic use can be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can be evaluated, for example, in an animal model system predictive of efficacy in human tumors. Alternatively, this property of the composition can be evaluated by in vitro assays known to those of skill in the art, by examining the ability of the humanized or chimeric antibody to inhibit cell growth or induce cytotoxicity. A therapeutically effective amount of a therapeutic compound, i.e., a humanized or chimeric antibody or pharmaceutical composition according to the present invention, can reduce tumor size, or otherwise ameliorate symptoms in a subject. A person of ordinary skill in the art can determine such amounts based on such factors as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0504] Exemplary, non-limiting ranges for a therapeutically effective amount of the humanized or chimeric antibody of the present invention are from 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, such as about 0.001 - 2 mg / kg, such as about 0.001 - 1 mg / kg, such as 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.

[0505] Administration can be, for example, intravenous, intramuscular, intraperitoneal or subcutaneous, and, for example, administered proximal to the target site.

[0506] Adjust the dosing regimen in the above-described methods and uses to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally decreased or increased as indicated by the exigencies of the therapeutic situation.

[0507] In one embodiment, the therapeutic efficacy is monitored during treatment, for example, at predetermined time points.

[0508] When needed, the effective daily dose of the pharmaceutical composition can be administered as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day, optionally in unit dosage forms. In another embodiment, the humanized or chimeric antibody, or the 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.

[0509] Although the humanized or chimeric antibodies of the present invention can be administered alone, it is preferred to administer the humanized or chimeric antibodies as a pharmaceutical composition as described above.

[0510] The effective dose of the humanized or chimeric antibody of the present invention can also be administered using a dosing period of once a week, once every two weeks or once every three weeks. The dosing period can be limited to, for example, 8 weeks, 12 weeks or until clinical progression has been confirmed. Optionally, the effective dose of the humanized or chimeric antibody of the present invention can be administered every second week, every third week or every fourth week.

[0511] In one embodiment, the humanized or chimeric antibody can be administered by infusion at a once-weekly dose calculated in mg / m 2 Such doses can be, for example, based on the mg / kg doses provided above: dose (mg / kg) x 70:1.8. Such administrations can be repeated, for example, 1 - 8 times, such as 3 - 5 times. The administration can be carried out by continuous infusion over a period of 2 - 24 hours, such as 2 - 12 hours. In one embodiment, the humanized or chimeric antibody can be administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.

[0512] In one embodiment, when administered once a week, the humanized or chimeric antibody can be administered at a once-weekly dose calculated as a fixed dose up to 8 times, such as 4 - 6 times. Such regimens can be repeated one or more times as needed, for example, after 6 months or 12 months. Such fixed doses can be, for example, based on the mg / kg doses provided above, where the body weight is estimated to be 70 kg. The dose can be determined or adjusted, for example, by taking a biological sample and measuring the amount of the humanized or chimeric antibody of the present invention in the blood after administration using an anti-idiotypic antibody targeting the binding region of the humanized or chimeric antibody of the present invention.

[0513] In one embodiment, the humanized or chimeric antibody can be administered by maintenance therapy, such as once a week for a period of 6 months or longer.

[0514] The humanized or chimeric antibody can also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence when cancer is in remission.

[0515] Parenteral compositions can be formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the present invention is dictated by and directly dependent on the following: (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 using such active compounds in the treatment of individuals with sensitivity.

[0516] Humanized or chimeric antibodies can also be prophylactically administered in order to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence when the cancer is in remission. This can be particularly useful in patients in whom it is difficult to localize known existing tumors due to other biological factors.

[0517] Diagnostic application

[0518] The humanized or chimeric antibodies of the present invention can also be used for diagnostic purposes, using compositions comprising the humanized or chimeric antibodies described herein. Accordingly, the present invention provides diagnostic methods and compositions using the humanized or chimeric antibodies described herein. Such methods and compositions can be used for purely diagnostic purposes, such as detecting or identifying diseases, as well as for monitoring the progress of a therapeutic treatment, monitoring disease progression, evaluating the post-treatment status, monitoring disease recurrence, assessing the risk of disease development, and the like.

[0519] In one aspect, the present invention relates to a method of diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, which comprises administering to a subject a humanized or chimeric antibody according to the present invention, a composition according to the present invention, or a therapeutic composition according to the present invention, optionally wherein the humanized or chimeric antibody is labeled with a detectable reagent.

[0520] In one aspect, the humanized or chimeric antibodies of the invention are used in vitro and then in vivo, for example, in the diagnosis of diseases by detecting the target level in a sample taken from a patient or the level of cells expressing the target of interest on their cell surface, wherein the cells expressing the specific target of interest and to which the humanized or chimeric antibody binds indicate the disease or are involved in the pathogenesis. This can be achieved, for example, by contacting the sample to be tested, optionally together with a control sample, with a humanized or chimeric antibody according to the invention under conditions allowing the antibody to bind to the target. Subsequently, complex formation can be detected (e.g., using ELISA). When using a control sample together with the test sample, the levels of the humanized or chimeric antibody or antibody - target complex are analyzed in both samples, and a statistically significantly higher level of the humanized or chimeric antibody or antibody - target complex in the test sample indicates a higher level of the target in the test sample compared to the control sample.

[0521] Examples of conventional immunoassays in which the humanized or chimeric antibodies of the invention can be used include, but are not limited to, ELISA, RIA, FACS assays, surface plasmon resonance analysis, chromatographic assays, tissue immunohistochemistry, Western blotting, and / or immunoprecipitation.

[0522] Thus, in one embodiment, the invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of CD3 - expressing cells, which comprises administering to a subject an antibody, bispecific antibody, composition, or pharmaceutical composition according to any aspect or embodiment described herein, optionally wherein the antibody is labeled with a detectable label.

[0523] In one embodiment, the invention relates to a method for detecting the presence of a target or cells expressing the target in a sample, which comprises:

[0524] - contacting the sample with a humanized or chimeric antibody of the invention under conditions allowing the humanized or chimeric antibody to bind to the target in the sample; and

[0525] - analyzing whether a complex has been formed. Typically, the sample is a biological sample.

[0526] In one embodiment, the sample is a tissue sample known or suspected to contain cells expressing a specific target or the target. For example, in situ detection of target expression can be accomplished by taking a histological sample from a patient and providing the humanized or chimeric antibody of the present invention to such a sample. The humanized or chimeric antibody can be provided by applying or overlaying the humanized or chimeric antibody to the sample, which is then detected using suitable means. Subsequently, not only the presence of the target or target-expressing cells can be determined, but also the distribution of the target or target-expressing cells in the tissue examined (e.g., in the context of evaluating cancer cell spread) can be determined. Using the present invention, one of ordinary skill in the art can readily appreciate that any of a wide variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0527] In the above assays, the humanized or chimeric antibody can be labeled with a detectable substance to permit detection of the bound antibody. Optionally, the bound (primary) specific humanized or chimeric antibody can be detected by an antibody that is labeled with a detectable substance and that binds to the primary specific humanized or chimeric antibody. In addition, in the above assays, a diagnostic composition comprising an antibody or bispecific antibody according to any aspect or embodiment described herein can 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 described herein.

[0528] The target level in a sample can also be evaluated by a competitive immunoassay that utilizes a target standard labeled with a detectable substance and an unlabeled target-specific humanized or chimeric antibody. In such an assay, a biological sample, the labeled target standard, and the target-specific humanized or chimeric antibody are combined, and the amount of the labeled target standard that binds to the unlabeled target-specific humanized or chimeric antibody is determined. The amount of target in the biological sample is inversely proportional to the amount of the labeled target standard that binds to the target-specific humanized or chimeric antibody.

[0529] Suitable labels for the target-specific humanized or chimeric antibody, secondary antibody, and / or target standard for use in in vitro diagnostic techniques include, but are not limited to, various enzymes, cofactors, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable cofactor complexes include streptavidin / biotin and biotin / avidin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; examples of luminescent materials include luminol; and examples of suitable radioactive materials include 125 I, 131 I, 35 S, and 3 H.

[0530] In one aspect, the target-specific humanized or chimeric antibodies of the invention are used in in vivo imaging of target-expressing tissues such as tumors. For in vivo methods, antibody fragments such as (Fab’)2, Fab, and Fab’ fragments are particularly advantageous due to their rapid distribution kinetics.

[0531] In vivo imaging can be performed by any suitable technique. For example, target-specific humanized or chimeric antibodies (such as antibodies or fragments) labeled with 99 Tc, 131 I, 111 In, or other gamma-ray emitting isotopes can be used to image the target-specific antibody accumulation or distribution in target-expressing tissues such as tumors, using a gamma scintillation camera (such as an Elscint Apex 409 ECT device), typically using a low-energy, high-resolution collimator or a low-energy general collimator. Optionally, labeling with 89 Zr, 76 Br, 18 F, or other positron-emitting radionuclides can be used to image the target-specific humanized or chimeric antibody or antibody fragment distribution in tumors (using positron emission tomography (PET)). Images obtained by using such techniques can be used to evaluate the target biodistribution in a patient, mammal, or tissue, for example, in the context of using the target as a biomarker for the presence of cancer / tumor cells. Variations on such techniques can include using magnetic resonance imaging (MRI) to improve imaging over gamma photographic techniques. Conventional immunoscintigraphy methods and principles are described, for example, in

[79] ,

[80] , and

[81] . In addition, such image techniques can also or optionally serve as the basis for surgical techniques for removing tumors. Furthermore, such in vivo imaging techniques can allow for the identification and localization of tumors in situations 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 conventional analytical techniques. All of these methods are features of the invention.

[0532] The in vivo imaging and other diagnostic methods provided by the invention are particularly useful in the detection of micrometastases in human patients (such as patients who have not been previously diagnosed with cancer or patients who are in a recovery / remission phase from cancer).

[0533] In one embodiment, the present invention provides an in vivo imaging method, wherein a target - specific humanized or chimeric antibody of the present invention is conjugated to a detection - facilitating radiopaque reagent, and the conjugated humanized or chimeric antibody is administered to a host, for example, by injection into the bloodstream, and the presence and localization of the labeled humanized or chimeric antibody in the host are determined. By this technique provided herein and any other diagnostic method, the present invention provides a method for screening for the presence of disease - related cells in a human patient or a biological sample taken from a human patient, and / or for evaluating the distribution of a target - specific humanized or chimeric antibody prior to target - specific ADC therapy.

[0534] For diagnostic imaging, a radioisotope can be directly or indirectly bound to the target - specific humanized or chimeric antibody through the use of an intermediate functional group. Useful intermediate functional groups include chelating agents such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, for example,

[82] ).

[0535] In addition to radioisotopes and radiopaque reagents, diagnostic methods can also be performed using target - specific antibodies conjugated to dyes (e.g., using a biotin - streptavidin complex), contrast agents, fluorescent compounds or molecules, and enhancers (e.g., paramagnetic ions) for magnetic resonance imaging (MRI) (see, for example,

[83] , which describes MRI techniques and the preparation of antibodies conjugated to MRI enhancers). Such diagnostic / detection reagents can be selected from reagents for MRI and fluorescent compounds. To load a target - specific humanized or chimeric antibody with a radioactive metal or paramagnetic ion, it may be necessary to react it with a reagent having a long tail to which multiple chelating groups are attached for binding the ion. Such tails can be polymers such as polylysine, polysaccharides, or another derivatized or derivatizable chain having pendant groups to which chelating groups such as porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and similar groups known for this use can be attached. The chelate can be conjugated to the target - specific humanized or chimeric antibody using standard chemical reagents.

[0536] Accordingly, the present invention provides diagnostic target - specific humanized or chimeric antibodies, wherein the target - specific humanized or chimeric antibody is conjugated to a contrast agent (e.g., a contrast enhancer for magnetic resonance imaging, computed tomography, or ultrasound) or a radionuclide, which can be, for example, a γ, β, α, Auger electron, or positron - emitting isotope.

[0537] In one aspect, the present invention relates to a diagnostic composition comprising an antibody or bispecific antibody according to the present invention.

[0538] In a further aspect, the present invention relates to a kit for detecting the presence of a target antigen or cells expressing a target in a sample, comprising:

[0539] - A target-specific humanized or chimeric antibody of the present invention; and

[0540] - Instructions for use of the kit.

[0541] Accordingly, in one aspect, the present invention provides a kit for detecting the presence of CD3 antigen or cells expressing CD3 in a sample, comprising the following steps:

[0542] a) contacting the sample with an antibody or bispecific antibody according to the present invention under conditions permitting the formation of a complex between the antibody or bispecific antibody and CD3; and

[0543] b) analyzing whether a complex has formed.

[0544] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container containing a target-specific humanized or chimeric antibody, and one or more reagents for detecting the binding of the target-specific humanized or chimeric antibody to the target. The reagents can include, for example, fluorescent tags, enzymatic tags, or other detectable tags. The reagents can also include secondary or tertiary antibodies or reagents for enzymatic reactions, where the enzymatic reaction produces a product that can be visualized. In one embodiment, the present invention provides a diagnostic kit comprising one or more target-specific humanized or chimeric antibodies of the present invention in labeled or unlabeled form in a suitable container, reagents for incubation for indirect assays, and substrates or derivatized reagents for detection in such assays, depending on the nature of the label. Control reagents and instructions for use can also be included.

[0545] The diagnostic kit can also be provided for use with a target-specific humanized or chimeric antibody, such as a labeled target-specific antibody, for detecting the presence of a target in a tissue sample or host. In such diagnostic kits, as well as in the kits for therapeutic use described elsewhere herein, the target-specific humanized or chimeric antibody can typically be provided in a lyophilized form in a container, alone or in combination with additional antibodies specific for target cells or peptides. Typically, a pharmaceutically acceptable carrier (such as an inert diluent) and / or its components, such as Tris, phosphate or carbonate buffers, stabilizers, preservatives, biocides, inert proteins such as serum albumin, etc. (usually in separate containers for mixing) and additional reagents (usually also in separate containers) are also included. In certain kits, a secondary antibody capable of binding to the target-specific humanized or chimeric antibody is also included, which is typically present in a separate container. The secondary antibody is typically conjugated to a label and formulated in a manner similar to the target-specific humanized or chimeric antibody of the present invention. Using the methods described above and elsewhere herein, the target-specific humanized or chimeric antibody can be used to define subsets of cancer / tumor cells and to characterize such cells and associated tumor tissue.

[0546] Anti-idiotypic antibody

[0547] In a further aspect, the invention relates to anti-idiotypic antibodies that bind to the humanized or chimeric antibodies of the invention as described herein.

[0548] Anti-idiotypic (Id) antibodies are antibodies that recognize idiotypic determinants that generally bind to the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing an animal of the same species and genetic type as the source of the anti-CD3 monoclonal antibody with the monoclonal antibody against which the anti-Id is to be prepared. The immunized animal can generally recognize and respond to the idiotypic determinants of the immunizing antibody by producing antibodies (anti-Id antibodies) against these idiotypic determinants. Such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of the invention.

[0549] Anti-Id antibodies can also be used as an "immunogen" to induce an immune response in yet another animal, generating so-called anti-anti-Id antibodies. The anti-anti-Id antibodies can be epitopically equivalent to the original monoclonal antibody that induced the anti-Id antibody. Thus, by using an antibody against the idiotypic determinant of a monoclonal antibody, other clones expressing antibodies with equivalent specificity can be identified. Anti-Id antibodies can be modified (thereby generating anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein with respect to the CD3-specific antibodies of the invention. For example, monoclonal anti-Id antibodies can be conjugated to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Serum from these mice typically contains anti-anti-Id antibodies that have binding properties similar (if not identical) to the original / parental CD3 antibody.

[0550] Sequence

[0551]

[0552]

[0553]

[0554] 。

[0555] The invention also relates to the following embodiments

[0556] 1. A humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region that comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 4, the sequence GTN, and the sequence shown in SEQ ID NO: 5, respectively.

[0557] 2. The antibody according to embodiment 1, wherein the VH region has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence shown in a VH sequence selected from the following:

[0558] a) The VH sequence shown in SEQ ID NO: 6;

[0559] b) The VH sequence shown in SEQ ID NO: 8;

[0560] c) The VH sequence shown in SEQ ID NO: 7; and

[0561] d) The VH sequence shown in SEQ ID NO: 9.

[0562] 3. The antibody according to any one of the preceding embodiments, wherein the VL region has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence shown in a VL sequence selected from the following:

[0563] a) The VL sequence shown in SEQ ID NO: 10;

[0564] b) The VL sequence shown in SEQ ID NO: 11; and

[0565] c) The VL sequence shown in SEQ ID NO: 12.

[0566] 4. The antibody according to any one of the preceding embodiments, wherein the VH region is selected from the following:

[0567] a) The VH sequence shown in SEQ ID NO: 6;

[0568] b) The VH sequence shown in SEQ ID NO: 8;

[0569] c) The VH sequence shown in SEQ ID NO: 7; and

[0570] d) The VH sequence shown in SEQ ID NO: 9.

[0571] 5. An antibody according to any one of the preceding embodiments, wherein the VL region is selected from the following:

[0572] a) the VL sequence as shown in SEQ ID NO: 10;

[0573] b) the VL sequence as shown in SEQ ID NO: 11; and

[0574] c) the VL sequence as shown in SEQ ID NO: 12.

[0575] 6. An antibody according to any one of the preceding embodiments, wherein the VH and VL regions are selected from the following:

[0576] a) the VH sequence as shown in SEQ ID NO: 6, and the VL sequence as shown in SEQ ID NO: 10;

[0577] b) the VH sequence as shown in SEQ ID NO: 8, and the VL sequence as shown in SEQ ID NO: 10;

[0578] c) the VH sequence as shown in SEQ ID NO: 9, and the VL sequence as shown in SEQ ID NO: 10;

[0579] d) the VH sequence as shown in SEQ ID NO: 6, and the VL sequence as shown in SEQ ID NO: 11;

[0580] e) the VH sequence as shown in SEQ ID NO: 6, and the VL sequence as shown in SEQ ID NO: 12;

[0581] f) the VH sequence as shown in SEQ ID NO: 7, and the VL sequence as shown in SEQ ID NO: 10;

[0582] g) the VH sequence as shown in SEQ ID NO: 7, and the VL sequence as shown in SEQ ID NO: 11;

[0583] h) the VH sequence as shown in SEQ ID NO: 7, and the VL sequence as shown in SEQ ID NO: 12;

[0584] i) the VH sequence as shown in SEQ ID NO: 8, and the VL sequence as shown in SEQ ID NO: 11;

[0585] j) the VH sequence as shown in SEQ ID NO: 8, and the VL sequence as shown in SEQ ID NO: 12;

[0586] k) A VH sequence as shown in SEQ ID NO:9, and a VL sequence as shown in SEQ ID NO:11; and

[0587] l) A VH sequence as shown in SEQ ID NO:9, and a VL sequence as shown in SEQ ID NO:12.

[0588] 7. The antibody according to any one of the preceding embodiments, wherein the binding region comprises a VH sequence and a VL sequence selected from the following:

[0589] a) A VH sequence as shown in SEQ ID NO:6, and a VL sequence as shown in SEQ ID NO:10;

[0590] b) A VH sequence as shown in SEQ ID NO:8, and a VL sequence as shown in SEQ ID NO:10; and

[0591] c) A VH sequence as shown in SEQ ID NO:9, and a VL sequence as shown in SEQ ID NO:10.

[0592] 8. The antibody according to any one of the preceding embodiments, wherein the antibody is a humanized antibody.

[0593] 9. The antibody according to embodiment 1, wherein the antibody is a chimeric antibody.

[0594] 10. The antibody according to any one of the preceding embodiments, wherein the antibody is a full-length antibody.

[0595] 11. The antibody according to any one of the preceding embodiments, wherein the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

[0596] 12. The antibody according to any one of the preceding embodiments, wherein the first and second heavy chains have an isotype selected from IgG1, IgG2, IgG3, and IgG4.

[0597] 13. The antibody according to any one of the preceding embodiments, wherein the antibody comprises an Fc region that has been modified such that, compared to a wild-type antibody, the binding of C1q to the antibody is reduced 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.

[0598] 14. An antibody according to any one of the foregoing embodiments, wherein the antibody comprises an Fc region that has been modified such that Fc-mediated T cell proliferation is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% compared to a wild-type antibody, wherein the T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

[0599] 15. An antibody according to any one of the foregoing embodiments, wherein the antibody comprises an Fc region that has been modified such that Fc-mediated CD69 expression is reduced 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 the Fc-mediated CD69 expression is determined in a functional assay based on PBMCs.

[0600] 16. An antibody according to any one of the foregoing embodiments, wherein the antibody comprises first and second immunoglobulin heavy chains, wherein in at least one of the first and second immunoglobulin heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in the human IgG1 heavy chain are not L, L, D, N and P, respectively.

[0601] 17. The antibody according to embodiment 16, wherein in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is not D.

[0602] 18. The antibody according to embodiment 16, wherein in at least one of the first and second heavy chains, the amino acid at the position corresponding to position N297 in the human IgG1 heavy chain is not N.

[0603] 19. The antibody according to embodiment 16, wherein in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are not L and L, respectively.

[0604] 20. The antibody according to any one of embodiments 16 and 19, wherein in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively; or A and A.

[0605] 21. The antibody according to embodiment 20, wherein in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively.

[0606] 22. The antibody according to embodiment 20, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to at least positions L234 and L235 in the human IgG1 heavy chain are A and A, respectively.

[0607] 23. The antibody according to any one of embodiments 1 - 16, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are not L, L, and D, respectively.

[0608] 24. The antibody according to embodiment 23, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A.

[0609] 25. The antibody according to embodiment 24, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0610] 26. The antibody according to embodiment 24, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are A, A, and A, respectively.

[0611] 27. The antibody according to embodiment 16, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are F, E, A, Q, and S, respectively.

[0612] 28. A bispecific antibody comprising a first binding region of an antibody according to any one of embodiments 1 - 12, and a second binding region that binds to a target different from the first antigen - binding region.

[0613] 29. The bispecific antibody according to embodiment 28, wherein the antibody comprises first and second heavy chains.

[0614] 30. The bispecific antibody according to embodiment 29, wherein

[0615] a) the bispecific antibody comprises an Fc region modified according to any one of embodiments 13 - 15; or

[0616] b) at least one of the first and second heavy chains comprises one or more amino acids modified as defined in any one of embodiments 16 - 27.

[0617] 31. A bispecific antibody according to any one of embodiments 28 - 30, wherein each of the first and second heavy chains comprises at least a hinge region, CH2, and CH3 regions, wherein in the first heavy chain, at least one of the amino acids at positions corresponding to positions selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain has been replaced, and in the second heavy chain, at least one of the amino acids at positions corresponding to positions selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain has been replaced, and wherein the first and the second heavy chains are not replaced at the same positions.

[0618] 32. The bispecific antibody according to embodiment 31, wherein the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain, and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain, or vice versa.

[0619] 33. The bispecific antibody according to any one of embodiments 28 - 32, wherein the first binding region is according to any one of embodiments 1 - 7, and the second binding region binds a target different from the first binding region.

[0620] 34. A nucleic acid construct encoding one or more of the amino acid sequences shown in Table 1.

[0621] 35. An expression vector comprising:

[0622] (i) a nucleic acid sequence encoding a heavy chain sequence of a humanized or chimeric antibody according to any one of embodiments 1 - 33;

[0623] (ii) a nucleic acid sequence encoding a light chain sequence of a humanized or chimeric antibody according to any one of embodiments 1 - 33; or

[0624] (iii) both (i) and (ii).

[0625] 36. A host cell comprising the expression vector of embodiment 35.

[0626] 37. The host cell according to embodiment 36, wherein the host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell.

[0627] 38. A composition comprising an antibody according to any one of embodiments 1 - 27 or a bispecific antibody according to any one of embodiments 28 - 33.

[0628] 39. A pharmaceutical composition comprising an antibody according to any one of embodiments 1 - 27 or a bispecific antibody according to any one of embodiments 28 - 33 and a pharmaceutically acceptable carrier.

[0629] 40. An antibody according to any one of embodiments 1 - 27, the bispecific antibody according to any one of embodiments 28 - 33, the composition according to embodiment 38, or the pharmaceutical composition according to embodiment 39, for use as a medicament.

[0630] 41. An antibody according to any one of embodiments 1 - 27, the bispecific antibody according to any one of embodiments 28 - 33, the composition according to embodiment 38, or the pharmaceutical composition according to embodiment 39, for use in the treatment of a disease.

[0631] 42. A method for treating a disease, comprising administering to a subject in need thereof an antibody according to any one of embodiments 1 - 27, the bispecific antibody according to any one of embodiments 28 - 33, the composition according to embodiment 38, or the pharmaceutical composition according to embodiment 39.

[0632] 43. The use or method according to any one of embodiments 40 - 42, wherein the disease is cancer, an infectious disease or an autoimmune disease.

[0633] 44. A method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, comprising administering to a subject an antibody according to any one of embodiments 1 - 27, the bispecific antibody according to any one of embodiments 28 - 33, the composition according to embodiment 38, or the pharmaceutical composition according to embodiment 39, optionally wherein the antibody or the bispecific antibody is labeled with a detectable reagent.

[0634] 45. A method for producing an antibody according to any one of embodiments 1 - 27 or a bispecific antibody according to any one of embodiments 28 - 33, comprising the following steps:

[0635] a) culturing a host cell according to any one of embodiments 36 - 37; and

[0636] b) purifying the antibody from the culture medium.

[0637] 46. A diagnostic composition comprising an antibody according to any one of embodiments 1 - 27 or a bispecific antibody according to any one of embodiments 28 - 33.

[0638] 47. A method for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, comprising the steps of:

[0639] a) contacting the sample with an antibody according to any one of embodiments 1 - 27, or a bispecific antibody according to any one of embodiments 28 - 33, under conditions allowing the formation of a complex between said antibody or bispecific antibody and CD3; and

[0640] b) analyzing whether a complex has been formed.

[0641] 48. A kit for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, comprising:

[0642] i) an antibody according to any one of embodiments 1 - 27, or a bispecific antibody according to any one of embodiments 28 - 33; and

[0643] ii) instructions for use of said kit.

[0644] 49. An anti-idiotypic antibody that binds to an antibody according to any one of embodiments 1 - 27.

[0645] Examples

[0646] Example 1 - Generation of Humanized CD3 Antibodies and Inactivated Antibody Variants

[0647] Humanization of CD3 antibody

[0648] Humanization of the murine CD3 antibody (US 8,236,308, described herein as IgG1-CD3) was performed by Antitope (Cambridge, UK) using its modified version of the germline humanization (CDR grafting) technique (EP 0 629 240). Using this technique, 4 different VH chains (SEQ ID NO:6, 7, 8, and 9) and 3 different VL chains (SEQ ID NO:10, 11, and 12) were designed. By combining these 4 VH with 3 VL chains, 12 different antibodies were generated. The humanized variants are described herein as huCD3. Thus, a humanized variant comprising the VH and VL according to the present invention is described, for example, as IgG1-huCD3-H1L1, meaning that said specific variant has an IgG1 isotype, is a humanized CD3, and comprises a VH amino acid sequence designated "H1" and defined according to SEQ ID NO:6, and a VL amino acid sequence designated "L1" and defined according to SEQ ID NO:10. Thus, H1 refers to variable heavy chain region VH1, L1 refers to variable light chain region VL1, and so on.

[0649] Specifically, the variants IgG1-huCD3-H1L1 (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO:6 and the VL1 sequence shown in SEQ ID NO:10), IgG1-huCD3-H1L2 (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO:6 and the VL2 sequence shown in SEQ ID NO:11), IgG1-huCD3-H1L3 (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO:6 and the VL3 sequence shown in SEQ ID NO:12), IgG1-huCD3-H3L3 (humanized CD3 comprising the VH3 sequence shown in SEQ ID NO:8 and the VL3 sequence shown in SEQ ID NO:12), IgG1-huCD3-H4L1 (humanized CD3 comprising the VH4 sequence shown in SEQ ID NO:9 and the VL1 sequence shown in SEQ ID NO:10), IgG1-huCD3-H3L1 (humanized CD3 comprising the VH3 sequence shown in SEQ ID NO:8 and the VL1 sequence shown in SEQ ID NO:10), IgG1-huCD3-H3L3 (humanized CD3 comprising the VH3 sequence shown in SEQ ID NO:8 and the VL3 sequence shown in SEQ ID NO:12), and IgG1-huCD3-H4L3 (humanized CD3 comprising the VH4 sequence shown in SEQ ID NO:9 and the VL3 sequence shown in SEQ ID NO:12) have been generated and tested in the examples described herein.

[0650] In some examples, an antibody comprising the heavy and light chain variable region sequences of huCLB-T3 / 4 (SEQ ID NOs: 17 and 18, respectively) was used as a control antibody (Labrijn et al., PNAS 2013, 110: 5145-50), and was used to validate different combinations of non-activating mutations in the Fc region (see Examples 8 - 10). huCBL-T3 / 4 is a humanized form of the murine CD3 antibody CLB-T3 / 4 (Parren et al., Res Immunol. 1991, 142(9):749-63). The two sequences (SEQ ID NOs: 17 and 18) were cloned into a relevant pcDNA3.3 (Invitrogen) expression vector and expressed by co-transfection in HEK293F cells. The resulting control antibody was designated IgG1-huCLB-T3 / 4.

[0651] In some examples, an antibody comprising the heavy and light chain variable region sequences of the CD20 antibody 7D8 (SEQ ID NO: 29 corresponds to the VH sequence and SEQ ID NO: 30 corresponds to the VL sequence) is used as a positive control. When used in the case of the positive control, it is referred to as "IgG1-CD20".

[0652] These IgG1-CD3 (i.e., chimeric, parental CD3 antibodies), IgG1-huCD3, and IgG1-huCLB-T3 / 4 antibodies are used in both monospecific and bispecific forms, where the bispecific antibodies are generated as described below.

[0653] HER2 antibody

[0654] In some examples, an antibody against HER2 is used. The VH and VL sequences of this HER2-specific antibody (antibody 169, SEQ ID NOs: 19 and 20, respectively) have been previously described (WO2012 / 143524 [Genmab]; Labrijn et al., PNAS 2013, 110: 5145-50). The antibody is used in both monospecific and bispecific forms and is named "IgG1-HER2".

[0655] b12 antibody

[0656] In some examples, the antibody b12, a gp120-specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5;230(3):812-23.) is used as a negative control and is named "IgG1-b12".

[0657] Expression

[0658] Antibodies are expressed as IgG1,κ or IgG1,λ, with or without the inactivating mutations described below, and having mutations in the CH3 domain that allow the generation of bispecific antibodies by the methods described below: IgG1-HER2-K409R, IgG1-b12-K409R, IgG1-CD3-F405L. Using 293fectin (Invitrogen, US), a mixture of plasmid DNAs encoding both the heavy and light chains of the antibody is transiently transfected into Freestyle HEK293F cells (Invitrogen, US) substantially as described by the manufacturer.

[0659] Purification of antibody

[0660] The culture supernatant was filtered through a 0.2 µm dead-end filter, loaded onto a 5 mL MabSelect SuRe column (GE Healthcare), and eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9 and dialyzed overnight against 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun). Optionally, after purification, the eluate was loaded onto a HiPrep Desalting column and the antibody was exchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun) buffer. After buffer dialysis or exchange, the sample was filtered through a 0.2 µm dead-end filter. Purity was determined by SDS-PAGE and concentration was measured by absorbance at 280 nm. The purified antibody was stored at 2-8 °C.

[0661] Generation of bispecific antibody

[0662] As described in WO 2011 / 147986 and Labrijn et al. (Labrijn et al., PNAS 2013, 110: 5145-50; Gramer et al., MAbs 2013, 5: 962-973), bispecific antibodies were generated in vitro using the DuoBody® platform technology, i.e., 2-MEA-induced Fab arm exchange. To allow the generation of bispecific antibodies by this method, IgG1 molecules carrying a single mutation in the CH3 domain were generated: an F405L mutation (i.e., IgG1-CD3 antibody) in one parental IgG1 antibody and a K409R mutation (i.e., HER2 or b12 antibody) in the other parental IgG1 antibody. To generate the bispecific antibody, the two parental antibodies (each at a final concentration of 0.5 mg / mL) were incubated with 25 or 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 500 μL TE at 31 °C for 5 h. The reduction reaction was stopped when the reducing agent 2-MEA was removed by using a PD-10 column (GE-healthcare, product #17-0851-01) equilibrated with 25 mL PBS. Before desalting, 2 mL PBS (B. Braun, product #3623140) was added to the sample to adjust the volume to 2.5 mL. Elution was done in 3.5 mL PBS. The sample was collected into an Amicon Ultra centrifugal unit (30 kD MWCO, Millipore, product #UFC803096) and concentrated by centrifugation at 3000x gCentrifuge at 8,000 rpm for 8 minutes for concentration. Adjust the volume to 500 µL with PBS (if needed), and filter the sample aseptically through a 0.2 µm filter (Millex-GV, product #SLGV004SL). Store the bispecific product at 2 - 8°C.

[0663] Optionally, to obtain the same bispecific antibody, mix 100 µg of the two parental antibodies and incubate with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 400 μL PBS (B.Braun, product #3623140) at 31°C for 5 hours. The reduction reaction is stopped when the reducing agent 2-MEA is removed by washing 4x with 400 µl PBS using an Amicon Ultra 0.5 ml centrifugal unit (10kD MWCO, Millipore, product #UFC501096) and centrifuging at 3,000 x g g for 10 minutes. The sample is collected into a new tube by inverting the filter and centrifuging at 1,000 g for 2 minutes. Adjust the volume to 200 µL with PBS (if needed). Measure the absorbance at 280 nm (A280) of the bispecific product to determine the final concentration. Perform HPLC cation exchange chromatography (HPLC-CEX) (as described in WO 2013 / 060867) to determine the amount of the bispecific product. Store the sample at 2 - 8°C.

[0664] The generated bispecific antibodies are described hereinafter as "K409R IgG1 backbone" and "F405L IgG1 backbone".

[0665] Inactivated mutation

[0666] Several antibody variants with one or more amino acid substitutions in the Fc region were generated. The inactivated Fc region prevents the antibody from interacting with Fc receptors present on blood cells such as monocytes, or with C1q to activate the classical complement pathway. The reduction of Fc activity was tested in antibody variants containing different combinations of amino acid substitutions in the Fc region. Up to five amino acid substitutions were introduced, including the mutations N297Q, L234A, L235A, L234F, L235E, D265A, and P331S. Substitutions in one or more of these five amino acid positions were introduced into the K409R and / or F405L IgG1 backbone. The following Fc region variants of the huCLB-T3 / 4 antibody were generated: N297Q (referring to the N297Q substitution, named IgG1-huCLB-T3 / 4-N297Q), LFLE (referring to the L234F / L235E substitution, named IgG1-huCLB-T3 / 4-LFLE), LALA (referring to the L234A / L235A substitution, named IgG1-huCLB-T3 / 4-LALA), LFLENQ (referring to the L234F / L235E / N297Q substitution, named IgG1-huCLB-T3 / 4-LFLENQ), LFLEDA (referring to the L234F / L235E / D265A substitution, named IgG1-huCLB-T3 / 4-LFLEDA), DA (referring to the D265A substitution, named IgG1-huCLB-T3 / 4-DA), DAPS (referring to the D265A / P331S substitution, named IgG1-huCLB-T3 / 4-DAPS), DANQ (referring to the D265A / N297Q substitution, named IgG1-huCLB-T3 / 4-DANQ), LFLEPS (referring to the L234F / L235E / P331S substitution, named IgG1-huCLB-T3 / 4-LFLEPS), and LFLEDANQPS (referring to the L234F / L235E / D265A / N297Q / P331S substitution, named IgG1-huCLB-T3 / 4-LFLEDANQPS).

[0667] In particular, in the IgG1-huCD3 antibody variant, a combination of three amino acid substitutions, including the mutations L234F, L235E, and D265A, and referred to as LFLEDA, was introduced into the K409R and F405L IgG1 backbone to generate an antibody with an inactivated Fc region. The resulting inactivated antibody variant was named with the suffix "-LFLEDA".

[0668] Example 2 - Binding of Humanized CD3 Antibodies and Their Inactivated Variants to Human and Cynomolgus Monkey T Cell Lines Expressing CD3

[0669] The binding of purified variants of humanized CD3 (huCD3) antibodies and bispecific (bs) IgG1-huCD3 x HER2 molecules with or without the LFLEDA mutation in the Fc region to the human T cell line Jurkat (Clone E6-1, ATCC ® TIB-152™, LGC Standards GmbH, Wesel, Germany) or the cynomolgus monkey T cell line HSC-F (Catalog No. JCRB1164; Health Science Research Resources Bank, Osaka, Japan) was analyzed by FACS. Except for the non-activating mutation LFLEDA, the antibody variants contained the F405L or K409R mutations as described in Example 1.

[0670] Cells (1x10 5 cells / well) were incubated with serial dilutions of the antibody preparation (3-fold dilution range from 5 - 10,000 ng / mL) in 100 µL of PBS / 0.1% BSA / 0.02% azide at 4 °C for 30 minutes in a polystyrene 96-well round bottom plate (Greiner bio-one 650101).

[0671] After washing twice in PBS / 0.1% BSA / 0.02% azide, the cells were incubated with the secondary antibody in 100 μL at 4 °C for 30 minutes. As the 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 / 100 in PBS / 0.1% BSA / 0.02% azide was used for all experiments. Next, the cells were washed twice in PBS / 0.1% BSA / 0.02% azide, resuspended in 150 μL of PBS / 0.1% BSA / 0.02% azide, and analyzed on a FACS Cantoll (BD Biosciences). Binding curves were analyzed using GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA) using non-linear regression (sigmoidal dose-response with variable slope).

[0672] Figure 1A shows the binding of IgG1λ-huCD3 variants IgG1-huCD3-H1L1 (SEQ ID NO:6 and 10 respectively), IgG1-huCD3-H1L2 (SEQ ID NO:6 and 11 respectively), IgG1-huCD3-H1L3 (SEQ ID NO:6 and 12 respectively), IgG1-huCD3-H3L3 (SEQ ID NO:8 and 12 respectively), and IgG1-huCD3-H4L1 (SEQ ID NO:9 and 10 respectively) with wild-type Fc regions to Jurkat cells, and the binding ability of IgG1-CD3-LFLEDA (the parental CD3 antibody with an inactivated LFLEDA mutation as described in Example 1) and IgG1-huCD3-H3L1-LFLEDA with an inactivated LFLEDA mutation is similar to that of huCD3 variants with wild-type Fc regions. Compared with the IgG1-huCD3 variants, the binding of IgG1-huCLB-T3 / 4 included as a positive control to Jurkat cells is strong. No binding was observed for the negative control antibody IgG1-b12.

[0673] Figure 6 A shows that the binding ability of IgG1-CD3-LFLEDA (the parental CD3 antibody with an inactivated LFLEDA mutation as described in Example 1), IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1-3huCD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA, and IgG1-huCD3-H4L3-LFLEDA with an inactivated LFLEDA mutation is similar to that of huCD3 variants with wild-type Fc regions. Compared with the IgG1-huCD3 variants, the binding of IgG1-huCLB-T3 / 4 included as a positive control to Jurkat cells is stronger at low antibody concentrations but similar at higher antibody concentrations. In summary, the humanized CD3 variants have maintained a binding ability to CD3 similar to that of IgG1-CD3 antibodies. No binding was observed for the negative control antibody IgG1-b12.

[0674] Figure 1Panel B shows that the bispecific antibody variants bsIgG1 CD3 x HER2, bsIgG1 CD3 x b12-LFLEDA, and bsIgG1 huCD3-H3L1 x HER2-LFLEDA also bind to Jurkat cells. The maximum binding values for these bispecific antibodies are higher than those of the monospecific antibodies. The EC50 concentrations of the bispecific antibodies are 6 - 10-fold higher. Again, no binding was observed for the negative control antibody IgG1-b12.

[0675] Figure 6 Panel B shows that the bispecific non-activated Fc antibody variants bsIgG1 huCD3-H3L1 x HER2-LFLEDA, bsIgG1-huCD3-H3L3 x HER2-LFLEDA, bsIgG1-huCD3-H1L1 x HER2-LFLEDA, bsIgG1-huCD3-H1L3 x HER2-LFLEDA, bsIgG1-huCD3-H4L1 x HER2-LFLEDA, and bsIgG1-huCD3-H4L3 x HER2-LFLEDA also bind to Jurkat cells. The maximum binding values for these bispecific antibodies are higher than those of the monospecific antibodies. The EC50 concentrations of the bispecific antibodies are 4 - 10-fold higher. Monovalent binding allows more antibodies to accumulate on the cell surface and thus allows higher binding values for the bispecific antibodies. Again, no binding was observed for the negative control antibody IgG1-b12.

[0676] Figure 2 Panel A shows that the binding of the IgG1-huCD3 variants IgG1-huCD3-H1L1, IgG1-huCD3-H1L2, IgG1-huCD3-H1L3, IgG1-huCD3-H3L3, and IgG1-huCD3-H4L1 with wild-type Fc region and IgG1-CD3-LFLEDA, IgG1-huCD3-H3L1-LFLEDA to the cynomolgus monkey T cell line HSC-F is similar. No binding was observed for the control antibody huCLB-T3 / 4 (which does not cross-react with cynomolgus monkey CD3) and the negative control antibody IgG1-b12.

[0677] Figure 7A shows that the binding of IgG1-huCD3 variants IgG1-CD3-LFLEDA, IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1-huCD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA, and IgG1-huCD3-H4L3-LFLEDA to cynomolgus monkey T cell line HSC-F is similar. No binding was observed for the negative control antibody IgG1-b12.

[0678] Figure 2 B shows that the bispecific antibody variants bsIgG1 CD3 x HER2 and bsIgG1 huCD3-H3L1-LFLEDA also bind to HSC-F cells. The maximum binding values for these bispecific antibodies are higher than those of the monospecific anti-CD3 variants. The EC50 concentrations of the bispecific antibodies are 10 - 12 times higher than those of the monospecific anti-CD3 antibodies. Again, no binding was observed for the negative control antibody IgG1-b12.

[0679] Figure 7 B shows that the bispecific non-activated Fc antibody variants bsIgG1 huCD3-H3L1 x HER2-LFLEDA, bsIgG1-huCD3-H3L3 x HER2-LFLEDA, bsIgG1-huCD3-H1L1 x HER2-LFLEDA, bsIgG1-huCD3-H1L3 x HER2-LFLEDA, bsIgG1-huCD3-H4L1 x HER2-LFLEDA, and bsIgG1-huCD3-H4L3 x HER2-LFLEDA also bind to HSC-F cells. The maximum binding values for these bispecific antibodies are higher than those of the monospecific anti-CD3 variants. The EC50 concentrations of the bispecific antibodies are 3 - 6 times higher than those of the monospecific anti-huCD3 antibodies. Again, no binding was observed for the negative control antibody IgG1-b12.

[0680] Example 3 – T Cell Activation by Humanized CD3 Antibody Variants

[0681] CD69 expression is an early marker of T cell activation. CD3 antibodies can mediate cross-linking of T cells and immune cells via the Fc region of the antibody, such as the IgG1 Fc region, through binding of CD3 expressed by T cells and Fc receptors expressed by immune cells. This can lead to T cell activation and induction of CD69. Antibody variants containing an inactivated Fc region (LFLEDA mutation) do not bind Fc receptors. Thus, it is expected that inactivated CD3 antibodies do not induce T cell activation and CD69 expression because the inactivated Fc region does not bind to immune cells expressing Fc receptors and thus cannot cross-link T cells and immune cells.

[0682] CD69 expression on T cells was evaluated by FACS analysis to determine early activation of T cells after incubation with humanized CD3 (huCD3) variants containing and not containing the LFLEDA mutation in the Fc region. In addition to the inactivated mutation, the LFLEDA variant contains the F405L or K409R mutation as described in Example 1.

[0683] PBMC were isolated from whole blood or buffy coat by density gradient separation using Leucosep tubes (#227290; Greiner Bio-one, Alphen a / d Rijn, the Netherlands), washed with PBS and resuspended in medium.

[0684] A dose-response series of huCD3 antibody variants, negative control (IgG1-b12), and positive controls (IgE-huCD3 and parental IgG1-CD3) (ranging from 0.1 - 1,000 ng / mL in 10-fold dilutions) were prepared in medium and added to wells of a 96-well round-bottom plate containing human or cynomolgus monkey PBMC. After incubation for 16 - 24 hours, the cells were pelleted by centrifugation, and the supernatant (containing cytokines) was collected and stored at -20 °C. The cells were then washed with PBS / 0.1% BSA / 0.02% azide and stained for 30 minutes at 4 °C with mouse anti-human CD28-PE (854.222.010; Sanquin, Amsterdam, the Netherlands; T cell marker) and mouse anti-human CD69-APC antibody (340560; BD Biosciences, Franklin Lakes, NJ), which cross-react with cynomolgus monkey CD28 and CD69, respectively. Unbound antibody was removed by washing twice with PBS / 0.1% BSA / 0.02% azide. The cells were resuspended at 150 µL / well and CD69 expression on CD28-positive cells was measured on a FACS CantoII (BD Biosciences).

[0685] Figure 3 shows that IgG1-CD3 (as described in Example 1) and a humanized IgG1-huCD3 variant with a wild-type IgG1 Fc region induce similar levels of CD69 expression on T cells from human Figure 3 A) and cynomolgus monkey Figure 3 B) origin. Non-activated (LFLEDA) IgG1-CD3-LFLEDA and IgG1-huCD3-H3L1 variants induce low levels of CD69 expression in human T cells. CD69 expression is not induced in cynomolgus monkey T cells by non-activated IgG1-huCD3 variants. The control antibody IgG1-b12 also does not induce CD69 expression in human or cynomolgus monkey T cells.

[0686] Figure 8 shows that non-activated (LFLEDA) IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1-3huCD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA and IgG1-huCD3-H4L3-LFLEDA variants induce low levels of CD69 expression in human T cells. Figure 8 A and 8B show the induction of CD69 expression on T cells from cynomolgus monkeys. The minor activation of the non-activated variants observed may be due to the cross-linking of CD3 molecules by the bivalent binding of CD3 antibodies. Such an explanation is supported by the observation that activation is reduced at the highest concentrations where antibody binding is monovalent. The control antibody IgG1-b12 also does not induce CD69 expression in human or cynomolgus monkey T cells.

[0687] Figure 8 C and 8D show that non-activated bispecific antibody variants bsIgG1-huCD3-H3L1 x HER2-LFLEDA, bsIgG1-huCD3-H3L3 x HER2-LFLEDA, bsIgG1-huCD3-H1L1 x HER2-LFLEDA, bsIgG1-huCD3-H1L3 x HER2-LFLEDA, bsIgG1-huCD3-H4L1 x HER2-LFLEDA and bsIgG1-huCD3-H4L3 x HER2-LFLEDA do not induce CD69 expression in T cells from human Figure 8 C) or cynomolgus monkey Figure 8 D). However, at higher antibody concentrations, some induction of CD69 expression is observed.

[0688] Example 4 – T cell proliferation induced by humanized CD3 antibody variants.

[0689] The effect of humanized CD3 (huCD3) antibody variants (described in Example 1) on the proliferation of human and cynomolgus monkey T cells was evaluated by a cell proliferation ELISA kit from Roche Applied Science (Cell Proliferation ELISA, BrdU kit, #11647229001; Roche Applied Science, Mannheim, Germany), which was performed according to the manufacturer's instructions.

[0690] Human or cynomolgus monkey PBMCs isolated from whole blood or buffy coat were incubated in 96-well culture plates with a dilution series of IgG1 huCD3 antibody variants (ranging from 0.1 - 1,000 ng / mL in 10-fold dilutions). IgE-CD3 and IgG1-huCLB-T3 / 4 were included as positive controls, and IgG1-b12 was included as a negative control. After 3 days of incubation with the antibody, BrdU (Roche Applied Science, Mannheim, Germany) was added to the medium, and the plates were incubated for 5 hours. The cells were then pelleted by centrifugation, and the supernatants were collected and stored at -20°C. The plates were dried and stored at 4°C until ELISA was performed.

[0691] BrdU incorporation into DNA was determined by ELISA according to the manufacturer's instructions (Roche Applied Science). The cells were fixed to the plates after the plates were incubated with anti-BrdU antibody conjugated to peroxidase for 90 minutes at RT. The plates were washed with PBST, and binding was detected using ABTS buffer (instead of the TMB solution provided with the kit). Color development was stopped after 30 minutes by adding 2% oxalic acid to the wells. OD405 nm was then measured on an EL808 ELISA reader.

[0692] Figure 4 It was shown that incubation of PBMCs with the parental IgG1-CD3 and humanized IgG1-huCD3 variants with wild-type IgG1 Fc region induced significant proliferation of human ( Figure 4 A) and cynomolgus monkey ( Figure 4 B) T cells even at very low concentrations of the antibody. Incubation with the non-activated LFLEDA variant of the IgG1-huCD3 antibody did not induce proliferation of human T cells ( Figure 4 A and 9A) or cynomolgus monkey T cells ( Figure 4Proliferation of B and 9B). Thus, although the non-activated variants of the IgG1-huCD3 antibody induced low levels of CD69 expression in human T cells (as shown in Example 3), proliferation of human T cells was not induced by these non-activated IgG1-huCD3 variants.

[0693] Figure 9 C and 9D show that the non-activated bispecific antibody variants bsIgG1-huCD3-H3L1 x HER2-LFLEDA, bsIgG1-huCD3-H3L3 x HER2-LFLEDA, bsIgG1-huCD3-H1L1 x HER2-LFLEDA, bsIgG1-huCD3-H1L3 x HER2-LFLEDA, bsIgG1-huCD3-H4L1 x HER2-LFLEDA, and bsIgG1-huCD3-H4L3 x HER2-LFLEDA do not induce proliferation of T cells isolated from human Figure 9 C) or cynomolgus monkeys ( Figure 9 D).

[0694] Example 5 – In Vitro T Cell-Mediated Cytotoxicity Induced by Humanized CD3 Antibody Variants

[0695] Tumor-specific T cell cytotoxicity can be mediated by bispecific antibodies that bind to CD3 with one arm and to a tumor-specific target such as HER2 with the other arm. Simultaneous binding of the bispecific antibody to both T cells and tumor cells will result in T cell activation and tumor cell-specific cytotoxicity. In this example, bispecific antibodies directed against CD3 (humanized variants) and HER2 were used to evaluate T cell-mediated cytotoxicity against HER2-positive tumor cells.

[0696] Thus, AU565 (human breast cancer) cells were cultured in RPMI 1640 supplemented with 10% (v / v) heat-inactivated CCS, 1.5 g / L sodium bicarbonate (Lonza), 1 mM sodium pyruvate, 4.5 g / L glucose (Sigma), 50 IU / mL penicillin, and 50 μg / mL streptomycin. The cell line was maintained at 37 °C in a 5% (v / v) CO2 humidified incubator. AU565 cells were cultured to near confluence, after which the cells were trypsinized, resuspended in medium, and passed through a cell strainer to obtain a single cell suspension. 5x10 4 cells were seeded into each well of a 96-well culture plate, and the cells were incubated at 37 °C, 5% CO2 for at least 3 hours to allow attachment to the plate.

[0697] Human or cynomolgus monkey PBMCs are isolated from whole blood or buffy coat. The isolated PBMCs are washed with PBS, resuspended in medium, and added to AU565 tumor cells in 96-well plates at a 1:1 ratio. The percentage of T cells present in the PBMCs is measured by FACS analysis using mouse anti-human CD3-PerCP (BD, #345766) antibody (for staining T cells), which cross-reacts with cynomolgus monkey CD3. The T cell content in the population of PBMCs used is typically 50 - 60%.

[0698] Dilution series (final concentration ranging from 0.001 to up to 10,000 ng / mL) of bispecific antibody variants bsIgG1 CD3 x HER2-LFLEDA, bsIgG1 CD3 x b12-LFLEDA, bsIgG1 huCD3-H3L1 x HER2-LFLEDA, bsIgG1-huCD3-H3L3 x HER2-LFLEDA, bsIgG1-huCD3-H1L1 x HER2-LFLEDA, bsIgG1-huCD3-H1L3 x HER2-LFLEDA, bsIgG1-huCD3-H4L1 x HER2-LFLEDA, and bsIgG1-huCD3-H4L3 x HER2-LFLEDA are prepared in medium and added to the plates. IgG1-HER2-LFLEDA and IgG1-b12 are included as controls. Except for the non-activating mutations, the LFLEDA antibody variants contain the F405L or K409R mutations for preparation in bispecific form (see Example 1). The plates are incubated at 37 °C, 5% CO2 for 3 days. Cells are incubated with 1 µM staurosporine (#S6942-200, Sigma) as a reference for 100% tumor cell killing. The plates are washed twice with PBS, and 150 μL of medium containing 10% Alamar blue is added to each well. The plates are incubated at 37 °C, 5% CO2 for 4 hours. Absorbance is measured at 590 nm (Envision, Perkin Elmer, Waltham, MA).

[0699] Bispecific CD3xHER2-LFLEDA antibody variants (bsIgG1-huCLB-T3 / 4xHER2-LFLEDA and bsIgG1-CD3xHER2-LFLEDA) induce killing of AU565 cells at low concentrations, using human effector cells ( Figure 5 A) or cynomolgus monkey effector cells ( Figure 5 B). When using human PBMCs ( Figure 5A), the CD3 bispecific control antibody huCLB-T3 / 4xHER2-LFLEDA (which did not show cross-reactivity with cynomolgus monkey CD3) only induced killing of AU565 cells. Thus, no killing of target cells was observed when cynomolgus monkey effector cells were used in the assay. Incubation with the monospecific IGG1-b12 or IgG1-HER2-LFLEDA or bsIgG1-CD3xb12-LFLEDA antibodies did not induce non-specific killing of target cells.

[0700] The bispecific antibody variants bsIgG1 huCD3-H3L1 x HER2-LFLEDA, bsIgG1-huCD3-H3L3 xHER2-LFLEDA, bsIgG1-huCD3-H1L1 x HER2-LFLEDA, bsIgG1-huCD3-H1L3 x HER2-LFLEDA, bsIgG1-huCD3-H4L1 x HER2-LFLEDA, and bsIgG1-huCD3-H4L3 x HER2-LFLEDA induced killing of AU565 cells at low concentrations, using human effector cells ( Figure 10 A) or cynomolgus monkey effector cells ( Figure 10 B). Incubation with the monospecific IgG1-b12 or IgG1-HER2-LFLEDA antibodies did not induce non-specific target cell killing ( Figure 10 A and B). Thus, the humanized CD3 variants containing the non-activated Fc region did not induce non-specific target cell killing, indicating that variants containing the non-activated Fc region can be used to ensure targeted T cell activation and thus avoid off-target T cell activation.

[0701] Example 6 – Activation of Rhesus Monkey T Cells by Humanized CD3 Antibody Variants

[0702] CD69 expression on rhesus monkey T cells was evaluated to determine early activation of T cells after incubation with humanized CD3 (huCD3) antibody variants with wild-type IgG1 Fc regions. Rhesus monkey PBMC isolation and CD69 expression assessment by flow cytometry were performed as described in Example 3.

[0703] Figure 11The humanized CD3 antibody variants IgG1-huCD3-H1L1, IgG1-huCD3-H1L2, IgG1-huCD3-H1L3, IgG1-huCD3-H3L3, and IgG1-huCD3-H4L1 were shown to induce CD69 expression on T cells of rhesus macaque origin to levels similar to those of IgG1-CD3 (as described in Example 1). The negative control antibody IgG1-b12 did not induce CD69 expression in rhesus macaque T cells. Thus, the huCD3 variants according to the present invention can be used in experiments involving rhesus macaque CD3. The huCD3 variants are cross-reactive with rhesus macaque CD3.

[0704] Example 7 - T cell activation by non-activated variants of huCLB-T3 / 4

[0705] CD69 expression on T cells was evaluated by FACS analysis to determine the early activation of T cells after incubation with IgG1-huCLB-T3 / 4 variants having mutations in the Fc region (see Example 1).

[0706] PBMC were isolated from whole blood or buffy coat by density gradient separation using Leucosep tubes (#227290; Greiner Bio-one, Alphen a / d Rijn, Netherlands), washed with PBS and resuspended in medium.

[0707] A dose-response series of IgG1-huCLB-T3 / 4 variants, negative control (IgG1-huCLB-T3 / 4-Fab), and positive control (IgE-huCLB-T3 / 4) (ranging from 1 - 1000 ng / mL in 3-fold dilutions) were prepared in medium and added to the wells of a 96-well round bottom plate containing PBMC. After incubation for 16 - 24 hours, the cells were pelleted by centrifugation, and the supernatant (containing cytokines) was collected and stored at -20°C. The cells were then washed with PBS / 0.1% BSA / 0.02% azide and stained for 30 minutes at 4°C with mouse anti-human CD28-PE (854.222.010; Sanquin, Amsterdam, Netherlands; T cell marker) and mouse anti-human CD69-APC antibody (340560; BD Biosciences, Franklin Lakes, NJ). Unbound antibody was removed by washing twice with PBS / 0.1% BSA / 0.02% azide. The cells were resuspended at 150 µL / well and CD69 expression on CD28-positive cells was measured on a FACS Canto II (BD Biosciences).

[0708] Figure 12 A shows that CD69 expression is high on cells incubated with IgE-huCLB-T3 / 4, IgG1-huCLB-T3 / 4, IgG1-huCLB-T3 / 4-DA, and IgG1-huCLB-T3 / 4-DAPS. Incubation with IgG1-huCLB-T3 / 4-N297Q induces a slightly lower CD69 expression level compared to wild-type IgG1-huCLB-T3 / 4, and incubation with IgG1-huCLB-T3 / 4-LFLE and IgG1-huCLB-T3 / 4-LFLEPS induces CD69 to a lesser extent. Incubation of PBMC with IgG1-CD3Fab, IgG1-huCLB-T3 / 4-LFLEDA, IgG1-huCLB-T3 / 4-LFLENQ, IgG1-huCLB-T3 / 4-DANQ, and IgG1-huCLB-T3 / 4-LFLEDANQPS antibodies does not induce any CD69 expression on T cells.

[0709] Figure 12 B shows that CD69 expression is high on cells incubated with IgE-huCLB-T3 / 4 and IgG1-huCLB-T3 / 4. Incubation with IgG1-huCLB-T3 / 4-LALA induces a slightly lower CD69 expression level compared to wild-type IgG1-huCLB-T3 / 4, and incubation with IgG1-huCLB-T3 / 4-LFLEDA and IgG1-b12 (negative control) does not induce any CD69 expression on T cells.

[0710] Example 8 – T Cell Proliferation by Non-Activating Variants of huCLB-T3 / 4

[0711] The effect of huCLB-T3 / 4 variants (described in Example 1) on the proliferation of T cells was evaluated by a cell proliferation ELISA kit from Roche Applied Science (Cell Proliferation ELISA, BrdU kit, #11647229001; Roche Applied Science, Mannheim, Germany), which was performed according to the manufacturer's instructions.

[0712] PBMCs isolated from whole blood or buffy coat were incubated in 96-well culture plates with a dilution series of IgG1-CD3 variants (ranging from 0.1 - 1000 ng / mL). IgG1-CD3 and IgE-CD3 were included as positive controls, and IgG1-b12 (with the K409R mutation for bispecific antibody generation) was included as a negative control. After 3 days of incubation with the antibodies, BrdU (Roche Applied Science, Mannheim, Germany) was added to the medium, and the plates were incubated for 5 hours. The cells were then pelleted by centrifugation, and the supernatants were collected and stored at -20°C. The plates were dried and stored at 4°C until ELISA was performed.

[0713] BrdU incorporation into DNA was determined by ELISA according to the manufacturer's instructions (Cell Proliferation ELISA, BrdU kit, #11647229001; Roche Applied Science). The cells were fixed to the plates after the plates were incubated with anti-BrdU antibody conjugated to peroxidase for 90 minutes at room temperature (RT). The plates were washed with PBST, and binding was detected using ABTS buffer (instead of the TMB solution provided with the kit). Color development was stopped after 30 minutes by adding 2% oxalic acid to the wells. OD405 nm was then measured on an EL808 ELISA reader.

[0714] Figure 13 A shows that incubation of PBMCs with IgG1-huCLB-T3 / 4, IgG1-huCLB-T3 / 4-DA, and IgG1-huCLB-T3 / 4-DAPS induces significant proliferation of T cells even at very low concentrations of the antibodies. Incubation with IgG1-huCLB-T3 / 4-N297Q induces dose-dependent proliferation, which is comparable to the IgE-huCLB-T3 / 4 positive control. Incubation of PBMCs with IgG1-huCLB-T3 / 4-Fab, IgG1-b12-N297Q, IgG1-huCLB-T3 / 4-LFLE, IgG1-huCLB-T3 / 4-LFLEDA, IgG1-huCLB-T3 / 4-LFLENQ, IgG1-huCLB-T3 / 4-LFLEPS, IgG1-huCLB-T3 / 4-DANQ, and IgG1-huCLB-T3 / 4-LFLEDANQPS antibodies does not induce proliferation of T cells.

[0715] Figure 13Panel B shows that incubation of PBMC with IgG1-huCLB-T3 / 4 induced significant proliferation of T cells even at very low concentrations of antibody. Incubation with IgE-huCLB-T3 / 4 (positive control) and IgG1-huCLB-T3 / 4-LALA induced dose-dependent proliferation. Incubation of PBMC with IgG1-huCLB-T3 / 4-LFLEDA did not induce proliferation of T cells.

[0716] Based on the results from Examples 7 and 8, further analysis was performed on the subset of mutants considered to be the least activated.

[0717] Example 9 – In vitro T cell-mediated cytotoxicity induced by non-activating antibody variant huCLB-T3 / 4

[0718] AU565 (human breast cancer) cells were cultured in RPMI 1640 supplemented with 10% (v / v) heat-inactivated CCS, 1.5 g / L sodium bicarbonate (Lonza), 1 mM sodium pyruvate, 4.5 g / L glucose (Sigma), 50 IU / mL penicillin, and 50 μg / mL streptomycin. The cell line was maintained at 37 °C in a 5% (v / v) CO2 humidified incubator. AU565 cells were cultured to near confluence. The cells were trypsinized, resuspended in medium, and passed through a cell strainer to obtain a single cell suspension. 5x10 4 cells were seeded into each well of a 96-well culture plate and the cells were incubated at 37 °C, 5% CO2 for at least 3 hours to allow attachment to the plate.

[0719] Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy volunteers using Leucosep 30 mL tubes according to the manufacturer's protocol (Greiner Bio-one). The isolated PBMC were washed with PBS, resuspended in medium, and added to the AU565 tumor cells in the 96-well plate at a 1:1 ratio. The percentage of T cells present in the PBMC was measured by FACS analysis using the mouse anti-human CD3-PerCP (BD, #345766) antibody (for staining T cells). The T cell content in the population of PBMC used was typically 50 - 60%.

[0720] Prepare dilution series (final concentration range 0.004 - 1000 ng / mL) of IgG1-b12, IgG1-huCLB-T3 / 4, IgG1-HER2, and bispecific huCLB-T3 / 4xb12 and huCLB-T3 / 4xHER2 antibodies expressed as different Fc variants, wild type, N297Q, LFLE, LALA, LFLENQ, LFLEDA, DANQ, and LFLEDENQPS in culture medium and add to plates. Plates are incubated at 37 °C, 5% CO2 for 3 days. Cells incubated with 1 μM staurosporine (#S6942-200, Sigma) are used as a reference for 100% tumor cell killing. After incubation, supernatants are removed and stored at -20 °C. Plates are washed twice with PBS and 150 μL of medium containing 10% Alamar Blue is added to each well. Plates are incubated at 37 °C, 5% CO2 for 4 hours. Absorbance is measured at 590 nm (Envision, Perkin Elmer, Waltham, MA).

[0721] Two experiments are performed using PBMC from different donors. In the first experiment, Fc variants N297Q, LFLE, LFLENQ, LFLEDA, DANQ, and LFLEDANQPS ( Figure 14 A - G) are tested. In the second experiment, Fc variants LFLEDA and LALA ( Figure 15 A - C) are tested. Antibodies with wild type Fc domains are included as a reference in both experiments. Incubation with wild type monospecific IgG1-huCLB-T3 / 4 or bispecific huCLB-T3 / 4xb12 antibodies induces non-specific killing of target cells ( Figure 14 A - G and 15A - C). Monospecific IgG1-huCLB-T3 / 4 and bsIgG1-huCLB-T3 / 4xb12 variants N297Q ( Figure 14 A - G) and LALA ( Figure 15 A - C) still induce some non-specific target cell killing, although to a lesser extent than the wild type antibodies tested in the same experiment. Non-specific target cell killing is not induced by any of the other tested IgG1-huCLB-T3 / 4 or bsIgG1-huCLB-T3 / 4xb12 antibodies with inactivated mutations ( Figure 14 A - G and 15A - C).

[0722] All bispecific huCLB-T3 / 4xHER2 antibodies induce dose-dependent killing of AU565 cells with at least comparable efficacy compared to wild type bispecific huCLB-T3 / 4xHER2 antibodies without inactivated mutationsFigure 14 A-G and 15A-C). Maximum killing occurs at very low concentrations.

[0723] Wild-type or non-activated variants of single-specific b12 or HER2 antibodies did not induce cytotoxicity, as expected.

[0724] Example 10 – Assessment of the binding of C1q to non-activated antibody variants of huCLB-T3 / 4

[0725] The interaction of C1q with antibodies that bind to target cells is the first step in the classical pathway of complement activation. Since wild-type IgG1 has an interaction site for C1q, the interaction of C1q with these non-activated IgG1 variants was evaluated by ELISA.

[0726] Dilution series (ranging from 7 - 30,000 ng / mL in 4-fold dilutions) of IgG1-huCLB-T3 / 4, bsIgG1-huCLB-T3 / 4xHER2, and IgG1-CD20 (positive control) and their non-activated antibody variants as described in Example 1 above were coated onto 96-well Microlon ELISA plates (Greiner, Germany) overnight at 4 °C. The plates were blocked with PBS supplemented with 0.025% Tween 20 and 0.1% gelatin. With washing between incubations, the plates were sequentially incubated with 3% pooled human serum (Sanquin, product #M0008) for 1 hour at 37 °C, with 100 μL / well of rabbit anti-human C1q (DAKO, product #A0136, 1 / 4,000) for 1 hour at RT, and with 100 μL / well of swine anti-rabbit IgG-HRP (DAKO, P0399, 1:10,000) as the detection antibody for 1 hour at RT. Detection was performed by adding 1 mg / mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany) for approximately 30 minutes. The reaction was stopped by adding 100 μL of 2% oxalic acid. Absorbance was measured at 405 nm in a microplate reader (Biotek, Winooski, VT). Log-transformed data were analyzed by fitting a sigmoidal response curve with variable slope using GraphPad Prism software.

[0727] Figure 16A shows that the antibodies IgG1-CD20 and IgG1-huCLB-T3 / 4 with wild-type IgG1 Fc regions show C1q binding. No C1q binding was detected on all evaluated antibody variants with inactivating mutations (N297Q, LFLE, LFLENQ, LFLEDA, DA, DAPS, DANQ, LFLEPS, LFLEDANQPS, LALA).

[0728] Figure 16 B shows that the antibody bsIgG1-huCLB-T3 / 4xHER2 with wild-type IgG1 Fc region shows C1q binding. No C1q binding was detected on all evaluated antibody variants with inactivating mutations (N297Q, LFLE, LFLENQ, LFLEDA, DA, DAPS, DANQ, LFLEPS, LFLEDANQPS, LALA).

[0729] Figure 16 C and Figure 16 D shows that the antibodies IgG1-CD20, IgG1-huCLB-T3 / 4 and bsIgG1-huCLB-T3 / 4xHER2 with wild-type IgG1 Fc regions show C1q binding. No C1q binding was detected on the antibody variants with inactivating mutations (LFLEDA and LALA).

[0730] Example 11 – Pharmacokinetic (PK) analysis of inactivated antibody variants

[0731] The mice in this study were housed in a barrier unit at the Central Laboratory Animal Facility (Utrecht, the Netherlands) and kept in filter-top cages with water and food provided ad libitum. All experiments were approved by the Animal Ethics Committee of Utrecht University. 7- to 10-week-old C.B-17 SCID mice (C.B-17 / Icr-Prkdc <scid> / IcrIcoCrl, Charles-River) were intravenously injected with 100 μg of wild-type antibody (IgG1-huCLB-T3 / 4, IgG1-HER2 or bsIgG-huCLB-T3 / 4xHER2) or its inactivated variants (LALA, LFLEDA, LFLENQ, DANQ or LFLEDANQPS), using 3 mice / group. At 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibody administration, 50 μL of blood samples were collected from the saphenous vein. The blood was collected into heparin-containing vials and centrifuged at 10,000 x g for 5 minutes. The plasma was stored at -20 °C until the antibody concentration was determined.

[0732] Human IgG concentration was determined using a total hIgG sandwich ELISA. For this assay, mouse mAb anti-human IgGκ clone MH16 (#M1268, CLB Sanquin, Netherlands) at a concentration of 2 μg / mL was coated onto a 96-well Microlon ELISA plate as the capture antibody. After blocking the plate with PBS supplemented with 0.2% bovine serum albumin, samples were added, serially diluted with ELISA buffer (PBS supplemented with 0.05% Tween 20 and 0.2% bovine serum albumin), and incubated for 1 hour at room temperature (RT) on a plate shaker. The plate was then incubated with goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, PA) and developed with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). The reaction was stopped after 30 minutes by adding 2% oxalic acid to the wells. Absorbance was measured at 405 nm in a microplate reader (Biotek, Winooski, VT).

[0733] Plasma clearance (mL / day / kg) was calculated based on the area under the curve (AUC) according to the following equation:

[0734]

[0735] Data analysis was performed using Graphpad prism software.

[0736] Figure 17 A shows that plasma human IgG concentration was lower for antibody variants N297Q, DANQ, LFLENQ and LFLEDANQPS compared to wild-type antibody, suggesting faster clearance. For antibody variants LFLEDA and LALA, the plasma human IgG concentrations were similar to those of the wild-type antibody.

[0737] Figure 17 B shows that the plasma clearance rates of antibody variants N297Q, DANQ, and LFLENQ are 2 - 3 times higher than those of the wild-type antibody. The clearance rate of antibody variant LFLEDANQPS is 3 - 5 times higher than that of the wild-type antibody. The plasma clearance rates of antibody variants LFLEDA and LALA are similar to those of the wild-type antibody.

[0738] Example 12 - In Vitro Immunogenicity Evaluation of the IgG1-LFLEDA Backbone

[0739] To determine the potential clinical immunogenicity of the IgG1-LFLEDA-K409R backbone, Antitope's EpiScreen™ platform was applied to IgG1-HER2-LFLEDA. Briefly, PBMCs were isolated from a cohort of 50 HLA-typed healthy individuals representing European and North American populations. After CD8+ T cell depletion, the PBMC preparations were cryopreserved individually and stored. Subsequently, the thawed PBMCs were cultured and incubated with either IgG1-HER2-LFLEDA-K409R or one of the control samples (IgG1-HER2 or IgG1-HER2-LFLE-K409R) for 5 - 8 days. The ability of the samples to induce CD4+ T cell responses was evaluated by measuring cell proliferation ([3 H -thymidine incorporation) and IL-2 production (ELISpot assay). Donors showing responses with a stimulation index (SI; signal / baseline signal) ≥ 1.9 in both assays were considered positive.

[0740] EpiScreen™ analysis showed that for IgG1-HER2-LFLEDA, 4 donors (8%) showed positive CD4+ T cell responses, which were comparable to the 4 (8%) and 3 (6%) positive responses for IgG1-HER2 and IgG1-HER2-LFLE, respectively ( Figure 18 ). Thus, IgG1-HER2-LFLEDA-K409R (as well as IgG1-HER2 and IgG1-HER2-LFLE-K409R) shows low potential for immunogenicity, with a frequency of responses below 10%. The positive control humanized A33 (e.g.

[84] ) was used as a clinical benchmark control antibody, which shows a high level of immunogenicity in clinical practice and routinely induces 20 - 30% T cell responses in the EpiScreen assay.

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[84] Ritter G et al.; 2001, Cancer Res., 61:6851-9。 Sequence Listing <110> Genmab A / S <120> Humanized or Chimeric CD3 Antibodies <130> P / 0082-WO <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Homo sapiens <400> 1 Gly Phe Thr Phe Asn Thr Tyr Ala 1 5 <210> 2 <211> 10 <212> PRT <213> Homo sapiens <400> 2 Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 1 5 10 <210> 3 <211> 16 <212> PRT <213> Homo sapiens <400> 3 Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 15 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <400> 4 Thr Gly Ala Val Thr Thr Ser Asn Tyr 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 6 <211> 125 <212> PRT <213> Homo sapiens <400> 6 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 125 <212> PRT <213> Homo sapiens <400> 7 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 8 <211> 125 <212> PRT <213> Homo sapiens <400> 8 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 9 <211> 125 <212> PRT <213> Homo sapiens <400> 9 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 10 <211> 109 <212> PRT <213> Homo sapiens <400> 10 Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 11 <211> 109 <212> PRT <213> Homo sapiens <400> 11 Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 12 <211> 109 <212> PRT <213> Homo sapiens <400> 12 Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 13 <211> 186 <212> PRT <213> Homo sapiens <400> 13 Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro 20 25 30 Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp 35 40 45 Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys 50 55 60 Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg 65 70 75 80 Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg 85 90 95 Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile 100 105 110 Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr 115 120 125 Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly 130 135 140 Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro 145 150 155 160 Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp 165 170 175 Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 180 185 <210> 14 <211> 150 <212> PRT <213> Homo sapiens <400> 14 Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys 1 5 10 15 Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser 20 25 30 Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly 35 40 45 Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr 50 55 60 Val Gln Val His Tyr Arg Met Cys Gln Ser Cys Val Glu Leu Asp Pro 65 70 75 80 Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val Ile Ala Thr Leu Leu 85 90 95 Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His Glu Thr Gly Arg Leu 100 105 110 Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg Asn Asp Gln Val Tyr 115 120 125 Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr Ser His Leu Gly Gly 130 135 140 Asn Trp Ala Arg Asn Lys 145 150 <210> 15 <211> 330 <212> PRT <213> Homo sapiens <400> 15 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 16 <211> 330 <212> PRT <213> Homo sapiens <400> 16 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 17 <211> 119 <212> PRT <213> Homo sapiens <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Arg Tyr Ser Arg Tyr Ile Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Pro Leu Tyr Gly Ser Ser Pro Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 18 <211> 106 <212> PRT <213> Homo sapiens <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Thr Tyr Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Leu Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Met Arg 100 105 <210> 19 <211> 121 <212> PRT <213> Homo sapiens <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Leu Ser Ala Tyr Ser Gly Asn Thr Ile Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Ile Val Val Arg Pro Asp Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 107 <212> PRT <213> Homo sapiens <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 21 <211> 177 <212> PRT <213> Homo sapiens <400> 21 Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr Gln 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu 20 25 30 Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser 35 40 45 Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly 50 55 60 Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His 65 70 75 80 His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp 85 90 95 Val Met Ala Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Leu 100 105 110 Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys 115 120 125 Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly 130 135 140 Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro 145 150 155 160 Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg 165 170 175 Ile <210> 22 <211> 177 <212> PRT <213> Homo sapiens <400> 22 Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr His 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu 20 25 30 Gly Ser Glu Val Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser 35 40 45 Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly 50 55 60 Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His 65 70 75 80 His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp 85 90 95 Val Met Ala Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Leu 100 105 110 Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys 115 120 125 Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly 130 135 140 Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro 145 150 155 160 Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg 165 170 175 Ile <210> 23 <211> 330 <212> PRT <213> Homo sapiens <400> 23 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 24 <211> 330 <212> PRT <213> Homo sapiens <400> 24 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 25 <211> 330 <212> PRT <213> Homo sapiens <400> 25 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 26 <211> 330 <212> PRT <213> Homo sapiens <400> 26 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Methionine, Threonine, Lysine, Asparagine, Glutamine, Valine, Serine, Leucine, Threonine, Cysteine, Leucine, Valine, Lysine, Glycine, Phenylalanine, Tyrosine 245 250 255 Proline, Serine, Aspartic Acid, Isoleucine, Alanine, Valine, Glutamic Acid, Tryptophan, Glutamic Acid, Serine, Asparagine, Glycine, Glutamine, Proline, Glutamic Acid, Asparagine 260 265 270 Asparagine, Tyrosine, Lysine, Threonine, Threonine, Proline, Proline, Valine, Leucine, Aspartic Acid, Serine, Aspa...

Claims

1. A humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region that comprises heavy chain variable (VH) region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 1, 2, and 3, respectively, and light chain variable (VL) region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO: 4, the sequence GTN, and SEQ ID NO: 5, respectively, and wherein the VH region is selected from the following: a) The VH sequence shown in SEQ ID NO: 6; b) The VH sequence shown in SEQ ID NO: 8; c) The VH sequence shown in SEQ ID NO: 7; and d) The VH sequence shown in SEQ ID NO:

9.

2. The antibody according to any one of the preceding claims, wherein the VL region is selected from the following: a) The VL sequence shown in SEQ ID NO: 11; and b) The VL sequence shown in SEQ ID NO:

12.

3. The antibody according to any one of claims 1-2, wherein the VH sequence is as shown in SEQ ID NO:

6.

4. The antibody according to any one of claims 1-2, wherein the VH and VL regions are selected from the following: a) The VH sequence shown in SEQ ID NO: 8, and the VL sequence shown in SEQ ID NO: 10; b) The VH sequence shown in SEQ ID NO: 9, and the VL sequence shown in SEQ ID NO: 10; c) The VH sequence shown in SEQ ID NO: 6, and the VL sequence shown in SEQ ID NO: 11; d) The VH sequence shown in SEQ ID NO: 6, and the VL sequence shown in SEQ ID NO: 12; e) The VH sequence shown in SEQ ID NO: 7, and the VL sequence shown in SEQ ID NO: 10; f) The VH sequence shown in SEQ ID NO: 7, and the VL sequence shown in SEQ ID NO: 11; g) The VH sequence shown in SEQ ID NO: 7, and the VL sequence shown in SEQ ID NO: 12; h) The VH sequence shown in SEQ ID NO: 8, and the VL sequence shown in SEQ ID NO: 11; i) The VH sequence shown in SEQ ID NO: 8, and the VL sequence shown in SEQ ID NO: 12; j) The VH sequence shown in SEQ ID NO: 9, and the VL sequence shown in SEQ ID NO: 11; and k) The VH sequence shown in SEQ ID NO: 9, and the VL sequence shown in SEQ ID NO:

12.

5. The antibody according to any one of claims 1-2, wherein the binding region comprises a VH sequence and a VL sequence selected from the following: a) The VH sequence as shown in SEQ ID NO:8, and the VL sequence as shown in SEQ ID NO:10; and b) The VH sequence as shown in SEQ ID NO:9, and the VL sequence as shown in SEQ ID NO:

10.

6. The antibody according to any one of claims 1-2, wherein the antibody is a humanized antibody.

7. The antibody according to claim 1, wherein the antibody is a chimeric antibody.

8. The antibody according to any one of claims 1-2 and 7, wherein the antibody is a full-length antibody.

9. The antibody according to any one of claims 1-2 and 7, wherein the antibody comprises an Fc region containing a first and a second immunoglobulin heavy chain.

10. The antibody according to any one of claims 1-2 and 7, wherein the first and second heavy chains have isotypes selected from IgG1, IgG2, IgG3, and IgG4.

11. The antibody according to any one of claims 1-2 and 7, wherein the antibody comprises a first and a second immunoglobulin heavy chain, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively; or A and A.

12. The antibody according to claim 11, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively.

13. The antibody according to claim 11, wherein in at least one of the first and second heavy chains, at least the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are A and A, respectively.

14. The antibody according to claim 11, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A.

15. The antibody according to claim 14, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

16. The antibody according to claim 14, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are A, A, and A, respectively.

17. The antibody according to claim 11, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are F, E, A, Q, and S, respectively.

18. A bispecific antibody comprising a first binding region of an antibody according to any one of claims 1-10, and a second binding region that binds to a target different from the first antigen-binding region.

19. The bispecific antibody according to claim 18, wherein the antibody comprises a first and a second heavy chain.

20. The bispecific antibody according to claim 19, wherein at least one of the first and second heavy chains comprises one or more amino acids modified as defined in any one of claims 11-17.

21. The bispecific antibody according to claim 20, wherein the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain, and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain.

22. The bispecific antibody according to claim 20, wherein the amino acid at the position corresponding to F409 in the human IgG1 heavy chain is R in the first heavy chain, and the amino acid at the position corresponding to K405 in the human IgG1 heavy chain is L in the second heavy chain.

23. The bispecific antibody according to any one of claims 18-21, wherein the first binding region is the binding region of an antibody according to any one of claims 1-5, and the second binding region binds to a target different from the first binding region.

24. An expression vector comprising: (i) a nucleic acid sequence encoding the heavy chain sequence of an antibody according to any one of claims 1-23; and (ii) a nucleic acid sequence encoding the light chain sequence of an antibody according to any one of claims 1-23.

25. A set of expression vectors consisting of: (i) a first expression vector comprising a nucleic acid sequence encoding the heavy chain sequence of an antibody according to any one of claims 1-23; and (ii) a second expression vector comprising a nucleic acid sequence encoding the light chain sequence of an antibody according to any one of claims 1-23.

26. A host cell comprising the expression vector of claim 24 or the set of expression vectors of claim 25.

27. The host cell according to claim 26, wherein the 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 1-17 or a bispecific antibody according to any one of claims 18-23.

29. A pharmaceutical composition comprising an antibody according to any one of claims 1-17 or a bispecific antibody according to any one of claims 18-23 and a pharmaceutically acceptable carrier.

30. A method for producing an antibody according to any one of claims 1-17 or a bispecific antibody according to any one of claims 18-23, comprising the steps of: a) culturing a host cell according to any one of claims 26-27; and b) purifying the antibody from the culture medium.

31. A diagnostic composition comprising an antibody according to any one of claims 1-17 or a bispecific antibody according to any one of claims 18-23.

32. A kit for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, comprising: i) an antibody according to any one of claims 1-17 or a bispecific antibody according to any one of claims 18-23; and ii) instructions for use of the kit.

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