Humanized or chimeric CD3 antibodies

Abstract

The present invention relates to humanized or chimeric antibodies that bind to CD3, and also to bispecific antibodies, compositions, pharmaceutical compositions, uses of the antibodies in treating diseases, and methods of treatment.

Description

Field of the Invention

[0001] The present invention relates to humanized or chimeric antibodies that bind to human CD3, compositions comprising the humanized or chimeric antibodies, and uses of the humanized or chimeric antibodies in treating diseases. Background of the Invention

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

[0004] In the absence of continued immunosuppressive drug therapy, treatment with the anti-CD3 monoclonal antibody hOKT3γ1(Ala-Ala) resulted in improved C-peptide responses and clinical parameters at least 2 years after the onset of type 1 diabetes [2].

[0005] A promising approach to improve targeted antibody therapy is by delivering cytotoxic cells specifically directed against cancer cells expressing antigens. This concept of using T cells to effectively kill tumor cells has been described [3]. However, initial clinical studies were quite disappointing, mainly due to low efficacy, severe side effects (cytokine burst) and immunogenicity of bispecific antibodies [4]. Advances in the design and application of bispecific antibodies have partially overcome the initial obstacles of cytokine burst and improved clinical effectiveness without dose-limiting toxicity [5].

[0006] For example, certain bispecific antibodies that target an antigen on tumor cells with one arm and CD3 on T cells, for example, with the other arm, provide Fc receptor binding via the Fc region. Upon binding, a complex of T cells, tumor cells, and effector cells bound to the Fc region of the antibody is formed, resulting in killing of the tumor cell [4]. Catumaxomab consists of a mouse IgG2a / rat IgG2b heterodimer and has been found to be successful in treating cancer-related ascites following intraperitoneal administration [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 have been attributed to symptoms associated with catumaxomab-induced cytokine-release (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) [8]-[9], which are related to the effector function of the Fc region of catumaxomab. Another antibody is ertumaxomab (HER2xCD3), which induces cytotoxicity in cell lines with low HER2 expression. Ermaconazomab is already in phase II clinical development for metastatic breast cancer

[10] -

[11] .

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

[12] -

[13] , however further improvements in such cross-reactive antibodies are needed.

[0008] Brief description of the invention

[0009] The object of the present invention is to provide a humanized or chimeric CD3 antibody. Therefore, 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 comprising a heavy chain variable (VH) region CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO: 1, 2 and 3, respectively, and a light chain variable (VL) region CDR1, CDR2 and CDR3 having the sequence shown in SEQ ID NO: 4, the sequence GTN and the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 60, respectively.

[0010] 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 comprising a heavy chain variable (VH) region CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO: 1, 2 and 3, respectively, and a light chain variable (VL) region CDR1, CDR2 and CDR3 having the sequence shown in SEQ ID NO: 4, the sequence GTN and the sequence shown in SEQ ID NO: 5, respectively.

[0011] In another aspect, the invention relates to a bispecific antibody comprising a first binding region of an antibody of the invention, and a second binding region that binds to a different target than the first antigen binding region.

[0012] In another aspect, the invention relates to nucleic acid constructs encoding one or more amino acid sequences of the invention.

[0013] In another aspect, the present invention relates to an expression vector comprising (i) a nucleic acid sequence encoding a heavy chain sequence of a humanized or chimeric antibody of the present invention, (ii) a nucleic acid sequence encoding a light chain sequence of a humanized or chimeric antibody of the present invention, or (iii) both (i) and (ii).

[0014] In another aspect, the present invention relates to a host cell comprising the expression vector of the present invention.

[0015] In another aspect, the invention relates to a composition comprising an antibody or bispecific antibody of the invention.

[0016] In another aspect, the invention relates to a pharmaceutical composition comprising an antibody or bispecific antibody of the invention and a pharmaceutically acceptable carrier.

[0017] In another aspect, the invention relates to an antibody or bispecific antibody, a composition or a pharmaceutical composition of the invention for use as a medicament.

[0018] In another aspect, the present invention relates to the antibody or bispecific antibody, composition or pharmaceutical composition of the present invention for use in treating a disease.

[0019] In another aspect, the invention relates to a method of treating a disease, comprising administering to a subject in need thereof an antibody or bispecific antibody, composition or pharmaceutical composition of the invention.

[0020] In one aspect, the invention relates to a method for diagnosing a disease characterized by involvement or accumulation of cells expressing CD3, comprising administering to a subject a humanized or chimeric antibody, composition or pharmaceutical composition of the invention, optionally wherein the humanized or chimeric antibody is labeled with a detectable agent.

[0021] In another aspect, the present invention relates to a method for producing the antibody or bispecific antibody of the present invention, comprising the steps of: a) culturing the host cell of the present invention, and b) purifying the antibody from the culture medium.

[0022] In another aspect, the invention relates to a diagnostic composition comprising the antibody or bispecific antibody of the invention.

[0023] In another aspect, the present invention relates to a method for detecting the presence of CD3 antigen or cells expressing CD3 in a sample, comprising the steps of: a) contacting the sample with an antibody or bispecific antibody of the present invention under conditions allowing formation of a complex between the antibody or bispecific antibody and CD3, and b) analyzing whether a complex has been formed.

[0024] In another aspect, the present invention relates to a kit for detecting the presence of CD3 antigen or cells expressing CD3 in a sample, comprising i) an antibody or bispecific antibody of the present invention, and ii) instructions for use of the kit.

[0025] In another aspect, the invention relates to anti-idiotypic antibodies that bind to an antibody of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 show( Figure 1 A) Monospecific antibody variants of IgG1-huCD3 and ( Figure 1 B) Binding curves of the bispecific antibody variant bsIgG1 huCD3xHER2 to the human T cell line Jurkat. 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 resulted in half-maximal binding (EC50).

[0028] Figure 2 show( Figure 2 A) Monospecific antibody variants of IgG1-huCD3 and ( Figure 2 B) Binding curve of the bispecific antibody variant bsIgG1 huCD3xHER2 to the cynomolgus monkey T cell line HSC-F. The data shown are the mean fluorescence intensity (MFI) of one representative experiment as described in Example 2.

[0029] Figure 3 : T cell activation by IgG1-huCD3 antibody variants. In PBMC cultures from human ( Figure 3 A) and cynomolgus monkeys ( Figure 3 B) Expression of CD69 on derived T cells was measured by FACS analysis as described in Example 3. These experiments were performed twice, and representative results from one experiment are shown.

[0030] 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.

[0031] Figure 5 : Induction of human ( Figure 5 A) and cynomolgus monkeys ( Figure 5 B) T cell-mediated cytotoxicity was determined as described in Example 5. Representative results from two independent experiments performed in duplicate are shown.

[0032] Figure 6 Monospecific antibody variants showing non-activated IgG1-huCD3 ( Figure 6 A) and the non-activated bispecific antibody variant bsIgG1-huCD3xHER2 ( Figure 6 B) Binding curves to the human T cell line Jurkat. Data shown are mean fluorescence intensity (MFI) of a representative experiment as described in Example 2. The table shows the antibody concentration (µg / mL) that resulted in half-maximal binding (EC50).

[0033] Figure 7 Monospecific antibody variants showing non-activated IgG1-huCD3 ( Figure 7 A) and the non-activated bispecific antibody variant bsIgG1-huCD3xHER2 ( Figure 7B) Binding curves to the cynomolgus monkey T cell line HSC-F. Data shown are mean fluorescence intensity (MFI) of a representative experiment as described in Example 2. The table shows the antibody concentration (µg / mL) that resulted in half-maximal binding (EC50).

[0034] Figure 8 : Through non-activated monospecific IgG1-huCD3 ( Figure 8 A and B) or non-activated bispecific bsIgG1-huCD3xHER2 antibody variants ( Figure 8 C and D) T cell activation. In PBMC cultures from human ( Figure 8 A and C) and cynomolgus monkeys ( Figure 8 B and D) Expression of CD69 on T cells derived from p361 cells was measured by FACS analysis as described in Example 3. These experiments were performed twice and representative results from one experiment are shown.

[0035] Fig. 9 : Through non-activated monospecific IgG1-huCD3 ( Fig. 9 A and B) or non-activated bispecific bsIgG1-huCD3xHER2 antibody variants ( Fig. 9 C and D) induced T-cell proliferation. Fig. 9 A and C) or cynomolgus monkeys ( Fig. 9 B and D) T-cell proliferation was measured in PBMCs incubated with various antibody variants for 3 days before proliferation was measured by cell proliferation ELISA as described in Example 4. Representative results from two independent experiments are shown.

[0036] Fig.10 : Induction of human ( Fig.10 A) and cynomolgus monkeys ( Fig.10 B) T cell-mediated cytotoxicity was determined as described in Example 5. Representative results from two independent experiments performed in duplicate are shown.

[0037] Fig.11 : Activation of rhesus macaque T cells by IgG1-huCD3 antibody variants. The expression of CD69 on T cells from rhesus macaque origin in PBMC cultures was measured by FACS analysis as described in Example 6.

[0038] Fig.12: T cell activation by variants of non-activated huCLB-T3 / 4 antibodies. IgG1-huCLB-T3 / 4 variants were titrated on PBMCs. Expression of CD69 on T cells in PBMC cultures was measured by FACS analysis as described in Example 7. Representative examples of 3 experiments are shown.

[0039] Fig.13 : T-cell proliferation by variants of non-activating huCLB-T3 / 4 antibodies. PBMCs were incubated with antibodies for 3 days, after which proliferation was measured by cell proliferation ELISA as described in Example 8. Representative results from two independent experiments are shown.

[0040] Fig.14 : In vitro T cell-mediated cytotoxicity induced by antibody variants of non-activated CD3 antibodies. Fig.14 AG]) induced T cell-mediated cytotoxicity, determined as described in Example 9. The average of two experiments performed in duplicate is shown.

[0041] Fig.15 : In vitro T cell-mediated cytotoxicity induced by non-activated huCLB-T3 / 4 variants. Fig.15 AC] induced T cell-mediated cytotoxicity, determined as described in Example 9. Mean values ​​from one experiment performed in duplicate are shown.

[0042] Fig.16 : Evaluation of the binding of C1q to non-activated huCLB-T3 / 4 antibody variants. C1q and monospecific IgG1huCLB-T3 / 4 ( Fig.16 Binding of IgG1-huCLB-T3 / 4xHER2 (Figures BD) and its non-activated antibody variants was evaluated by ELISA as described in Example 10. The results in the figures are representative of n=2 experiments.

[0043] Fig.17 : Pharmacokinetic (PK) analysis of non-activated huCLB-T3 / 4 antibody variants compared to wild-type IgG1-huCLB-T3 / 4 antibody as described in Example 11. Plasma concentrations of human IgG1 were plotted against time ( Fig.17 A). Plasma clearance was calculated as described in Example 11 ( Fig.17 B). The horizontal dashed line indicates the mean clearance of human IgG1 antibody in SCID mice (10 mL / day / kg).

[0044] Fig.18 : The frequency of positive T cell responses in healthy HLA-type donors. An SI index of ≥1.9 was considered a positive response in both the proliferation and IL-2 secretion assays. Humanized A33 was used as a clinical benchmark control antibody, which clinically showed high levels of immunogenicity and routinely induced 20-30% T cell responses in the EpiScreen assay. KLH reactions were included to check PBMC quality (after thawing).

[0045] Fig.19 : Sequence alignment of the heavy chain (VH) and light chain (VL) variable regions of the humanized CD3 antibodies of the present invention.

[0046] Fig. 20 : Expression levels of IgG1-huCD3-H1L1 variants in Expi293F supernatants as measured by Octet RED using an anti-human IgG immobilized sensor.

[0047] Fig.21 : Binding of IgG1-huCD3-H1L1-LFLEDA mutants to Jurkat cells.

[0048] Fig. 22 : Binding of CD3 bispecific antibody variants to human T cells. Circles represent binding of the huCD3-H1L1-LFLEDA variant, triangles represent binding of the huCD3-H1L1-LFLEDA-LK55N variant, and squares represent binding of the CD3-hmAb286-LFLEDA variant.

[0049] Fig.23 : Effect of huCD3 x HER2 antibody affinity variants on A431 cells (low HER2 expressing cells) treated with CD3 and HER2 affinity variants ( Fig.23 A) and AU565 cells (high HER2 expression cells) ( Fig.23 B) Cytotoxicity.

[0050] Fig.24 : huCD3-H1L1xHER2-LFLEDA was used in NOD-SCID mice at 7, 14, 21 and 28 days after tumor inoculation ( Fig.24 A) and on day 29 ( Fig.24 B) Resulting reduction in tumor size.

[0051] Fig.25 : huCD3-H1L1xCD20-LFLEDA was used in NOD-SCID mice at 7, 14 and 21 days after tumor inoculation ( Fig.25 A) and on day 21 ( Fig.25 B) Resulting reduction in tumor size. DETAILED DESCRIPTION OF THE INVENTION

[0053] 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 comprising a heavy chain variable (VH) region CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO: 1, 2 and 3, respectively, and a light chain variable (VL) region CDR1, CDR2 and CDR3 having the sequence shown in SEQ ID NO: 4, the sequence GTN and the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 60, respectively.

[0054] In one embodiment, the present invention relates to a humanized or chimeric antibody that binds to human CD3, wherein the antibody comprises a binding region comprising a heavy chain variable (VH) region CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO: 1, 2 and 3, respectively, and a light chain variable (VL) region CDR1, CDR2 and CDR3 having the sequence shown in SEQ ID NO: 4, the sequence GTN and the sequence shown in SEQ ID NO: 5, respectively.

[0055] The term "antibody" as used herein means an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, which has the ability to specifically bind to an antigen under typical 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 1 hour, at least about 2 hours, at least about 4 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 time (e.g., sufficient to induce, promote, enhance and / or regulate the physiological response associated with antibodies that bind to antigens and / or sufficient for antibodies to recruit effector activity). The binding region (or binding domain, which can also be used herein, has the same meaning) that interacts with an antigen comprises the variable regions of both the heavy and light chains of an immunoglobulin molecule. The constant region of an antibody (Ab) can mediate the binding of an 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 (e.g., C1q, the first complement of the classical pathway of complement activation). As noted above, the term antibody as used herein, unless otherwise indicated or clearly contradicted by the context, includes fragments of antibodies that retain the ability to specifically 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 included within the term "antibody" include (i) Fab' or Fab fragments, a V-binding fragment of a L 、V H , C L and CH 1 domain, or a monovalent antibody as described in WO2007059782 (Genmab A / S); (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) a monovalent fragment consisting essentially of V H and C H 1 domain; and (iv) a V domain consisting essentially of a single arm of the antibody L and V H In addition, although the two domains V L and V H are encoded by separate genes, but they can be joined using recombinant methods by a synthetic linker that enables them to be made into a single protein chain, where V L The VH region is paired with the VH region to form a monovalent molecule (called a single-chain antibody or single-chain Fv (scFv), see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are included in the term antibody, unless otherwise noted or the context clearly indicates. Although such fragments are generally included in the meaning of antibodies, they are collectively and independently unique features of the present invention, showing different biological properties and functions. These and other useful antibody fragments in the context of the present invention are further discussed herein. It should also be understood that the term antibody, unless otherwise indicated, also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen provided by any known technology, such as enzymatic cleavage, peptide synthesis and recombinant technology. The antibodies produced may have any isotype.

[0056] As used herein, the terms "immunoglobulin heavy chain", "heavy chain of an immunoglobulin" or "heavy chain" refer to one of the chains of an immunoglobulin. A heavy chain is typically composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that defines the isotype of the immunoglobulin. The heavy chain constant region is typically composed of three domains, CH1, CH2 and CH3. The heavy chain constant region may further comprise a hinge region. As used herein, the term "immunoglobulin" refers to 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 possibly 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 by disulfide bonds in the so-called "hinge region". Like the heavy chain, each light chain is typically composed of several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region typically comprises one domain, CL. In addition, the VH and VL regions can be further subdivided into regions of hypervariability (or hypervariable regions, which can be highly variable in sequence and / or form of structurally defined loops), also called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see

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

[16] -

[17] .

[0057] 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] encoded by a heavy chain constant region gene. Thus, in one embodiment, the antibody comprises a heavy chain of an immunoglobulin of the IgG1 type or any allotype thereof. In addition, each heavy chain isotype may be combined with a kappa (κ) or lambda (λ) light chain.

[0058] The term "chimeric antibody" as used herein refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the constant region is derived from a different species, such as a human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a term widely used for different types of antibody modifications, and it is a method well known to technicians. In particular, chimeric antibodies can be produced using standard DNA techniques described in

[18] . Thus, chimeric antibodies can be recombinant antibodies that have been genetically or enzymatically engineered. The production of chimeric antibodies is within the knowledge of technicians, and thus the production of chimeric antibodies of the present invention can be achieved by other methods than those described herein. Chimeric monoclonal antibodies are developed for therapeutic applications to reduce antibody immunogenicity. They can generally contain a non-human (e.g., mouse) variable region that is specific for the target antigen, and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" used in the context of chimeric antibodies refers to a region containing the CDRs and framework regions of both the heavy and light chains of an immunoglobulin.

[0059] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology with a human variable domain. This can be achieved by transplanting the six non-human antibody complementary determining regions (CDRs) that together form the antigen binding site onto a homologous human receptor framework region (FR) (see

[19] -

[20] ). In order to fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the framework residues of the parent antibody (i.e., the non-human antibody) into the human framework region (back mutation). Structural homology modeling can help identify amino acid residues in the framework region that are important for the binding properties of the antibody. Therefore, a humanized antibody may comprise a non-human CDR sequence, a substantially human framework region optionally comprising one or more amino acid back mutations mutated to a non-human amino acid sequence, and a completely human constant region. Optionally, additional amino acid modifications that are not necessarily back mutations may be applied to obtain a humanized antibody with preferred characteristics (e.g., affinity and biochemical properties).

[0060] A humanized or chimeric antibody according to any aspect or embodiment of the invention may be referred to as a "humanized or chimeric CD3 antibody," "a humanized or chimeric antibody of the invention," "a CD3 antibody," or "a CD3 antibody of the invention," all of which have the same meaning and purpose unless otherwise contradicted by the context.

[0061] The amino acid sequence of an antibody of non-human origin is different from that of an antibody of human origin, and therefore the non-human antibody is likely to produce immunogenicity when administered to a human patient. However, despite the non-human origin of the antibody, its CDR segment is responsible for the ability of the antibody to bind to its target antigen, and the goal of humanization is to maintain the specificity and binding affinity of the antibody. Therefore, humanization of non-human therapeutic antibodies is performed to minimize their immunogenicity in humans, while at the same time such humanized antibodies maintain the specificity and binding affinity of the antibody of non-human origin.

[0062] As used herein, the term "binding region" refers to a region of an antibody that is capable of binding to any molecule, such as a polypeptide, for example, present on a cell, bacterium, or virion.

[0063] As used herein, the term "binding" refers to the binding of an antibody to a predetermined antigen or target, typically with a relative affinity corresponding to about 10 -6 M or smaller, e.g. 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or smaller, about 10 -10 M or less or about 10 -11 M or even smaller K D The affinity of the antibody to the antigen is at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, and at least 100,000 times lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) that is not the predetermined antigen or a closely related antigen. D The affinity of the antibody is lower depending on the K D , so that when the antibody K D When the affinity for an antigen is very low (i.e., the antibody is highly specific), the affinity for the antigen may be at least 10,000 times lower than the affinity for a nonspecific antigen. D " (M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction.

[0064] The term "human CD3" used herein refers to human cluster of differentiation 3 protein, which is part of the T cell co-receptor protein complex and is composed of four different chains. CD3 also exists in other species, and therefore the term "CD3" can be used herein and is not limited to human CD3, unless the context is contradictory. In mammals, the complex contains a CD3γ(gamma) chain (human CD3γ chain Swissprot P09693 or cynomolgus monkey CD3γ Swissprot Q95LI7), a CD3δ(delta) chain (human CD3δ Swissprot P04234 or cynomolgus monkey CD3δ Swissprot Q95LI8), two CD3ε(epsilon) chains (human CD3ε Swissprot P07766; or cynomolgus monkey CD3ε Swissprot Q95LI5), rhesus monkey CD3ε (Swissprot G7NCB9), and a CD3ζ-chain (zeta) chain (human CD3ζ Swissprot P20963, cynomolgus monkey CD3ζ Swissprot Q09TK0). These chains associate with a molecule called T-cell receptor (TCR) and generate activation signals in T lymphocytes. TCR and CD3 molecules together constitute the TCR complex.

[0065] It is within the knowledge of the technician that the amino acid sequence mentioned by the Swissprot number includes a signal peptide, which is removed after the protein is translated. Therefore, proteins present on the cell surface, such as CD3, do not include a signal peptide. In particular, the amino acid sequences listed in Table 1 do not contain such a signal peptide. Such proteins listed in Table 1 can be referred to as "mature proteins". Therefore, 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: 22 shows the amino acid sequence of mature rhesus monkey CD3ε. Therefore, the term "mature" used herein refers to a protein that does not contain any signal or leader sequence.

[0066] It is well known that signal peptide sequence homology, length and cleavage site position 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 from http: / / www.cbs.dtu.dk / services / SignalP / ).

[0067] In a specific embodiment, the humanized or chimeric antibody of the invention binds to the epsilon chain of CD3, e.g., the epsilon chain of human CD3 (SEQ ID NO: 13). In yet another specific embodiment, the humanized or chimeric antibody binds to an epitope within amino acids 1-27 of the N-terminal portion of human CD3 epsilon (epsilon) (SEQ ID NO: 13). In such a specific embodiment, the antibody may even further cross-react with other non-human primate species such as cynomolgus monkeys (cynomolgus monkey CD3 epsilon SEQ ID NO: 21) and / or rhesus monkeys (rhesus monkey CD3 epsilon SEQ ID NO: 22).

[0068] The term "cross-reactivity" as used herein refers to the ability of an antibody, such as a humanized or chimeric antibody of the present invention, to bind to its target on a different species. Specifically, the humanized CD3 antibodies exemplified in the embodiments described herein have the ability to bind to human (Example 2), cynomolgus monkey (Example 2) and rhesus monkey CD3.

[0069] Compared to the original antibody, the antibodies of the present invention comprising the CDR sequences defined herein and further comprising framework regions may differ in sequence outside the CDR sequences, but still retain full binding ability. Therefore, the present invention also relates to antibodies comprising amino acid sequences of variable regions having certain sequence identity with any sequence described herein.

[0070] The term "sequence identity" as used in the context of the present invention refers to the percent identity between two sequences as a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller

[21] . In addition, the percent 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] (e.g., as used in Vector NTI Advance® software version 11.5; Invitrogen Inc.).

[0071] Thus, in one embodiment, the VH region has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to at least one amino acid sequence shown in a VH sequence selected from:

[0072] a) the VH sequence shown in SEQ ID NO: 6;

[0073] b) the VH sequence shown in SEQ ID NO: 8;

[0074] c) the VH sequence shown in SEQ ID NO:7; and

[0075] d) The VH sequence shown in SEQ ID NO:9.

[0076] In a specific embodiment, the VH region has at least 96% amino acid sequence identity to at least one amino acid sequence shown in the VH sequence selected from:

[0077] a) the VH sequence shown in SEQ ID NO: 6;

[0078] b) the VH sequence shown in SEQ ID NO: 8;

[0079] c) the VH sequence shown in SEQ ID NO:7; and

[0080] d) The VH sequence shown in SEQ ID NO:9.

[0081] In one embodiment, the VL region has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to at least one amino acid sequence shown in a VL sequence selected from:

[0082] a) the VL sequence shown in SEQ ID NO: 10;

[0083] b) the VL sequence shown in SEQ ID NO: 11; and

[0084] c) the VL sequence shown in SEQ ID NO:12.

[0085] In a specific embodiment, the VL region has at least 95% amino acid sequence identity to at least one amino acid sequence shown in a VL sequence selected from:

[0086] a) the VL sequence shown in SEQ ID NO: 10;

[0087] b) the VL sequence shown in SEQ ID NO: 11; and

[0088] c) the VL sequence shown in SEQ ID NO:12.

[0089] In one embodiment, the VH region is selected from:

[0090] a) the VH sequence shown in SEQ ID NO: 6;

[0091] b) the VH sequence shown in SEQ ID NO: 8;

[0092] c) the VH sequence shown in SEQ ID NO:7; and

[0093] d) The VH sequence shown in SEQ ID NO:9.

[0094] In one embodiment, the VL region is selected from:

[0095] a) the VL sequence shown in SEQ ID NO: 10;

[0096] b) the VL sequence shown in SEQ ID NO: 11; and

[0097] c) the VL sequence shown in SEQ ID NO:12.

[0098] In one embodiment, only one of the VH or VL sequences is 100% identical to one of the sequences disclosed herein, while 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.

[0099] In a specific embodiment, the VH sequence has at least 97% amino acid sequence identity to at least one amino acid sequence shown in a VH sequence selected from:

[0100] a) the VH sequence shown in SEQ ID NO: 6;

[0101] b) the VH sequence shown in SEQ ID NO:7;

[0102] c) the VH sequence shown in SEQ ID NO: 8; and

[0103] d) the VH sequence shown in SEQ ID NO:9;

[0104] and a VL sequence having at least 95% amino acid sequence identity with at least one amino acid sequence shown in a VL sequence selected from the group consisting of:

[0105] i. the VL sequence shown in SEQ ID NO: 10;

[0106] ii. the VL sequence shown in SEQ ID NO: 11; and

[0107] iii. the VL sequence shown in SEQ ID NO:12.

[0108] In one embodiment, the VH and VL sequences are selected from:

[0109] a) VH and VL sequences that are at least 90% identical to the sequences set forth in SEQ ID NOs: 6 and 10, respectively; 7 and 10, respectively; 8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively; 7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively; 6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively; and 9 and 12, respectively;

[0110] b) VH and VL sequences that are at least 95% identical to the sequences set forth in SEQ ID NOs: 6 and 10, respectively; 7 and 10, respectively; 8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively; 7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively; 6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively; and 9 and 12, respectively;

[0111] c) VH and VL sequences that are at least 97% identical to the sequences set forth in SEQ ID NOs: 6 and 10, respectively; 7 and 10, respectively; 8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively; 7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively; 6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively; and 9 and 12, respectively;

[0112] d) VH and VL sequences that are at least 99% identical to the sequences set forth in SEQ ID NOs: 6 and 10, respectively; 7 and 10, respectively; 8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively; 7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively; 6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively; and 9 and 12, respectively;

[0113] e) VH and VL sequences that are at least 100% identical to the sequences set forth in SEQ ID NOs: 6 and 10, respectively; 7 and 10, respectively; 8 and 10, respectively; 9 and 10, respectively; 6 and 11, respectively; 7 and 11, respectively; 8 and 11, respectively; 9 and 11, respectively; 6 and 12, respectively; 7 and 12, respectively; 8 and 12, respectively; and 9 and 12, respectively;

[0114] f) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0115] g) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0116] h) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0117] i) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0118] j) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0119] k) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0120] 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;

[0121] m) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0122] 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;

[0123] o) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0124] 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;

[0125] q) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0126] r) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0127] 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;

[0128] t) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0129] u) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0130] v) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0131] x) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0132] y) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO: 6 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO: 10, 11 or 12;

[0133] z) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:6 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0134] aa) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0135] ab) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0136] ac) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0137] ad) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0138] ae) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0139] af) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0140] ag) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0141] ah) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0142] ai) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0143] aj) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0144] ak) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0145] a1) 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;

[0146] am) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0147] 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;

[0148] ao) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0149] ap) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0150] aq) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0151] ar) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0152] as) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0153] at) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:7 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0154] ba) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 8 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 10, 11 or 12;

[0155] bb) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0156] bc) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0157] bd) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0158] 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;

[0159] bf) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0160] bg) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0161] bh) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0162] bi) a VH sequence having at least 97% 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;

[0163] bj) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0164] bk) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0165] bl) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0166] bm) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0167] bn) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0168] bo) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0169] bp) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0170] bq) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO: 8 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 10, 11 or 12;

[0171] br) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO: 8 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO: 10, 11 or 12;

[0172] bs) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0173] bt) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:8 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0174] ca) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0175] cb) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0176] cc) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0177] cd) a VH sequence that is at least 90% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0178] ce) a VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:9 and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0179] cf) a VH sequence having at least 95% identity to the sequence shown in SEQ ID NO:9 and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0180] cg) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0181] ch) a VH sequence that is at least 95% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0182] ci) a VH sequence having at least 97% identity to the sequence shown in SEQ ID NO:9 and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO:10, 11 or 12;

[0183] cj) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0184] ck) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0185] cl) a VH sequence that is at least 97% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0186] cm) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0187] cn) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0188] co) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0189] cp) a VH sequence that is at least 99% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 100% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0190] cq) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 90% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0191] cr) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 95% identical to the sequence shown in SEQ ID NO:10, 11 or 12;

[0192] cs) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 97% identical to the sequence shown in SEQ ID NO:10, 11 or 12; and

[0193] ct) a VH sequence that is at least 100% identical to the sequence shown in SEQ ID NO:9 and a VL sequence that is at least 99% identical to the sequence shown in SEQ ID NO:10, 11 or 12.

[0194] In one embodiment, the binding region comprises a VH and a VL selected from:

[0195] a) the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:10;

[0196] b) the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:10;

[0197] c) the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:10;

[0198] d) the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:11;

[0199] e) the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:12;

[0200] f) the VH sequence shown in SEQ ID NO:7, and the VL sequence shown in SEQ ID NO:10;

[0201] g) the VH sequence shown in SEQ ID NO:7, and the VL sequence shown in SEQ ID NO:11;

[0202] h) the VH sequence shown in SEQ ID NO:7, and the VL sequence shown in SEQ ID NO:12;

[0203] i) the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:11;

[0204] j) the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:12;

[0205] k) the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:11; and

[0206] l) the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:12.

[0207] In specific embodiments, the binding region comprises a VH sequence and a VL sequence selected from the group consisting of:

[0208] a) the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:10;

[0209] b) the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:10; and

[0210] c) the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:10.

[0211] The humanized antibodies of the present invention can be produced by comparing the heavy chain and light chain variable region amino acid sequences with a database of human germline variable region sequences to identify heavy chain and light chain human sequences with a suitable degree of homology for use as human variable framework regions. A series of humanized heavy chain and light chain variable regions can be designed by transplanting, for example, mouse CDRs onto the framework regions (identified as described above) and, if necessary, by backmutation (mutation of one or more human amino acid residues at specified positions in the framework regions back to non-human amino acids) to specific mouse residue sequences identified as being critical for restoring antibody binding efficacy. Then, according to the application of computer technology: iTope TM and TCED TM The variant sequence with the lowest occurrence of possible T-cell epitopes as determined by (

[24] ,

[25] and

[26] ) was selected.

[0212] In addition, the humanized antibodies of the present invention may also be "deimmunized". Deimmunization may be desirable because the presence of human T cell epitopes within protein sequences such as the humanized antibodies of the present invention may increase the immunogenicity risk profile when they have the potential to activate helper T cells. Such activation of helper T cells may be avoided by deimmunization. Deimmunization may be performed by introducing mutations into the amino acid sequence of the humanized antibody to remove T cell epitopes without significantly reducing the binding affinity of the antibody.

[0213] Therefore, 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 fully variable heavy chain sequence and / or a fully variable light chain sequence to 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 back mutation if necessary, and (iv) expressing the sequence in a suitable expression system.

[0214] Therefore, the full-length antibodies of the present invention can be produced by a method comprising the following steps: (i) comparing non-human variable heavy chain sequences and variable light chain sequences with a database of human germline sequences, (ii) selecting the human germline sequence with the highest homology to the non-human sequence, (iii) transplanting the non-human CDR to the selected human germline to obtain a humanized sequence, (iv) if necessary, optimizing the humanized sequence by back mutation, (v) identifying constant heavy chain and light chain sequences, and (vi) expressing the complete heavy chain sequence and the complete light chain sequence in a suitable expression system. The full-length antibodies of the present invention can therefore be produced as described in Example 1. It is within the knowledge of the technician to produce full-length antibodies starting from CDR sequences or complete variable region sequences. Therefore, the technician will know how to produce full-length antibodies of the present invention.

[0215] 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.

[0216] The term "complete light chain sequence" as used herein refers to a sequence consisting of a variable light chain and a constant light chain sequence.

[0217] Back mutations can be introduced by standard DNA mutagenesis. Such standard techniques for DNA mutagenesis are described in

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

[0218] Thus, in one embodiment, the antibody is a humanized antibody.

[0219] Chimeric antibodies can be produced by replacing all constant region sequences of non-human (e.g., mouse) antibodies with constant region sequences of human origin. Therefore, the complete non-human variable region sequence is retained in the chimeric antibody. Therefore, the chimeric antibody of the present invention can be produced by a method comprising the following steps: expressing non-human variable heavy chain (SEQ ID NO: 27), non-human variable light chain sequence (SEQ ID NO: 28), human constant heavy chain and human constant light chain sequence in a suitable expression system, and thereby producing full-length chimeric antibodies. Alternative methods can be used. Such methods for producing chimeric antibodies are within the knowledge of the technician, and therefore the technician will know how to produce chimeric antibodies of the present invention.

[0220] Thus, in one embodiment, the antibody is a chimeric antibody.

[0221] In one embodiment, the antibody is a full length antibody.The term "full length antibody" as used herein refers to an antibody (eg, a parent or variant antibody) that comprises all heavy and light chain constant and variable domains corresponding to those naturally occurring in a wild-type antibody of that isotype.

[0222] In one embodiment, the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

[0223] The term "Fc region" used herein refers to a region including 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 may further include a CH1 region at the N-terminus of the hinge region.

[0224] 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 Eu numbering as described in Kabat.

[0225] Unless otherwise indicated or contradicted by 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 according to Kabat", "Eu numbering according to Kabat" or "according to the Eu numbering system".

[0226] The term "CH1 region" or "CH1 domain" as used herein refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering system. However, the CH1 region may also be any other subtype described herein.

[0227] The term "CH2 region" or "CH2 domain" as used herein refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering system. However, the CH2 region may also be any other subtype described herein.

[0228] The term "CH3 region" or "CH3 domain" as used herein refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering system. However, the CH3 region may also be any other subtype described herein.

[0229] 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). Therefore, in a specific embodiment, the isotype of the immunoglobulin heavy chain is IgG1 or any allotype thereof, such as IgG1m(f) (SEQ ID NO: 15).

[0230] When targeting the antigen CD3 as part of the T-cell receptor (TCR), a T cell-specific mechanism of cell killing is desirable. Other effector functions, such as complement activation, may be undesirable, and therefore reducing effector function is desirable. Clq binding is the first step in the complement cascade, and therefore serves as an indicator of the complement dependent cytotoxicity (CDC) ability of an antibody. If binding of Clq to an antibody can be avoided, activation of the complement cascade can also be avoided.

[0231] Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that binding of CIq 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% compared to the wild-type antibody, wherein CIq binding is determined by ELISA.

[0232] As used herein, the term "modified" refers to an amino acid sequence of an Fc region that is not identical to the amino acid sequence of a wild-type Fc region. That is, amino acid residues at specified positions in a wild-type Fc region have been replaced, deleted or inserted to alter, for example, the binding site for C1q, the binding site for other effector molecules, or the binding to an Fc receptor (FcR). Such modifications of an amino acid sequence may be prepared by replacing one or more amino acids with conservative amino acids, or may be prepared by replacing 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. Replacements may also be prepared by replacement with non-conservative amino acids.

[0233] In the context of the present invention, amino acids can be described as conservative or non-conservative amino acids, and can be classified accordingly. Amino acid residues can also be divided into types defined by optional physical and functional properties. Therefore, the type of amino acid can be reflected in one or two of the following tables:

[0234] Conservative amino acid residues

[0235] Acidic residue D and E Basic residues K, R and H Hydrophilic uncharged residues S, T, N and Q Aliphatic uncharged residue G, A, V, L, and I Nonpolar uncharged residues C, M and P Aromatic residues F, Y and W

[0236] Alternative physical and functional classifications of amino acid residues

[0237] Alcohol-containing residues S and T Aliphatic residues I, L, V, and M Cycloalkenyl-related residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K and R Small residues A, C, D, G, N, P, S, T and V Minimal residue A, G, and S Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible residues Q, T, K, S, G, P, D, E, and R

[0238] In the context of the present invention, substitutions in an antibody, such as a humanized or chimeric antibody, are represented as follows:

[0239] Original amino acid – Position – Replaced amino acid;

[0240] Any amino acid residue is indicated with reference to the generally accepted amino acid nomenclature using either the three letter code or the one letter code, including the codes Xaa and X. Thus, the notation "L234F" or "Leu234Phe" means that the antibody comprises a substitution of leucine at amino acid position 234 with phenylalanine.

[0241] The substitution of an amino acid at a given position for any other amino acid is indicated as follows:

[0242] Original amino acid – position; or for example "L234".

[0243] For modifications in which the original amino acid and / or the replaced amino acid may include more than one but not all amino acids, the more than one amino acid may be separated by "," or " / ". For example, replacing leucine with phenylalanine, arginine, lysine or tryptophan at position 234:

[0244] “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”.

[0245] In the context of the present invention such nomenclature may be used interchangeably but have the same meaning and purpose.

[0246] In addition, the term "replacement" includes being replaced into any one of other 19 kinds of natural amino acids, or other amino acids, such as non-natural amino acids. For example, the amino acid L on the replacement position 234 includes the following various replacements: 234A, 234C, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P and 234Y. That is, in this way, it is equivalent to naming 234X, wherein X indicates any amino acid except the original amino acid. These replacements may also be referred to as L234A, L234C etc., or L234A, C etc., or L234A / C / etc. The naming is similarly applicable to each and all positions mentioned herein, and any one of such replacements is particularly included herein.

[0247] Antibodies of the present invention may also include deletions of amino acid residues. Such deletions may be represented by "del", and include, for example, L234del. Thus, in such embodiments, the leucine at position 234 is deleted from the amino acid sequence.

[0248] The terms "amino acid" and "amino acid residue" are used interchangeably herein.

[0249] As used herein, the term "CIq binding" refers to the binding of CIq to an antibody when the antibody is bound to its antigen. As used herein, the term "binding to its antigen" refers to the binding of an antibody to its antigen both in vivo and in vitro.

[0250] As used herein, the term "reduce" when referring to CIq binding refers to the ability of an antibody of the invention to reduce, minimize or even completely inhibit the binding of CIq to the antibody when compared to the binding of CIq to the wild-type antibody.

[0251] As used herein, the term "wild-type antibody" refers to an antibody that is identical to the antibody to be tested except that it is not inactive when used in a comparative assay of the antibodies of the invention. In this context, the term "inactive" refers to a modified Fc region that has reduced or no C1q binding, as determined in Example 10, i.e., reduced or no Fc-mediated T-cell proliferation as determined in Example 4 (i.e., T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay when C1q binding is determined by ELISA; and / or reduced or no Fc-mediated CD69 expression as determined in Example 3 (i.e., Fc-mediated CD69 expression is measured in a PBMC-based functional assay). Thus, a wild-type antibody comprises amino acids that naturally occur in an immunoglobulin heavy chain, i.e., an antibody that does not comprise any amino acid modifications that may alter or reduce the ability of the antibody to interact with, e.g., C1q, an Fc receptor, etc. Thus, such a wild-type antibody will remain an activating antibody capable of binding, e.g., C1q. Wild-type antibodies and antibodies of the present invention may contain amino acid modifications other than those that affect the ability of the antibody to induce effector function, to prepare the antibody into a bispecific antibody or the like.

[0252] The term "ELISA" as used herein refers to enzyme-linked immunosorbent assay, which is a test for identifying substances using antibodies and color changes. A first specific antibody is attached to the surface of a plate. Thereby a protein from a sample is added, wherein the binding to the first specific antibody is tested. A second antibody is added that binds to the antibody from the sample. The second antibody is attached to an enzyme, and in a final step a substance comprising a substrate for the enzyme is added. The subsequent reaction produces a detectable signal, the most common of which is a color change of the substrate. The concept of the ELISA method is well known in the art, and various ways of performing an ELISA are considered to be part of the method for evaluating the antibodies of the present invention. Therefore, this explanation should not be construed as limiting, as various forms of ELISA can be performed, such as described in Example 4.

[0253] In particular, the ability of the antibody of the present invention to bind to C1q can be determined by ELISA, comprising the steps of: (i) coating the antibody onto a 96-well plate, (ii) adding 3% serum, (iii) adding anti-human C1q antibody, (iv) developing the plate, and (v) measuring OD 405 nm. Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that 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% compared to a wild-type antibody, wherein C1q binding is determined by 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 OD405 Thus, in a specific embodiment, the binding of C1q is assessed as described in Example 10.

[0254] As used herein, the term "Fc receptor" or "FcR" refers to a protein present on the surface of certain cells. An FcR binds to the Fc region of an antibody. There are several different types of FcRs, which are classified according to the type of antibody they recognize. For example, the Fcγ (gamma) receptor binds to an IgG type antibody.

[0255] The term "Fcγ receptor", "Fc gamma receptor" or "FcγR" as used herein 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. The family includes several members, FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), FcγRIIIb (CD16b), which differ in antibody affinity due to different molecular structures.

[0256] 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 binding of various effector molecules to the Fc region. In the context of the present invention, "Fc binding", "Fc receptor binding", "FcR binding" and "antibody Fc region binding to FcR" refer to the binding of the Fc region to Fc receptors (FcR) or effector molecules. The terms "FcγR binding" and "FcγRI binding" refer to binding to or binding to Fc gamma receptors and Fc gamma receptor I with the Fc region, respectively. When the CD3 antibody binds to T cells, the wild-type Fc region of the CD3 antibody binds to FcR present on other cells (e.g., monocytes), which results in nonspecific Fc-mediated T cell activation. Such nonspecific Fc-mediated T cell activation may be undesirable. T cells may also be activated by targeted or target-specific T cell activation. For many indications such as the treatment of cancer, such targeted T cell activation may be highly desirable. The term "targeted T cell activation" used herein refers to guiding T cells to specific cells such as tumor cells by using bispecific antibodies, wherein the bispecific antibodies include a first binding region that binds to a specific target such as a tumor target on a tumor cell, and a second binding region that binds to a T cell-specific target such as CD3. Therefore, T cell targeting to specific cells such as tumor cells can be promoted by using bispecific antibodies, wherein one of the binding regions binds to CD3 present on T cells and another binding region binds to a target-specific antigen, such as a target-specific antigen on a tumor cell. Nevertheless, nonspecific Fc-mediated T cell activation may still exist, and therefore such unwanted nonspecific Fc-mediated T cell activation by Fc-mediated cross-linking should be avoided, and may be required by preparing an Fc region that is inactive for such activity. Therefore, the interaction between the inactive Fc region and the Fc receptor present is prevented. The humanized antibodies of the present invention have been shown to be inactive when tested in several different assays (i.e., referring to Examples 3-5). Another tested CD3 antibody huCLB-T3 / 4 comprising amino acid modifications of the Fc region also proved to be inactive when tested in different assays (i.e., see Examples 7-10). The humanized CD3 antibodies of the present invention comprising amino acid substitutions L234F, L235E and D265A as described in the Examples showed low levels of CD69 expression on T cells (Example 3), abrogation of Fc-mediated T-cell proliferation (Example 4) and no non-specific target killing when in the form of a bispecific antibody (Example 5). Therefore, the humanized antibodies of the present invention showed excellent results in several assays when compared to wild-type antibodies.

[0257] The antibodies of the present invention may comprise modifications of the Fc region. When an antibody comprises such modifications, it may become an inactive or non-activated antibody. The terms "inactive", "inactive" or "non-activated" as used herein refer to an Fc region that is at least unable to bind to any Fcγ receptor, induce Fc-mediated FcR cross-linking, or induce FcR-mediated target antigen cross-linking through two Fc regions of a single antibody, or is unable to bind to C1q. The inactivity of the Fc region of a humanized or chimeric CD3 antibody is suitably tested using a monospecific form of the antibody, but the inactive Fc region thus identified can be used with a bispecific or other humanized or chimeric multispecific CD3 antibody.

[0258] Several variants can be constructed to prepare the Fc region of antibodies inactive for interaction with Fc gamma receptors and C1q for use in therapeutic antibody development. Examples of such variants are described herein.

[0259] Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that the antibody mediates reduced Fc-mediated T-cell proliferation 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 peripheral blood mononuclear cell (PBMC) based functional assay.

[0260] As used herein, the term "reduce" refers to a reduction in activity or expression when compared to a control protein, such as an antibody. Specifically, the term "reduce" when it relates to T-cell proliferation refers to the ability of the antibody of the present invention to reduce, minimize or even completely inhibit T-cell proliferation when compared to T-cell proliferation bound by a wild-type antibody. The ability of the antibody to reduce T-cell proliferation can be evaluated by a functional assay based on PBMC, as described in Example 4 and Example 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. Because the antibodies of the present invention have cross-reactivity, the PBMC-based assay described herein can be performed with PBMCs of any species to show the reduction of T-cell proliferation, as long as the PBMCs of the species used are within the cross-reactivity spectrum of the antibody, such as humans, cynomolgus monkeys or rhesus monkeys.

[0261] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay for evaluating the functional characteristics of an antibody of the 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. Thus, in one embodiment, T-cell proliferation is measured by a method comprising the following steps: incubating PBMCs with an antibody in the range of 1-1000 ng / mL for three days at 37°C in a 5% (vol / vol) CO2 humidified incubator; adding a compound, such as BrdU, which is incorporated into the DNA of the proliferating cells; incubating for 5 hours, pelleting the cells, drying the cells, optionally storing the cells at 4°C, coating the cells onto an ELISA plate, incubating at room temperature with anti-BrdU-peroxidase for 90 minutes, developing with 1 mg / mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) for about 30 minutes, adding 100 μL 2% oxalic acid to terminate the reaction, and measuring the absorbance at 405 nm in a suitable microplate reader.

[0262] As used herein, the term "proliferation" refers to cell growth in the context of cell division.

[0263] The term "BrdU" as used herein refers to 5-bromo-2'-deoxyuridine, which is a homologue 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, the incorporated BrdU can be detected in an ELISA using anti-BrdU-peroxidase. BrdU incorporation is therefore a measure of proliferation.

[0264] In one embodiment, the antibody comprises an Fc region that has been modified such that 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% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0265] The term "reduction" as used herein refers to a reduction in activity or expression compared to a control protein such as an antibody. Specifically, the term "reduction" when it comes to the expression level of T cell activation marker CD69 refers to a reduction in the expression level of CD69 compared to the expression level of CD69 when T cells are bound by wild-type antibodies (provided that both binding regions of the antibody are bound to CD3). The ability of the antibody to reduce CD69 expression can be evaluated by a functional assay based on PBMC, as described in Example 3 and Example 7. Therefore, in one embodiment, the expression of CD69 is measured by a method comprising the following steps: incubating PBMCs for 16-24 hours at 37°C in a 5% (vol / vol) CO2 humid incubator with an antibody in the range of 1-1000ng / mL, washing the cells, staining the cells with mouse anti-human CD28-PE and mouse-anti-human CD69-APC antibodies at 4°C, and measuring CD69 expression on CD28 positive cells by flow cytometry.

[0266] The term "CD69" as used herein refers to cluster of differentiation 69, which is CD69 Gene encoding human transmembrane C-type lectin protein. Activation of T lymphocytes and natural killer (NK) cells in vivo and in vitro induces the expression of CD69. CD69 is used as a signaling receptor involved in cell activation events including proliferation, as a signaling receptor in lymphocytes including natural killer cells and platelets, and induces specific genes.

[0267] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay for evaluating the functional characteristics of an antibody of the 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. The PBMC-based functional assays described in Examples 3, 4, 5, and 7 include the following steps: (i) incubating PBMCs with antibodies at 37° C. in a 5% (vol / vol) 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, measuring CD69 expression on CD28-positive cells by flow cytometry. Thus, in one embodiment, CD69 expression can be measured as described in Example 3, 4, 5, or 7.

[0268] Therefore, the amino acids in the Fc region that play a major role in the interaction with C1q and Fc Gamma receptors can be modified. Examples of amino acid positions that can be modified include positions L234, L235, and P331. Combinations thereof, such as L234F / L235E / P331S, can cause a significant reduction in binding to human CD64, CD32A, CD16, and C1q.

[0269] Thus, in one embodiment, the amino acid at at least one of the positions corresponding to L234, L235 and P331 may be A, A and S, respectively ([1],

[28] ). In addition, L234F and L235E amino acid substitutions may result in an Fc region with abolished interactions with Fc gamma receptors and C1q (

[29] -

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

[31] ). Thus, in one embodiment, the amino acid at the position corresponding to D265 may be A. Binding to C1q may be abolished by mutating positions D270, K322, P329 and P331. Mutating any of these positions to D270A or K322A or P329A or P331A may render the antibody lacking CDC activity (

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

[0270] An alternative approach to minimizing the interaction of the Fc region with the Fc gamma receptor and C1q is by removing glycosylation sites from the antibody. Mutating position N297 to, for example, Q, A and E removes glycosylation sites that are critical for IgG-Fc gamma receptor interactions. Thus, in one embodiment, the amino acid at the position corresponding to N297 may be G, Q, A or E (

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

[31] ).

[0271] Alternatively, although human IgG2 and IgG4 subclasses are thought to be naturally deficient in their interactions with C1q and Fc gamma receptors, interactions with Fc gamma receptors (Fc gamma receptors) have been reported (

[34] -

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

[0272] Other approaches to further minimize interactions with Fc gamma receptors and C1q in IgG2 antibodies include those described in

[36] and

[37] .

[0273] The hinge region of antibodies may also be important for interactions with Fc gamma receptors and complement (

[38] -

[39] ). Therefore, mutations or deletions in the hinge region may affect the effector function of antibodies.

[0274] The term "cross-linking" as used herein refers to the indirect bridging of an antibody Fab arm (monovalent or bivalent) that binds to a target antigen through the Fc region of a cell-bound antibody bearing an FcR. Thus, an antibody that binds to its target antigen on a cell bearing the target antigen can cross-link to another cell expressing an FcR.

[0275] As used herein, the term "non-specific killing" refers to killing of cells by the cytotoxic function of T cells or other effector cells through tumor target antigen-independent activation of the cells. Thus, non-specific killing means that cells with tumor targets can be killed by, for example, cytotoxic T cells, rather than by antibodies that bind to the tumor target, for example, by inducing CDC.

[0276] The present inventors have shown (see Examples 3-5, 7-10) that an inactivated Fc region can be obtained by modifying one or more of at least five specific amino acid positions of the Fc region.

[0277] Thus, in one embodiment, the antibody comprises a first and a second immunoglobulin heavy chain, 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 of a human IgG1 heavy chain are not L, L, D, N and P, respectively.

[0278] In one embodiment, in both the first and the second heavy chain one or more of the amino acids at the positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are other than L, L, D, N and P, respectively.

[0279] In another embodiment, in at least one of the first and second heavy chains, one or more amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions corresponding to N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0280] As used herein, the term "amino acid corresponding to a position" refers to the amino acid position numbering of a human IgG1 heavy chain. 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] ). Thus, an amino acid or segment in one sequence that "corresponds" to an amino acid or segment in another sequence is an amino acid or segment that is aligned with the other amino acids or segments using a standard sequence alignment program such as ALIGN, ClustalW or similar programs, typically with default settings, and has at least 50%, at least 80%, at least 90% or at least 95% identity with a human IgG1 heavy chain. It is well known in the art how to align sequences or segments within a sequence and thereby determine the position in a sequence that corresponds to an amino acid position of the present invention.

[0281] In the context of the present invention, amino acids may be defined as described above.

[0282] The term "amino acid is not" or similar terms when referring to an amino acid in a heavy chain should be understood to mean that the amino acid is any other amino acid than the specific amino acid mentioned. For example, the amino acid at the position corresponding to L234 of a human IgG1 heavy chain is not L, meaning that the amino acid may be any other naturally or non-naturally occurring amino acid except L.

[0283] In one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is not D.

[0284] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain is not D, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0285] In one embodiment, in at least one of said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is a hydrophobic or polar amino acid.

[0286] The term "hydrophobic" as used herein with respect to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0287] The term "polar" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S and T.

[0288] In another embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgGl heavy chain is an aliphatic uncharged, aromatic or acidic amino acid.

[0289] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L and V.

[0290] The term "aromatic" as used herein with respect to amino acid residues refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of F, T and W.

[0291] The term "acidic" as used herein 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 said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from D and E.

[0292] In a specific embodiment, in at least one of said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V and W.

[0293] In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is not D.

[0294] In one embodiment, in both the first and the second heavy chain, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain is not D, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0295] In one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgGl heavy chain is a hydrophobic or polar amino acid.

[0296] The term "hydrophobic" as used herein with respect to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0297] The term "polar" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S and T. Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S and T. In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0298] In one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S and T.

[0299] In another embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgGl heavy chain is an aliphatic uncharged, aromatic or acidic amino acid.

[0300] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L and V.

[0301] The term "aromatic" as used herein with respect to amino acid residues refers to any amino acid residue selected from F, T and W. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from F, T and W.

[0302] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from D and E. Thus, in one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from D and E.

[0303] In a specific embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgGl heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V and W.

[0304] In a further embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position N297 in a human IgG1 heavy chain is not N.

[0305] In one embodiment, in at least one of the first and second heavy chains the amino acid at the position corresponding to N297 in a human IgG1 heavy chain is not N, and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain is P.

[0306] In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position N297 in a human IgG1 heavy chain is not N.

[0307] In one embodiment, in both the first and the second heavy chain the amino acid at the position corresponding to N297 in a human IgG1 heavy chain is not N, and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain is P.

[0308] In a further embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 of a human IgG1 heavy chain are not L and L, respectively.

[0309] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 of a human IgG1 heavy chain are not L and L, respectively, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0310] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of a 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.

[0311] In one embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 of a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0312] The term "hydrophobic" as used herein with respect to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y.

[0313] The term "polar" with respect to an amino acid residue as used herein refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of amino acids C, D, E, H, K, N, Q, R, S and T.

[0314] In a specific 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 a 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.

[0315] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.

[0316] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 of a human IgG1 heavy chain are not L and L, respectively, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0317] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to L234 and L235 of a human IgGl heavy chain are hydrophobic or polar amino acids.

[0318] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from A, C, F, G, H, I, M, R, T, V, W and Y.

[0319] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each chosen from the group consisting of C, D, E, H, K, N, Q, R, S and T.

[0320] In a specific embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a 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.

[0321] In another embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 of a human IgG1 heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0322] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, G, I and V.

[0323] The term "aromatic" with respect to amino acid residues as used herein refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of F, T and W.

[0324] The term "acidic" as used herein 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 said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 of a human IgG1 heavy chain are each selected from D and E.

[0325] In a specific embodiment, in at least one of said first and second heavy chains, 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.

[0326] In one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E; or A and A, respectively.

[0327] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 of a 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 of a human IgG1 heavy chain are N and P, respectively.

[0328] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E; or A and A, respectively.

[0329] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 of a 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 of a human IgG1 heavy chain are N and P, respectively.

[0330] In a specific embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

[0331] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

[0332] In one embodiment, in at least one of said first and second heavy chains, at least the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively.

[0333] In one embodiment, in both said first and second heavy chains at least the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively.

[0334] In one embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are not L, L and D, respectively.

[0335] 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 of the human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 of the human IgG1 heavy chain are N and P, respectively.

[0336] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 of a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V and W, and the amino acid corresponding to position D265 is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V and W.

[0337] In one embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 of a human IgGl heavy chain are hydrophobic or polar amino acids.

[0338] The term "hydrophobic" as used herein with respect to amino acid residues refers to amino acid residues selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of 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 a human IgG1 heavy chain are each selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y.

[0339] The term "polar" with respect to amino acid residues as used herein refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S and T. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of C, D, E, H, K, N, Q, R, S and T, and the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S and T.

[0340] In a specific 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 a 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, and the amino acid at the position corresponding to position D265 in a 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.

[0341] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to L234, L235 and D265 of a human IgGl heavy chain are hydrophobic or polar amino acids.

[0342] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of 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 a human IgG1 heavy chain are each selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y.

[0343] 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 each selected from the group consisting of 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 the group consisting of C, E, H, K, N, Q, R, S and T.

[0344] In a specific embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgGl heavy chain are each selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W and Y, and the amino acid at the position corresponding to position D265 in a human IgGl heavy chain is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y.

[0345] In another embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 of a human IgGl heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0346] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L and V, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, G, I and V.

[0347] The term "aromatic" with respect to amino acid residues as used herein refers to any amino acid residue selected from the group consisting of F, T and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are each selected from the group consisting of F, T and W.

[0348] The term "acidic" as used herein 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 said first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are each selected from D and E.

[0349] In a specific embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a 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.

[0350] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgGl heavy chain are not L, L and D, respectively.

[0351] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235 and D265 of a human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 of a human IgG1 heavy chain are N and P, respectively.

[0352] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to L234, L235 and D265 of a human IgGl heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

[0353] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a 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 a human IgG1 heavy chain are each selected from A, G, I and V.

[0354] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are each selected from D and E.

[0355] In a specific embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a 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.

[0356] In one embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A; or A, A and A, respectively.

[0357] 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 of a 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 of a human IgG1 heavy chain are N and P, respectively.

[0358] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A; or A, A and A, respectively.

[0359] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235 and D265 of a 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 of a human IgG1 heavy chain are N and P, respectively.

[0360] In a specific embodiment, in at least one of said first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively.

[0361] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgGl heavy chain are F, E and A respectively.

[0362] In one embodiment, in at least one of said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are A, A and A, respectively.

[0363] In one embodiment, in both said first and second heavy chains the amino acids at the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are A, A and A, respectively.

[0364] In another embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are F, E, A, Q and S, respectively.

[0365] In one embodiment, in both said first and second heavy chains the amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgGl heavy chain are F, E, A, Q and S respectively.

[0366] In a specific embodiment, the antibody of the present invention comprises the VH sequence shown in SEQ ID NO:8, the VL sequence shown in SEQ ID NO:10, and the amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one heavy chain are F, E and A, respectively.

[0367] In another embodiment, the antibody of the present invention comprises the VH sequence shown in SEQ ID NO:8, the VL sequence shown in SEQ ID NO:12, and the amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one heavy chain are F, E and A, respectively.

[0368] In another embodiment, the antibody of the present invention comprises the VH sequence shown in SEQ ID NO:6, the VL sequence shown in SEQ ID NO:10, and the amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one heavy chain are F, E and A, respectively.

[0369] In another embodiment, the antibody of the present invention comprises the VH sequence shown in SEQ ID NO:6, the VL sequence shown in SEQ ID NO:12, and the amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one heavy chain are F, E and A, respectively.

[0370] In another embodiment, the antibody of the present invention comprises the VH sequence shown in SEQ ID NO:9, the VL sequence shown in SEQ ID NO:10, and the amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one heavy chain are F, E and A, respectively.

[0371] In another embodiment, the antibody of the present invention comprises the VH sequence shown in SEQ ID NO:9, the VL sequence shown in SEQ ID NO:12, and the amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one heavy chain are F, E and A, respectively.

[0372] In one aspect, an antibody of the invention comprises the human IgLC2 / IgLC3 constant domain lambda light chain of SEQ ID NO:31.

[0373] In one aspect, the antibodies of the present invention can be modified in the light chain (LC) and / or the heavy chain (HC) to increase expression levels and / or production yields. In one embodiment, the antibodies of the present invention can be modified in the light chain (LC). Such modifications are known in the art and can be performed according to the methods described in, for example, Zheng, L., Goddard, J.-P., Baumann, U., & Reymond, J.-L. (2004) Expression improvement and mechanistic study of theretro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis. Journal of Molecular Biology, 341(3), 807–14. doi:10.1016 / j.jmb.2004.06.014.

[0374] In one embodiment, an antibody of the invention comprises a light chain (LC) wherein the amino acid at the position corresponding to position T41 of the lambda light chain of SEQ ID NO: 10 is not T.

[0375] In one embodiment, an antibody of the invention comprises a light chain (LC) wherein the amino acid at the position corresponding to position T41 of the lambda light chain of SEQ ID NO: 10 is selected from H, I, K, L, Q, R and V.

[0376] In one embodiment, an antibody of the invention comprises a light chain (LC) wherein the amino acid at the position corresponding to position T41 of the lambda light chain of SEQ ID NO: 10 is H, K or R, eg, K.

[0377] In one embodiment, an antibody of the invention comprises a light chain (LC) in which the amino acid at the position corresponding to position F10 of the lambda light chain of SEQ ID NO: 10 is not F, and wherein one or more of the amino acid positions corresponding to positions T41, K55 and L97 of the lambda light chain of SEQ ID NO: 10 are not T, K and L, respectively.

[0378] In one embodiment, an antibody of the invention comprises a light chain (LC) in which the amino acids at positions corresponding to positions F10, T41, K55 and L97 of the lambda light chain of SEQ ID NO: 10 are not F, T, K and L, respectively.

[0379] In one embodiment, an antibody of the invention comprises a light chain (LC) in which the amino acid at the position corresponding to position T41 is selected from H, I, K, L, Q, R or V, e.g., selected from H, K and R, e.g., K, and wherein the amino acids at the positions corresponding to positions F10, T41, K55 and L97 of the lambda light chain of SEQ ID NO: 10 are L, K, N and H, respectively.

[0380] In one embodiment, an antibody of the invention comprises a light chain (LC) wherein the amino acids at the positions corresponding to positions R23 and A35 are not R and A, respectively.

[0381] In one embodiment, an antibody of the invention comprises a light chain (LC) wherein the amino acid at the position corresponding to position R23 is selected from the group consisting of A, G, H, K, Q, S and T, e.g., selected from A and G, and wherein the amino acid at the position corresponding to A35 is selected from the group consisting of I, L, M, P, V, G, F and W, e.g., selected from the group consisting of I, L, M, P and V.

[0382] In one embodiment, an antibody of the invention comprises a light chain (LC) wherein the amino acid at the position corresponding to position R23 is A or G, such as A, and the amino acid at the position corresponding to position A35 is P.

[0383] In one embodiment, an antibody of the invention comprises a light chain (LC) in which the amino acids at positions F10, R23, A35, R47, D71, A82, D83, S86, I87 and F89 corresponding to the lambda light chain of SEQ ID NO: 10 are not F, R, A, R, D, A, D, S, I and F, respectively.

[0384] In one embodiment, an antibody of the invention comprises a light chain (LC) wherein the amino acid at the position corresponding to position R23 is selected from A, G, H, K, Q, S and T, e.g., selected from A and G, wherein the amino acid at the position corresponding to A35 is selected from I, L, M, P, V, G, F and W, e.g., selected from I, L, M, P, and wherein the amino acids at the positions corresponding to positions F10, R47, D71, A82, D83, S86, I87 and F89 of the lambda light chain of SEQ ID NO: 10 are L, T, G, P, E, A, E and Y, respectively.

[0385] In one embodiment, an antibody of the invention comprises a light chain (LC) in which the amino acid at the position corresponding to position R23 is A or G, and wherein the amino acids at the positions corresponding to positions F10, A35, R47, D71, A82, D83, S86, I87 and F89 of the lambda light chain of SEQ ID NO: 10 are L, P, T, G, P, E, A, E and Y, respectively.

[0386] In one aspect, the antibodies of the invention may be modified in the light chain (LC) to increase the affinity of the antibody. Modifications that result in increased affinity of antibodies are known in the art.

[0387] In one aspect, the antibodies of the invention may be modified in the light chain (LC) to reduce the affinity of the antibody. This may be advantageous in some cases and result in increased efficacy. Specifically, a low affinity CD3 arm may have an effect on the motility of circulating T cells in the tumor site, resulting in better engagement of tumor cells with T cells, see MølhØj et al., Molecular Immunology 44 (2007). Specifically, this can be used in a bispecific format in which a CD3 antibody is used as one of the binding arms. Modifications that result in reduced antibody affinity are known in the art, see, e.g., Webster et al. Int J Cancer Suppl. 1988; 3: 13-6.

[0388] In one embodiment, an antibody of the invention comprises a light chain (LC) wherein

[0389] (i) the amino acid at position corresponding to position F10 of the lambda light chain of SEQ ID NO: 10 is not F, or

[0390] (ii) the amino acid at the position corresponding to position K55 of the lambda light chain of SEQ ID NO: 10 is not K, or

[0391] (iii) the amino acid at the position corresponding to position F10 of the lambda light chain of SEQ ID NO: 10 is not F, and the amino acid at the position corresponding to position K55 of the lambda light chain of SEQ ID NO: 10 is not K.

[0392] In one embodiment, an antibody of the invention comprises a constant light chain (LC) wherein

[0393] (i) the amino acid at position F10 corresponding to the lambda light chain of SEQ ID NO: 10 is L, or

[0394] (ii) the amino acid at the position corresponding to position K55 of the lambda light chain of SEQ ID NO: 10 is N, or

[0395] (iii) the amino acid at the position corresponding to position F10 of the lambda light chain of SEQ ID NO: 10 is L, and the amino acid at the position corresponding to position K55 of the lambda light chain of SEQ ID NO: 10 is N.

[0396] In one embodiment, an antibody of the invention comprises a light chain (LC) in which the amino acids at positions corresponding to positions F10, T41, K55, and L97 of the lambda light chain of SEQ ID NO: 10 are not F, T, K, and L, respectively. Such modifications are used to both increase expression levels and decrease affinity.

[0397] The antibodies of the present invention comprise a light chain (LC) in which the amino acids at positions F10, T41, K55 and L97 corresponding to the lambda light chain of SEQ ID NO: 10 are L, K, N and H, respectively. Such modifications are used to both increase expression levels and reduce affinity.

[0398] In one embodiment, an antibody of the present invention comprises a heavy chain (HC) and a light chain (LC), wherein the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain of SEQ ID NO: 15 are F, E and A, respectively, and wherein the position corresponding to F405 of the human IgG1 heavy chain of SEQ ID NO: 15 is L, and wherein (i) the positions corresponding to positions F10, T41, K55 and L97 of the lambda light chain of SEQ ID NO: 10 are L, K, N and H, respectively, or (ii) the position corresponding to position T41 of the lambda light chain of SEQ ID NO: 10 is K.

[0399] In one embodiment, an antibody of the invention comprises a heavy chain (HC) of SEQ ID NO:25 and a light chain (LC) of SEQ ID NO:32.

[0400] In one embodiment, an antibody of the invention comprises a heavy chain (HC) of SEQ ID NO:25 and a light chain (LC) of SEQ ID NO:33.

[0401] In one aspect, the present invention relates to a multispecific antibody comprising at least a first binding region of an antibody of any aspect or embodiment described herein, and one or more binding regions that bind to one or more different targets than the first binding region. Such a multispecific antibody may be a bispecific antibody.

[0402] Thus, in one aspect, the invention relates to a bispecific antibody comprising a first binding region of the antibody of any aspect or embodiment described herein, and a second binding region that binds a different target than the first binding region.

[0403] The term "multispecific antibody" refers to an antibody that is specific for at least two different, e.g., at least three, typically non-overlapping epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types.

[0404] The term "bispecific antibody" refers to an antibody that is specific for at least two different, usually non-overlapping epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types.

[0405] In one embodiment, the bispecific antibody comprises a first and a second heavy chain.

[0406] Embodiments involving modifications of the Fc region and embodiments involving specific amino acid replacements are contemplated as part of any bispecific antibody of 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 embodiment described herein (e.g., those described with respect to providing an inactive Fc region). In one embodiment, both the first and second heavy chains comprise one or more amino acids modified as defined in any embodiment described herein (e.g., those described with respect to providing an inactive Fc region). Thus, the bispecific antibody comprises an Fc region modified in accordance with any aspect or embodiment described herein; or at least one of the first and second heavy chains comprises one or more amino acids modified in accordance with any aspect or embodiment defined herein.

[0407] Thus, in one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10; and wherein the Fc region has been modified such that binding of C1q to said antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA.

[0408] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:12; and wherein the Fc region has been modified such that binding of C1q to said antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA.

[0409] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; and wherein the Fc region has been modified such that binding of C1q to said antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA.

[0410] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12; and wherein the Fc region has been modified such that binding of C1q to said antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA.

[0411] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10; and wherein the Fc region has been modified such that binding of C1q to said antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA.

[0412] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12; and wherein the Fc region has been modified such that binding of C1q to said antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA.

[0413] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10; and wherein the Fc region has been modified such that the antibody mediates Fc-mediated T-cell proliferation 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 using a peripheral blood mononuclear cell (PBMC) based functional assay.

[0414] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO: 8 and the VL sequence shown in SEQ ID NO: 12; and wherein the Fc region has been modified such that the antibody mediates Fc-mediated T-cell proliferation 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 using a peripheral blood mononuclear cell (PBMC) based functional assay.

[0415] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; and wherein the Fc region has been modified such that the antibody mediates Fc-mediated T-cell proliferation 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 using a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0416] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO: 6 and the VL sequence shown in SEQ ID NO: 12; and wherein the Fc region has been modified such that the antibody mediates Fc-mediated T-cell proliferation 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 using a peripheral blood mononuclear cell (PBMC) based functional assay.

[0417] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10; and wherein the Fc region has been modified such that the antibody mediates Fc-mediated T-cell proliferation 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 using a peripheral blood mononuclear cell (PBMC) based functional assay.

[0418] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12; and wherein the Fc region has been modified such that the antibody mediates Fc-mediated T-cell proliferation 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 using a peripheral blood mononuclear cell (PBMC) based functional assay.

[0419] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10; and wherein the Fc region has been modified such that 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% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined using a PBMC-based functional assay.

[0420] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:12; and wherein the Fc region has been modified such that 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% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined using a PBMC-based functional assay.

[0421] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; and wherein the Fc region has been modified such that 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% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined using a PBMC-based functional assay.

[0422] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12; and wherein the Fc region has been modified such that 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% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined using a PBMC-based functional assay.

[0423] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10; and wherein the Fc region has been modified such that 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% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined using a PBMC-based functional assay.

[0424] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12; and wherein the Fc region has been modified such that 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% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined using a PBMC-based functional assay.

[0425] In a specific embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence 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 of the human IgG1 heavy chain are F, E and A, respectively.

[0426] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence 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 of the human IgG1 heavy chain are F, E and A, respectively.

[0427] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence 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 of the human IgG1 heavy chain are F, E and A, respectively.

[0428] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence 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 of the human IgG1 heavy chain are F, E and A, respectively.

[0429] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence 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 of the human IgG1 heavy chain are F, E and A, respectively.

[0430] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence 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 of the human IgG1 heavy chain are F, E and A, respectively.

[0431] Examples of bispecific antibody molecules that can be used in the present invention include (i) a single antibody having two arms containing different antigen binding regions, (ii) a single chain antibody specific for two different epitopes, such as two scFvs connected in series by an additional peptide linker; (iii) a dual variable domain antibody (DVD-Ig TM ), in which each light and heavy chain comprises two variable domains connected in series by a short peptide bond (

[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) flexibodies, which are a combination of scFv and diabodies, resulting in multivalent molecules; (vii) so-called "dock-and-lock" molecules (Dock-and-Lock®), based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fab, can produce trivalent bispecific binding proteins consisting of two identical Fab fragments connected to different Fab fragments; (viii) so-called Scorpion molecules, which comprise, for example, two scFvs fused to the two ends of a human Fab arm; and (ix) diabodies.

[0432] In one embodiment, the bispecific antibody of the present invention is a diabody, a cross-body or a bispecific antibody obtained by controlled Fab arm exchange, such as DuoBody® (e.g. described in

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

[0433] Examples of different types of bispecific antibodies include, but are not limited to (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, wherein each side of the molecule comprises at least two different antibody Fab fragments or a portion of a Fab fragment; (iii) IgG fusion molecules, wherein a full-length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment; (iv) Fc fusion molecules, wherein a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc-region or a portion thereof; (v) Fab fusion molecules, wherein different Fab-fragments are fused together, to a heavy chain constant domain, an Fc-region or a portion thereof; and (vi) ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, Nanobodies®), wherein different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, Nanobodies®) are fused to each other, or to another protein or carrier molecule fused to a heavy chain constant domain, an Fc-region or a portion thereof.

[0434] Examples of IgG-like molecules with complementary CH3 domain molecules include, but are not limited to, Triomab® (TrionPharma / Fresenius Biotech,

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

[43] ), CrossMAbs (Roche,

[44] ) and electrostatic pairing (Amgen,

[45] -

[46] ; Chugai,

[47] ; Oncomed,

[48] ), LUZ-Y (Genentech), DIG bodies and PIG bodies (Pharmabcine), 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] ).

[0435] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, dual targeting (DT)-Ig (GSK / Domantis), 2-in-1 antibodies (Genentech), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star,

[54] ), ZybodiesTM (Zyngenia), using a shared light chain approach (Crucell / Merus,

[55] ), κλBodies (NovImmune), and CovX-body® (CovX / Pfizer).

[0436] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig TM (Abbott,

[56] ), dual-domain bispecific antibodies (Unilever; Sanofi Aventis,

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

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

[59] ) and TvAb (Roche,

[59] ,

[60] ).

[0437] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), dual affinity redirecting technology (Fc-DART TM ) (MacroGenics,

[62] ,

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

[0438] Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), Dual-Acting or Bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0439] Examples of ScFv-based, diabody-based antibodies and domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE®) (Micromet, Tandem Diabody (Tandab) (Affimed), dual affinity redirecting 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 nanobodies® (Ablynx), dual-targeting heavy chain domain-only antibodies.

[0440] It is also contemplated that any monospecific antibody that 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 has been tested in a functional assay and meets the requirements described herein. Such bispecific antibodies can be provided by the methods described in

[41] , which is incorporated herein by reference.

[0441] Thus, in a specific embodiment, each of the first and second heavy chains comprises at least a hinge region, a CH2 and a CH3 region, wherein at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 of a human IgG1 heavy chain in the first heavy chain is replaced, and at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 of a human IgG1 heavy chain in the second heavy chain is replaced, and wherein the first and second heavy chains are not replaced at the same position. In this case, the term "replacement" refers to the replacement of an amino acid at a specific amino acid position by another naturally or non-naturally occurring amino acid. Thus, a "replaced" amino acid at a position corresponding to a position in a 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.

[0442] In one embodiment, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain in the first 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 the amino acid at at least one position corresponding to a position selected from T366, L368, K370, D399, F405 and Y407 in the human IgG1 heavy chain in the second heavy chain is replaced.

[0443] In one embodiment, the amino acid at the position corresponding to K409 of human IgG1 heavy chain in the first heavy chain is not K, L or M, and the amino acid at the position corresponding to F405 of human IgG1 heavy chain in the second heavy chain is not F and optionally the amino acid at the position corresponding to K409 of human IgG1 heavy chain is K.

[0444] In one embodiment, the amino acid at the position corresponding to F405 of human IgG1 heavy chain in the first heavy chain is not F, R and G, and the amino acid at the position corresponding to the position selected from T366, L368, K370, D399, Y407 and K409 of human IgG1 heavy chain in the second heavy chain is replaced.

[0445] In one embodiment, the amino acid in said first heavy chain at the position corresponding to K409 of a human IgG1 heavy chain is not K, L or M, and the amino acid at the position corresponding to F405 of a human IgG1 heavy chain is not F.

[0446] In a further embodiment, the amino acid at the position corresponding to F405 of human IgG1 heavy chain in said first heavy chain is L, and the amino acid at the position corresponding to K409 of human IgG1 heavy chain in said second heavy chain is R, or vice versa.

[0447] Thus, in one embodiment, the amino acid at the position corresponding to K409 of a human IgG1 heavy chain in the first heavy chain is R, and the amino acid at the position corresponding to F405 of a human IgG1 heavy chain in the second heavy chain is L.

[0448] In a further embodiment, the humanized or chimeric CD3 antibody of the invention comprises one or more inactive substitutions disclosed in any of the above embodiments, such as L234F, L235E and D265A, in at least one of the first and second heavy chains; 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 comprises one or more inactive substitutions disclosed in any of the above embodiments, such as L234F, L235E and D265A, in at least one of the first and second heavy chains; and an additional substitution at the K409 position, such as K409R. Specifically, in one embodiment, the humanized or chimeric CD3 antibody of the invention comprises one or more inactive substitutions disclosed in any of the above embodiments, such as L234F, L235E and D265A, in both the first and second heavy chains; and a substitution at the F405 position, such as F405L. In one embodiment, the humanized or chimeric CD3 antibody of the invention comprises one or more inactive substitutions disclosed in any of the above embodiments, such as L234F, L235E and D265A, and an additional substitution at position K409, such as K409R, in both the first and second heavy chains. Such antibodies can be used to generate bispecific antibodies.

[0449] Therefore, in a further embodiment, the amino acids at the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain in the second heavy chain is R.

[0450] In one embodiment, the amino acids at the positions corresponding to L234, L235, D265, N297 and P331 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E, A, N and P, respectively, the amino acid at the position corresponding to F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0451] In an alternative embodiment, the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively, the amino acid at the position corresponding to K409 of the human IgG1 heavy chain in the first heavy chain is R, and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain in the second heavy chain is L.

[0452] In one embodiment, the amino acids at the positions corresponding to L234, L235, D265, N297 and P331 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E, A, N and P, respectively, the amino acid at the position corresponding to K409 of the human IgG1 heavy chain in the first heavy chain is R, and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain in the second heavy chain is L.

[0453] In another embodiment, the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively, the amino acid at the position corresponding to F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0454] In one embodiment, the amino acids at the positions corresponding to L234, L235, D265, N297 and P331 of the human IgG1 heavy chain in both the first and the second heavy chains are F, E, A, N and P, respectively, the amino acid at the position corresponding to F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0455] In an alternative embodiment, the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively, the amino acid at the position corresponding to K409 of the human IgG1 heavy chain in the first heavy chain is R, and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain in the second heavy chain is L.

[0456] In one embodiment, the amino acids at the positions corresponding to L234, L235, D265, N297 and P331 of the human IgG1 heavy chain in both the first and the second heavy chains are F, E, A, N and P, respectively, the amino acid at the position corresponding to K409 of the human IgG1 heavy chain in the first heavy chain is R, and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain in the second heavy chain is L.

[0457] As described herein, T cells are recruited to specific target cells, such as cancer or tumor cells, providing a method for killing target cells. The inventors have demonstrated that bispecific CD3xHER2 antibodies comprising specific amino acid substitutions L234F, L235E and D265A in two heavy chains can induce killing of AU565 cells, as described in Example 5. T cell-mediated killing can be obtained by a bispecific antibody that targets CD3 by a first binding region and another target by a second binding region. Therefore, in one embodiment, the first binding region is in accordance with any embodiment described herein for humanized or chimeric CD3 antibodies, and the second binding region binds to different targets 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., one of the heavy and light chain pairs of the antibody, is a humanized or chimeric antibody as described herein. Therefore, half of the bispecific antibody is a humanized or chimeric antibody in accordance with the present invention in combination with CD3 and the other half can be a humanized, chimeric, fully non-human or fully human antibody in combination with a second target. Thus, in one embodiment, the antibody comprises a first and a second heavy chain, a first and a second light chain, wherein the first heavy chain and the first light chain are humanized or chimeric, and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are fully human, and are linked by a disulfide bridge to form a second binding region, wherein the first binding region is in accordance with any aspect or embodiment described herein, and the second binding region binds to a different target. In one embodiment, the antibody comprises a first and a second heavy chain, a first and a second light chain, wherein the first heavy chain and the first light chain are humanized or chimeric, and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are humanized or chimeric, and are linked by a disulfide bridge to form a second binding region, wherein the first binding region is in accordance with an aspect or embodiment described herein, and the second binding region binds to a different CD3 epitope than the first binding region.

[0458] Therefore, in one embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0459] In one embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234, L235 and D265 of 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 of the human IgG1 heavy chain is L, and in the second heavy chain the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain is R.

[0460] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0461] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0462] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0463] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0464] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0465] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:8, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0466] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0467] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0468] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0469] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0470] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0471] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0472] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0473] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:6, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0474] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0475] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0476] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0477] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:10; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0478] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0479] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0480] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:19, and the VL sequence shown in SEQ ID NO:20; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0481] In another embodiment, the bispecific antibody comprises: a first binding region comprising the VH sequence shown in SEQ ID NO:9, and the VL sequence shown in SEQ ID NO:12; a second binding region comprising the VH sequence shown in SEQ ID NO:29, and the VL sequence shown in SEQ ID NO:30; wherein the amino acids at the positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in both the first and second heavy chains are F, E and A, respectively; and wherein the amino acid at the position corresponding to position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at the position corresponding to position K409 of the human IgG1 heavy chain in the second heavy chain is R.

[0482] As used herein, the term "disulfide bridge" refers to a covalent bond between two cysteine ​​residues, ie, the interaction may also be referred to as a Cys-Cys interaction.

[0483] The term "target" as used herein refers to a molecule to which a binding region of an antibody of the invention binds. When used in the context of antibody binding, the term includes any antigen against which an antibody is generated.

[0484] In a specific embodiment, the first heavy chain and the first light chain are humanized or chimeric, and are connected by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are fully human and are connected by a disulfide bridge to form a second binding region, wherein the first binding region binds to a different target according to any aspect or embodiment described herein; and wherein the amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively.

[0485] In a specific embodiment, the first heavy chain and the first light chain are humanized or chimeric and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and the light chain are fully human and are linked by 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 CD3 epitope than the first binding region; and wherein the amino acids at positions corresponding to positions L234, L235 and D265 of the human IgG1 heavy chain in at least one of the first and second heavy chains are F, E and A, respectively.

[0486] In a specific embodiment, the first heavy chain and the first light chain are humanized or chimeric and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and the light chain are fully human and are linked by a disulfide bridge to form a second binding region, wherein the first binding region binds to a different target according to any aspect or embodiment described herein, and the second binding region; and wherein the amino acids at positions L234, L235 and D265 in both the first and second heavy chains corresponding to positions of human IgG1 heavy chain are F, E and A, respectively.

[0487] In a specific embodiment, the first heavy chain and the first light chain are humanized or chimeric and are linked by a disulfide bridge to form a first binding region; and the second heavy chain and light chain are fully human and are linked by 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 CD3 epitope than the first binding region; and wherein the amino acids at positions L234, L235 and D265 in both the first and second heavy chains corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively.

[0488] In one aspect, the bispecific antibody of the invention comprises a second binding region that binds to human HER2 or human CD20.

[0489] In one embodiment, the bispecific antibody of the invention comprises a second binding region that binds human CD20.

[0490] In one embodiment, the second binding region may be derived from a full-length monoclonal CD20 antibody, such as the antibodies disclosed in WO2004035607 (Genmab A / S), including ofatumumab (2F2), 11B8 and 7D8; the antibodies disclosed in WO2005103081 (Genmab A / S), including 2C6; AME-133; TRU-015; IMMU-106; ocrelizumab (Gazyva®); tositumomab (Bexxar®); and rituximab (Rituxan® / MabThera®).

[0491] In one embodiment, the second binding region can be derived from a full-length monoclonal CD20 antibody that binds to an epitope on CD20 that does not contain or require amino acid residues alanine at position 170 or proline at position 172, but that contains or requires amino acid residues asparagine at position 163 and asparagine at position 166.

[0492] In one embodiment, the bispecific antibody of the invention comprises a second binding region that binds to human CD20, said binding region comprising:

[0493] (i) a VH CDR1 region of SEQ ID NO:34, a VH CDR2 region of SEQ ID NO:35, a VH CDR3 region of SEQ ID NO:36, a VL CDR1 region of SEQ ID NO:37, a VL CDR2 region of DAS, and a VL CDR3 region of SEQ ID NO:38,

[0494] (ii) the VH CDR1 region of SEQ ID NO: 41, the VH CDR2 region of SEQ ID NO: 42, the VH CDR3 region of SEQ ID NO: 43, the VL CDR1 region of SEQ ID NO: 44, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO: 45,

[0495] (iii) the VH CDR1 region of SEQ ID NO:48, the VH CDR2 region of SEQ ID NO:49, the VH CDR3 region of SEQ ID NO:50, the VL CDR1 region of SEQ ID NO:51, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:52, or

[0496] (iv) the VH CDR1 region of SEQ ID NO:55, the VH CDR2 region of SEQ ID NO:56, the VH CDR3 region of SEQ ID NO:57, the VL CDR1 region of SEQ ID NO:58, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:59.

[0497] In one embodiment, the bispecific antibody of the invention comprises a second binding region that binds to human CD20, said binding region comprising:

[0498] (i) a VH sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:29, and a VL sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:30,

[0499] (ii) a VH sequence that has at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:39, and a VL sequence that has at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:40,

[0500] (iii) a VH sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:46, and a VL sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:47, or

[0501] (iv) a VH sequence that has at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:53, and a VL sequence that has at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:54.

[0502] In one embodiment, the bispecific antibody of the invention comprises a second binding region that binds to human CD20, wherein the second binding region comprises:

[0503] (i) the VH sequence of SEQ ID NO: 29, and the VL sequence of SEQ ID NO: 30,

[0504] (ii) the VH sequence of SEQ ID NO: 39, and the VL sequence of SEQ ID NO: 40,

[0505] (iii) the VH sequence of SEQ ID NO: 46, and the VL sequence of SEQ ID NO: 47, or

[0506] (iv) the VH sequence of SEQ ID NO: 53, and the VL sequence of SEQ ID NO: 54.

[0507] Nucleic acid constructs, expression vectors and host cells

[0508] In one aspect, the present invention relates to a nucleic acid construct encoding one or more sequences shown in Table 1. Therefore, the present 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.

[0509] In a further aspect, the present invention relates to a nucleic acid construct encoding a sequence of a humanized or chimeric CD3 antibody of the present invention, an expression vector comprising the nucleic acid construct of the present invention, a host cell comprising the expression vector, and a method of producing the antibody by culturing the host cell under suitable conditions for producing and optionally recovering the antibody. Humanized CD3 antibodies are also referred to as "huCD3".

[0510] 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 of the present invention, (ii) a nucleic acid sequence encoding a light chain sequence of a humanized or chimeric antibody of the present invention, or (iii) both (i) and (ii). Thus, the expression vector comprises one or more nucleic acid constructs or nucleic acid sequences of any aspect or embodiment described herein.

[0511] In one embodiment, the expression vector of the invention comprises a nucleic acid sequence encoding one or more of the heavy and light chain CDR sequences selected from SEQ ID NO: 1, 2, 3, 4 and 5 and the sequence GTN.

[0512] 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 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 a 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 an amino acid sequence according to SEQ ID NO: 15, 16, 23, 24, 25 and 26.

[0513] In a specific embodiment, the expression vector comprises a nucleic acid sequence encoding a variant of one or more of the above amino acid sequences, the 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 modifications, such as deletions or insertions, preferably substitutions, such as conservative or non-conservative substitutions, or having at least 80% identity with any of the sequences, such as at least 85% identity or 90% identity or 95% identity with any of the above amino acid sequences, such as 96% identity or 97% identity or 98% identity or 99% identity. The present invention also relates to nucleic acid sequences that are different from the above nucleic acid sequences but encode the same amino acid sequence as the antibody of the present invention due to differences in the genetic code. For example, the nucleic acid sequence may be different, but produce the same amino acid sequence as any amino acid sequence described herein. How to identify such additional nucleic acid sequences based on the genetic code is well known to the technician.

[0514] 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 (eg, a human antibody).

[0515] Such expression vectors described above can be used to recombinantly produce the antibodies of the present invention.

[0516] In the context of the present invention, the expression vector can be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (comprising a nucleic acid sequence of a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the humanized or chimeric CD3 antibody-encoding nucleic acid is contained in a naked DNA or RNA vector, including, for example, a linear expression element (as described in, for example,

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

[65] and / or

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

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

[68] ,

[69] ,

[70] and

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

[72] and

[73] ).

[0517] 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), etc.

[0518] The expression vector may additionally or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH (reviewed in

[75] and

[76] ).

[0519] Nucleic acid constructs and / or vectors may further comprise nucleic acid sequences encoding secretion / localization sequences that can target polypeptides (e.g., nascent polypeptide chains) to the cytoplasmic space or cell culture medium. Such sequences are known in the art and include secretion leader sequences or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transport sequences, chloroplast transport sequences), membrane localization / anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), etc., which are well known in the art.

[0520] In the expression vector of the present invention, the humanized or chimeric CD3 antibody-encoding nucleic acid 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., human CMV IE promoter / enhancer and RSV, SV40, SL3-3, MMTV and HIV LTR promoters), effective poly (A) termination sequences, origins of replication of plasmid products in E. coli, antibiotic resistance genes as selective markers and / or suitable cloning sites (e.g., polymeric linkers). Nucleic acid constructs and / or vectors may also contain inducible promoters relative to constitutive promoters, such as CMV IE (technicians will know that these terms are actually descriptors of the degree of gene expression under certain conditions).

[0521] In one embodiment, the humanized or chimeric CD3 antibody-encoding expression vector is located in a host cell or a host animal, and / or is delivered to the host cell or the host animal via a viral vector.

[0522] Such expression vectors can be used to recombinantly produce humanized or chimeric CD3 antibodies.

[0523] In one aspect, the invention provides a host cell comprising an expression vector of the invention.

[0524] In one aspect, the humanized or chimeric CD3 antibody of any aspect or embodiment described herein is provided by using a recombinant eukaryotic, recombinant prokaryotic or recombinant microbial host cell that produces the antibody. Therefore, the present invention provides a recombinant eukaryotic, recombinant prokaryotic or recombinant microbial host cell that produces 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 comprises a nucleic acid sequence stably integrated into the cell genome, which comprises a sequence encoding the expression of a humanized or chimeric CD3 antibody as described herein. In another embodiment, the host cell comprises a non-integrated nucleic acid sequence, such as a plasmid, a cosmid, a phagemid or a linear expression element, which comprises a sequence encoding the expression of a humanized or chimeric CD3 antibody as described herein.

[0525] The term "recombinant host cell" (or simply "host cell") used herein means a cell into which an expression vector or nucleic acid construct or sequence is introduced. It should be understood that the term refers not only to the specific subject cell, but also to the progeny of the cell. Because certain modifications may occur during passage due to mutations or environmental influences, the progeny may in fact be different from the parent cell, but are still included in the scope of the term "host cell" used herein. Recombinant host cells include, for example: eukaryotic host cells, such as CHO cells, HEK-293 cells, PER.C6, NS0 cells and lymphocytes; and prokaryotic cells, such as Escherichia coli; and other eukaryotic hosts, such as plant cells and fungi.

[0526] In a further aspect, the present invention relates to a method for producing a humanized or chimeric CD3 antibody of the present invention, said method comprising the following steps:

[0527] a) cultivating the host cell of the present invention as described above, and

[0528] b) recovering and / or purifying the antibody of the invention from the culture medium.

[0529] In a further aspect, the nucleotide sequence encoding the sequence of a humanized or chimeric CD3 antibody further encodes a second portion, 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:

[0530] a) culturing a host cell comprising an expression vector comprising the nucleotide sequence, and

[0531] b) recovering and / or purifying the humanized or chimeric CD3 antibody fusion protein from the culture medium.

[0532] Composition

[0533] In one aspect, the invention provides a composition comprising an antibody or bispecific antibody according to any aspect and embodiment described herein.

[0534] 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.

[0535] Pharmaceutical compositions may be formulated according to conventional techniques, such as those described in

[77] , using a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients.

[0536] The pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients should be suitable for the humanized or chimeric antibodies of the present invention and the selected mode of administration. The suitability of the carrier and other components of the pharmaceutical composition is determined by the absence of a significant negative effect on the biological properties required for antigen binding of the selected compound or pharmaceutical composition of the present invention (e.g., less than a substantial effect (10% or less relative inhibition, 5% or less relative inhibition, etc.)).

[0537] The pharmaceutical compositions 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 pharmaceutical compositions.

[0538] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration and is non-toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors well known in the medical field, including the activity of the specific composition of the present invention or its amide used, the route of administration, the number of administrations, the excretion rate of the specific compound used, the duration of treatment; other drugs, compounds and / or materials used in combination with the specific composition used; the age, sex, weight, condition, general health and previous medical history of the patient being treated, and other factors.

[0539] The pharmaceutical composition can be administered by any suitable route and mode. Suitable routes for in vivo and in vitro administration of the humanized or chimeric antibodies of the present invention are well known in the art and can be selected by a person of ordinary skill in the art.

[0540] In one embodiment, the pharmaceutical compositions of the invention are administered parenterally.

[0541] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion.

[0542] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0543] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents and the like that are physiologically compatible with the humanized or chimeric antibodies of the invention.

[0544] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, glucose, polyols (e.g., 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 colloid solution, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical field.

[0545] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For pharmaceutically active substances, the use of such media and agents is known in the art. Unless any conventional media or agents are incompatible with the active compound, it is contemplated that they are used in the pharmaceutical compositions of the present invention. When referring to "active compound", it is contemplated that it also refers to humanized or chimeric antibodies of the present invention.

[0546] Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0547] The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant, such as (1) a water-soluble antioxidant, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) an oil-soluble antioxidant, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) a metal chelator, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0548] The pharmaceutical compositions of the present invention may also comprise isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0549] The pharmaceutical compositions of the invention may also contain one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which may increase the half-life or efficacy of the pharmaceutical composition. The humanized or chimeric antibodies of the invention may be prepared together 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 such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid alone or with wax, 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] ).

[0550] In one embodiment, the humanized or chimeric antibodies of the present invention can be formulated to ensure appropriate in vivo distribution. 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. For pharmaceutically active substances, the use of such media and agents is known in the art. Unless any conventional media or agents are incompatible with the active compound, it is expected that they will be used in the pharmaceutical compositions of the present invention. Other active or therapeutic compounds may also be incorporated into the composition.

[0551] Pharmaceutical compositions for injection must generally be sterile and stable under the conditions of preparation and storage. The composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. The carrier can be an aqueous or non-aqueous solvent or dispersion medium, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof; vegetable oils such as olive oil, and injectable organic esters, such as ethyl oleate. Suitable fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersants, and by using surfactants. In many cases, it is preferred to include isotonic agents such as sugars, polyols such as glycerol, mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of injectable compositions can be produced by including agents that delay absorption in the composition, such as monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent together with one or a combination of ingredients listed above as needed, followed by aseptic microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion matrix and the desired other ingredients, such as those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any other desired ingredients from a previously sterile-filtered solution thereof.

[0552] Sterile injection solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent together with a composition or combination of compositions listed above as required, followed by aseptic microfiltration. Generally speaking, dispersions are prepared by incorporating the active compound into a sterile solvent comprising a basic dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which obtain a powder of the active ingredient plus any other required components from a sterile filtered solution thereof before.

[0553] Therapeutic applications

[0554] In another aspect, the present invention relates to a humanized or chimeric antibody or a pharmaceutical composition of the invention as defined in any aspect or embodiment described herein for use as a medicament.

[0555] In another aspect, the present invention relates to a humanized or chimeric antibody or a pharmaceutical composition of the present invention as defined in any aspect or embodiment described herein for use in treating a disease.

[0556] The humanized or chimeric antibodies or pharmaceutical compositions of the present invention can be used to treat any cancer in which the effector mechanism of cytotoxic T cells is required. For example, the humanized or chimeric antibodies can be administered to cells cultured in vitro or in vitro, or to human subjects in vivo, for example, to treat or prevent conditions such as cancer, inflammatory or autoimmune conditions. The term "subject" as used herein is generally a person who responds to the humanized or chimeric antibodies or pharmaceutical compositions. Subjects may, for example, include human patients with conditions that can be corrected or improved by regulating target function or by directly or indirectly causing killing of cells.

[0557] In another aspect, the invention provides a method for treating or preventing a disorder such as cancer, wherein the recruitment of T cells will aid in the treatment or prevention, the method comprising administering a therapeutically effective amount of a humanized or chimeric antibody or pharmaceutical composition of the invention to a subject in need thereof. The method generally comprises administering to a subject a humanized or chimeric antibody in an amount effective to treat or prevent the disorder.

[0558] In a specific aspect, the invention relates to a method of treating cancer comprising administering to a subject in need thereof a humanized or chimeric antibody or a pharmaceutical composition of the invention as defined in any aspect and embodiment described herein.

[0559] In another aspect, the invention relates to the 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 both CD3 and a cancer-specific target or a target that is overexpressed in cancer or is associated with cancer, 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 squamous cell carcinoma of the head and neck, cervical cancer, pancreatic cancer, testicular cancer, malignant melanoma, soft tissue cancer (e.g. synovial sarcoma), indolent or aggressive forms of B-cell lymphoma, chronic lymphocytic leukemia or acute lymphocytic leukemia.

[0560] The effective dosage and dosage regimen of the humanized or chimeric antibody depends on the disease or condition to be treated, and can be determined by one skilled in the art.

[0561] Doctors with ordinary skills in the art can easily determine and prescribe the effective amount of pharmaceutical composition needed. For example, the doctor can start the dosage of the humanized or chimeric antibody used in the pharmaceutical composition below the level required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, the suitable dosage of the composition of the present invention is the amount of the humanized or chimeric antibody at the minimum dose that effectively produces a therapeutic effect according to a specific dosage regimen. Such an effective dose generally depends on the factors described above.

[0562] 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 that predicts efficacy in human tumors. Alternatively, this property of the composition can be evaluated by examining the ability of humanized or chimeric antibodies to inhibit cell growth or induce cytotoxicity by in vitro assays known to the skilled person. A therapeutically effective amount of a therapeutic compound, i.e., a therapeutic humanized or chimeric antibody or pharmaceutical composition of the present invention, can reduce tumor size, or otherwise improve the symptoms of a subject. One of ordinary skill in the art will be able to determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and a particular composition or selected route of administration.

[0563] An exemplary, non-limiting range for a therapeutically effective amount of a humanized or chimeric antibody of the invention is about 0.001-30 mg / kg, e.g., about 0.001-20 mg / kg, e.g., about 0.001-10 mg / kg, e.g., about 0.001-5 mg / kg, e.g., about 0.001-2 mg / kg, e.g., about 0.001-1 mg / kg, e.g., 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.

[0564] Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and, for example, administered in the vicinity of the target site.

[0565] The dosage regimen in the above-mentioned treatment methods and uses is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be given, several divided doses may be given over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0566] In one embodiment, during treatment, eg, at predetermined time points, the efficacy of the treatment is monitored.

[0567] If desired, the effective daily dose of the pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In another embodiment, the humanized or chimeric antibody or pharmaceutical composition is administered by slow continuous infusion over a long period of time, for example, more than 24 hours, to minimize unwanted side effects.

[0568] While it is possible to administer the humanized or chimeric antibody of the present invention alone, it is preferred to administer the humanized or chimeric antibody as a pharmaceutical composition as described above.

[0569] An effective dose of the humanized or chimeric antibody of the present invention may also be administered using a weekly, biweekly, or triweekly dosing period. The dosing period may be limited to, for example, 8 weeks, 12 weeks, or until clinical progression is determined. Alternatively, an effective dose of the humanized or chimeric antibody of the present invention may be administered every other week, two weeks, or three weeks.

[0570] In one embodiment, the humanized or chimeric antibody may be administered by infusion at a dose of mg / m 2 The calculated weekly dose is given. Such dosage can be, for example, based on the mg / kg dosage provided above, according to the following: dosage (mg / kg) x70: 1.8. Such administration can be repeated for example 1-8 times, for example 3-5 times. Administration can be carried out for a period of time of 2-24 hours, for example 2-12 hours by continuous infusion. In one embodiment, humanized or chimeric antibodies can be given for a long time by slow continuous infusion, for example more than 24 hours, to reduce toxic side effects.

[0571] In one embodiment, when administered once a week, the humanized or chimeric antibody can be administered up to 8 times, e.g., 4-6 times, as a weekly dose calculated as a fixed dose. Such a regimen can be repeated one or more times as needed, e.g., after 6 or 12 months. Such a fixed dose can be, for example, based on the mg / kg dose provided above, with a body weight estimate of 70 kg. The dose can be determined or adjusted by measuring the amount of the humanized or chimeric antibody of the present invention in the blood after administration, e.g., by obtaining a biological sample and using an anti-idiotypic antibody targeting the binding region of the humanized or chimeric antibody of the present invention.

[0572] In one embodiment, the humanized or chimeric antibody can be administered by maintenance therapy, for example once a week for a period of 6 months or more.

[0573] Humanized or chimeric antibodies can also be administered prophylactically 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.

[0574] For ease of administration and uniformity of dosage, parenteral compositions may be formulated in dosage unit form. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such an active compound for treatment of individual sensitivities.

[0575] Humanized or chimeric antibodies can also be given prophylactically 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 may be particularly useful in patients where it is difficult to locate a tumor known to exist due to other biological factors.

[0576] Diagnostic Applications

[0577] 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. Thus, 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 a disease, as well as for monitoring the progress of therapeutic treatments, monitoring disease progression, evaluating status after treatment, monitoring recurrence of a disease, evaluating the risk of developing a disease, etc.

[0578] In one aspect, the invention relates to a method for diagnosing a disease characterized by involvement or accumulation of CD3-expressing cells, comprising administering to a subject a humanized or chimeric antibody of the invention, a composition of the invention, or a pharmaceutical composition of the invention, optionally wherein the humanized or chimeric antibody is labeled with a detectable agent.

[0579] In one aspect, the humanized or chimeric antibodies of the present invention are used in vitro, for example, for diagnosing a cell indicating a disease or a disease involving pathogenesis in which a specific target of interest is expressed and is bound by a humanized or chimeric antibody by detecting the level of a target in a sample obtained from a patient or by expressing the target of interest on its cell surface. This can be achieved, for example, by contacting a humanized or chimeric antibody of the present invention under conditions that allow the antibody to bind to the target, optionally together with a control sample, under conditions that allow the antibody to bind to the target. Complex formation can then be detected (e.g., using ELISA). When a control sample is used together with a test sample, the level of a humanized or chimeric antibody or antibody-target complex is analyzed in two samples, and a statistically significant higher level of a humanized or chimeric antibody or antibody-target complex in the test sample indicates a higher level of target in the test sample compared to the control sample.

[0580] Examples of conventional immunoassays in which the humanized or chimeric antibodies of the invention can be used include, without limitation, ELISA, RIA, FACS analysis, plasmon resonance analysis, chromatography, tissue immunohistochemistry, Western blotting, and / or immunoprecipitation.

[0581] Thus, in one embodiment, the invention relates to a method of diagnosing a disease characterized by involvement or accumulation of CD3-expressing cells, comprising administering to a subject the antibody, bispecific antibody, composition or pharmaceutical composition of any aspect or embodiment described herein, optionally wherein the antibody is labeled with a detectable label.

[0582] In one embodiment, the present invention relates to a method for detecting the presence of a target or a cell expressing a target in a sample, comprising:

[0583] - contacting the sample with a humanized or chimeric antibody of the present invention under conditions that allow the humanized or chimeric antibody to bind to a target in the sample; and

[0584] - Analyzing whether a complex is formed. Typically, the sample is a biological sample.

[0585] In one embodiment, the sample is a tissue sample known or suspected to contain a specific target and / or a cell expressing the target. For example, in situ detection of target expression can be achieved by removing a histological sample from a patient and providing a humanized or chimeric antibody of the present invention to such a sample. Humanized or chimeric antibodies can be provided by applying or coating humanized or chimeric antibodies to a sample, and then detecting it using a suitable tool. It is then possible to detect not only the presence of a target or a cell expressing the target, but also the distribution of a target or a cell expressing the target in the examined tissue (for example, in the case of evaluating the spread of cancer cells). Using the present invention, it is readily understood by those of ordinary skill that any of the various histological methods (for example, a staining procedure) can be modified to achieve such in situ detection.

[0586] In the above-mentioned assay, humanized or chimeric antibodies can be labeled with detectable substances to allow the antibody to be detected. Alternatively, the (first) specific humanized or chimeric antibody of the combination can be detected by labeling with a detectable substance and combining the antibody detection of the first specific humanized or chimeric antibody. In addition, in the above-mentioned assay, a diagnostic composition comprising the antibody or bispecific antibody of any aspect or embodiment described herein can be used. Therefore, in one aspect, the present invention relates to a diagnostic composition comprising the antibody or bispecific antibody of any aspect or embodiment described herein.

[0587] The target level in the sample can also be evaluated by competitive immunoassay using a target standard labeled with a detectable substance and an unlabeled target-specific humanized or chimeric antibody. In this type of assay, a biological sample, a labeled target standard, and a target-specific humanized or chimeric antibody are combined, and the amount of the labeled target standard bound to the unlabeled target-specific humanized or chimeric antibody is determined. The amount of the target in the biological sample is inversely proportional to the amount of the labeled target standard bound to the target-specific humanized or chimeric antibody.

[0588] Suitable labels for target-specific humanized or chimeric antibodies, secondary antibodies and / or target standards for in vitro diagnostic techniques include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase and acetylcholinesterase; Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; Examples of luminescent materials include luminol; and Examples of suitable radioactive materials include 125 I. 131 I. 35 S and 3 H.

[0589] In one aspect, the target-specific humanized or chimeric antibodies of the invention are used for in vivo imaging of tissues expressing the target, such as tumors.For in vivo methods, antibody fragments, such as (Fab')2, Fab and Fab' fragments, are particularly advantageous because of their rapid distribution kinetics.

[0590] In vivo imaging can be performed by any suitable technique. 99 Tc, 131 I. 111 In or other gamma-emitting isotope-labeled target-specific humanized or chimeric antibodies (e.g., antibodies or fragments) can be used to image target-specific antibody accumulation or distribution in tissues expressing the target, such as tumors, using a gamma scintillation camera (e.g., Elscint Apex 409 ECT device), typically using a low-energy high-resolution collimator or a low-energy universal collimator. Alternatively, using 89 Zr, 76 Br, 18F or other positron-emitting radionuclide labels can be used in tumors to image the distribution of target-specific humanized or chimeric antibodies or antibody fragments using positron emission tomography (PET). Images obtained using such techniques can be used to evaluate the biodistribution of the target in a patient, mammal or tissue, for example, in the case of using the target as a biomarker for the presence of cancer / tumor cells. Variations on this technique can include the use of magnetic resonance imaging (MRI) to improve imaging via gamma camera technology. Conventional immunoscintigraphy methods and principles are described, for example, in

[79] ,

[80] and

[81] . In addition, such images can also or alternatively be used as the basis for surgical techniques to remove tumors. In addition, such in vivo imaging techniques can allow identification and localization of tumors in cases where a patient is identified as having a tumor (due to the presence of other biomarkers, metastases, etc.) but the tumor cannot be identified by traditional analytical techniques. All of these methods are features of the present invention.

[0591] The in vivo imaging and other diagnostic methods provided by the present invention are particularly useful for detecting micrometastases in human patients (eg, patients not previously diagnosed with cancer or patients in recovery / remission from cancer).

[0592] In one embodiment, the present invention provides an in vivo imaging method, wherein the target-specific humanized or chimeric antibody of the present invention is conjugated to a radiopaque agent that facilitates detection, the conjugated humanized or chimeric antibody is administered to a host, such as by injection into the bloodstream, and the presence and location of the labeled humanized or chimeric antibody in the host is analyzed. Through this technology and any other diagnostic methods provided herein, the present invention provides methods for screening for the presence of disease-associated cells in a biological sample obtained from a human patient or from a human patient and / or for evaluating the distribution of target-specific humanized or chimeric antibodies prior to target-specific ADC therapy.

[0593] For diagnostic imaging, the radioisotope can be conjugated to the target-specific humanized or chimeric antibody either directly or indirectly through the use of an intermediate functional group. Useful intermediate functional groups include chelators such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, e.g.,

[82] ).

[0594] In addition to radioisotopes and radiopaque agents, diagnostic methods can be performed using target-specific antibodies conjugated to dyes (e.g., biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancers (e.g., paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g.,

[83] , which describes MRI techniques and the preparation of antibodies conjugated to MRI enhancers). Such diagnostic / detection agents can be selected from agents for MRI and fluorescent compounds. In order to load the 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 to bind the ions. Such a tail can be a polymer such as polylysine, a polysaccharide, or another derivatized or derivatized chain with pendant groups to which the chelating groups can be attached, such as porphyrins, polyamines, crown ethers, dithiosemicarbazones, polyoximes, etc., which are known for this purpose. The chelating agent can be coupled to the target-specific humanized or chimeric antibody using standard chemistry.

[0595] Thus, 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., for magnetic resonance imaging, computed tomography or ultrasound contrast enhancement) or a radionuclide (which may be, for example, a γ-, β-, α-, Auger electron- or positron-emitting isotope).

[0596] In one aspect, the invention relates to a diagnostic composition comprising an antibody or a bispecific antibody of the invention.

[0597] In a further aspect, the present invention relates to a kit for detecting the presence of a target antigen or a cell expressing the target in a sample, comprising:

[0598] - a target-specific humanized or chimeric antibody of the invention; and

[0599] - Instructions for use of the kit.

[0600] Therefore, 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:

[0601] a) contacting the sample with the antibody or bispecific antibody of the invention under conditions that allow formation of a complex between the antibody or bispecific antibody and CD3; and

[0602] b) Analyze whether a complex is formed.

[0603] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container comprising 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. Reagents may include, for example, fluorescent labels, enzyme labels or other detectable labels. Reagents may also include a second or third antibody or a reagent for an enzyme reaction, wherein the enzyme reaction produces a product that can be visualized. In one embodiment, the present invention provides a diagnostic kit, which is included in a suitable container in a labeled or unlabeled form of one or more target-specific humanized or chimeric antibodies of the present invention, a reagent for indirect assay incubation, and a substrate or derivatization reagent (depending on the nature of the label) for detection in such an assay. Control reagents and instructions for use may also be included.

[0604] Diagnostic kits for use with target-specific humanized or chimeric antibodies, such as labeled target-specific antibodies, can also be provided for detecting the presence of a target in a tissue sample or a host. In such diagnostic kits and in kits for therapeutic use described elsewhere herein, target-specific humanized or chimeric antibodies can generally be provided in a lyophilized form in a container alone or in combination with another antibody specific for a target cell or peptide. Typically, a pharmaceutically acceptable carrier (e.g., an inert diluent) and / or its components, such as Tris, phosphate or carbonate buffer, stabilizers, preservatives, biocides, inert proteins such as serum albumin, etc. (usually in separate containers for mixing) and additional reagents (also typically in separate containers) can also be included. In some kits, a second antibody capable of binding to a target-specific humanized or chimeric antibody is also included, which is typically present in a separate container. The second antibody is typically conjugated to a label and formulated in a manner similar to the target-specific humanized or chimeric antibodies of the present invention. Using the methods described above and elsewhere herein, target-specific humanized or chimeric antibodies can be used to define subsets of cancer / tumor cells and characterize such cells and associated tumor tissue.

[0605] Anti-idiotypic antibodies

[0606] In a further aspect, the invention relates to anti-idiotypic antibodies in combination with the humanized or chimeric antibodies of the invention as described herein.

[0607] Anti-idiotypic (Id) antibodies are antibodies that recognize unique determinants that are usually associated with the antigen binding site of an antibody. Anti-Id antibodies can be prepared by immunizing an animal of the same species and genotype as the source of the CD3 monoclonal antibody with a monoclonal antibody for which the anti-Id is prepared. The immunized animal can usually recognize and respond to the idiotypic determinants of the immunizing antibody by producing antibodies (anti-Id antibodies) to these idiotypic determinants. Such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of the present invention.

[0608] The anti-Id antibody can also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id antibody can be epitopically identical to the original monoclonal antibody that induced the anti-Id antibody. Thus, by using antibodies directed against the idiotypic determinants of the monoclonal antibody, it is possible to identify other clones expressing antibodies of the same specificity. The anti-Id antibody can be altered (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein for the CD3-specific antibodies of the invention. For example, the monoclonal anti-Id antibody can be fused to a carrier such as a keyhole limpet. Hemocyanin (KLH) is coupled and used to immunize BALB / c mice. Sera from these mice typically contain anti-anti-Id antibodies with binding properties similar to (if not identical to) the original / parental CD3 antibody.

[0609] sequence

[0610] Table 1

[0611]

[0612]

[0613]

[0614]

[0615]

[0616] The CDR regions were annotated according to the IMGT definition. Example

[0617] Example 1 - Generation of humanized CD3 antibodies and non-activating antibody variants

[0618] Humanization of CD3 Antibody

[0619] The humanization of the mouse CD3 antibody (US 8,236,308, described herein as IgG1-CD3) was performed by Antitope (Cambridge, UK) using its improved form of germline humanization (CDR-grafting) technology (EP 0 629 240). Using this technology, 4 different VH chains (SEQ ID NOs: 6, 7, 8 and 9) and 3 different VL chains (SEQ ID NOs: 10, 11 and 12) were designed. By combining these 4 VH with 3 VL chains, 12 different antibodies were produced. The humanized variants are described herein as huCD3. Therefore, the humanized variants of the present invention comprising VH and VL are described as, for example, IgG1-huCD3-H1L1, which means that the specific variant has an IgG1 isotype, is a humanized CD3 and comprises a VH amino acid sequence called "H1" and defined according to SEQ ID NO: 6 and a VL amino acid sequence called "L1" and defined according to SEQ ID NO: 10. Thus, H1 refers to the variable heavy chain region VH1, L1 refers to the variable light chain region VL1, and so on.

[0620] 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 VL3 sequence shown in SEQ ID NO: 12), and IgG1-huCD3-H4L2 (humanized CD3 comprising the VH3 sequence shown in SEQ ID NO: 8 and the VL3 sequence shown in SEQ ID NO: 12). : The present invention relates to a humanized CD3 comprising a VH3 sequence shown in SEQ ID NO:8 and a humanized CD3 comprising a VL1 sequence shown in SEQ ID NO:10), IgG1-huCD3-H3L3 (humanized CD3 comprising a VH3 sequence shown in SEQ ID NO:8 and a VL3 sequence shown in SEQ ID NO:12), and IgG1-huCD3-H4L3 (humanized CD3 comprising a VH4 sequence shown in SEQ ID NO:9 and a VL3 sequence shown in SEQ ID NO:12), and tested in the embodiments described herein.

[0621] In some embodiments, antibodies comprising the heavy and light chain variable region sequences of huCLB-T3 / 4 (SEQ ID NO: 17 and 18, respectively) are used as control antibodies (Labrijn et al., PNAS 2013, 110: 5145-50) and to verify different non-activating mutation combinations 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 NO: 17 and 18) are cloned into the relevant pcDNA3.3 (Invitrogen) expression vector and expressed in HEK293F cells by co-transfection. The resulting control antibody is described as IgG1-huCLB-T3 / 4.

[0622] In some embodiments, an antibody comprising the heavy and light chain variable region sequences of 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 context of a positive control, it is referred to as "IgG1-CD20".

[0623] These IgG1-CD3 (ie, chimeric parent CD3 antibody), IgG1-huCD3, and IgG1-huCLB-T3 / 4 antibodies were used in monospecific and bispecific formats, with bispecific antibodies being generated as described below.

[0624] HER2 Antibodies

[0625] In certain embodiments, antibodies against HER2 are used. The VH and VL sequences (antibody 169, SEQ ID NO: 19 and 20, respectively) for the HER2-specific antibodies are described previously (WO2012 / 143524 [Genmab]; Labrijn et al., PNAS 2013, 110: 5145-50). The antibody is used in both monospecific and bispecific forms, and is referred to as "IgG1-HER2".

[0626] b12 antibody

[0627] In some embodiments, antibody b12, a gp120-specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5;230(3):812-23.) was used as a negative control and is referred to as "IgG1-b12".

[0628] Express

[0629] Antibodies were expressed as IgG1, κ or IgG1, λ, with or without the non-activating mutations described below and with mutations in the CH3 domain that enabled the generation of bispecific antibodies by the methods described below: IgG1-HER2-K409R, IgG1-b12-K409R, IgG1-CD3-F405L. A mixture of plasmid DNA encoding both the heavy and light chains of the antibodies was transiently transfected into Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer.

[0630] Antibody purification

[0631] Culture supernatants were filtered through a 0.2 µm blank filter loaded onto a 5 mL MabSelect SuRe column (GE Health Care) and eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluate was immediately neutralized with 2M Tris-HCl, pH 9, and dialyzed overnight into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun). Alternatively, 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 dialysis or buffer exchange, the samples were sterile filtered through a 0.2 µm blank filter. Purity was determined by SDS-PAGE, and concentration was measured by absorbance at 280 nm. Purified antibodies were stored at 2-8°C.

[0632] Generation of bispecific antibodies

[0633] Bispecific antibodies were generated in vitro using DuoBody® platform technology, i.e. 2-MEA-induced Fab-arm exchange, 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). In order to be able to generate bispecific antibodies by this method, IgG1 molecules with single mutations in the CH3 domain were generated: F405L mutation in one parent IgG1 antibody (i.e. IgG1-CD3 antibody) and K409R mutation in the other parent IgG1 antibody (i.e. HER2 or b12 antibody). To generate bispecific antibodies, the two parent 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 hours. The reduction reaction was terminated 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 performed in 3.5 mL PBS. Samples were collected in an Amicon Ultra centrifugal device (30 kD MWCO, Millipore, product#UFC803096) and centrifuged at 3000x g Concentrate by centrifugation for 8 min. Adjust volume to 500 µL with PBS (when needed) and sterile filter the sample through a 0.2 µm filter (Millex-GV, Catalog No. SLGV004SL). Store the bispecific product at 2-8 °C.

[0634] In an alternative approach to obtain the same bispecific antibody, to generate the bispecific antibody, 100 µg of the two parent antibodies were mixed and incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 400 µL PBS (B. Braun, product number 3623140) at 31°C for 5 hours. The mixture was centrifuged using an Amicon Ultra 0.5 ml centrifuge device (10 kDMWCO, Millipore, product number UFC501096) and centrifuged with 400 µl PBS at 13000x gThe reduction reaction was terminated by washing 4 times by centrifugation for 10 min to remove the reducing agent 2-MEA. The sample was collected in a new tube by inverting the filter and centrifuging at 1000 g for 2 min. The volume was adjusted to 200 µL with PBS (when necessary). The absorbance at 280 nm (A280) of the bispecific product was measured to determine the final concentration. HPLC cation exchange chromatography (HPLC-CEX) (described in WO 2013 / 060867) was performed to determine the amount of bispecific product. The samples were stored at 2-8 °C.

[0635] The resulting bispecific antibodies are described below as "K409R IgG1 backbone" and "F405L IgG1 backbone".

[0636] Non-activating mutations

[0637] Several antibody variants were produced with one or more amino acid replacements in the Fc region. Non-activated Fc regions prevent antibodies from interacting with Fc-receptors present on hemocytes (e.g., monocytes), or prevent antibodies from interacting with C1q to activate the classical complement pathway. The reduction of Fc activity was tested in antibody variants comprising different combinations of amino acid replacements in the Fc region. Up to 5 amino acid replacements were introduced, including mutations N297Q, L234A, L235A, L234F, L235E, D265A, and P331S. Replacements in one or more of these 5 amino acid positions were introduced into K409R and / or F405L IgG1 skeletons. The following Fc region variants of huCLB-T3 / 4 antibodies were generated: N297Q (referring to N297Q substitution, referred to as IgG1-huCLB-T3 / 4-N297Q), LFLE (referring to L234F / L235E substitution, referred to as IgG1-huCLB-T3 / 4-LFLE), LALA (referring to L234A / L235A substitution, referred to as IgG1-huCLB-T3 / 4-LALA), LFLENQ (referring to L234F / L235E / N297Q substitution, referred to as IgG1-huCLB-T3 / 4-LFLENQ), LFLEDA (referring to L234F / L235E / D265A substitution, referred to as IgG1-huCLB-T3 / 4-LFLEDA), DA (referring to D265A substitution, referred to as IgG1-huCLB-T3 / 4-DA), DAPS (referring to D265A / P331S substitution, called IgG1-huCLB-T3 / 4-DAPS), DANQ (referring to D265A / N297Q substitution, called IgG1-huCLB-T3 / 4-DANQ), LFLEPS (referring to L234F / L235E / P331S substitution, called IgG1-huCLB-T3 / 4-LFLEPS) and LFLEDANQPS (referring to L234F / L235E / D265A / N297Q / P331S substitution, called IgG1-huCLB-T3 / 4-LFLEDANQPS).

[0638] Specifically, in the IgG1-huCD3 antibody variant, a combination of 3 amino acid substitutions, including mutations L234F, L235E and D265A and termed LFLEDA, were introduced into the K409R and F405L IgG1 backbone to generate an antibody with a non-activated Fc region. The resulting non-activated antibody variant was named with the suffix "-LFLEDA".

[0639] Example 2 - Binding of humanized CD3 antibodies and their non-activating variants to human and cynomolgus monkey T cell lines expressing CD3

[0640] Purified variants of humanized CD3 (huCD3) antibodies and bispecific (bs) IgG1-huCD3 x HER2 molecules (see Example 1), with or without LFLEDA mutations in the Fc region, were incubated with the human T cell line Jurkat (Clone E6-1, ATCC ® Binding to TIB-152™, LGC Standards GmbH, Wesel, Germany) or the cynomolgus monkey T cell line HSC-F (cat. no. JCRB1164; Health Science Research Resources Bank, Osaka, Japan) was analyzed by FACS analysis. In addition to the non-activating mutant LFLEDA, the antibody variants contained F405L or K409R mutations as described in Example 1.

[0641] Cells (1x10 5 Cells / well) were incubated in polystyrene 96-well round-bottom plates (Greiner bio-one 650101) with serially diluted antibody preparations (range 5-10,000 ng / mL, in 3-fold dilutions) in 100 µL PBS / 0.1% BSA / 0.02% azide at 4°C for 30 min.

[0642] After washing twice with PBS / 0.1% BSA / 0.02% azide, cells were incubated with secondary antibody in 100 μL at 4°C for 30 min. As secondary antibody, R-phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab')2 (109-116-098, Jackson ImmunoResearchLaboratories, Inc., West Grove, PA) diluted 1 / 100 in PBS / 0.1% BSA / 0.02% azide was used for all experiments. Next, cells were washed twice in PBS / 0.1% BSA / 0.02% azide, resuspended in 150 μL PBS / 0.1% BSA / 0.02% azide and analyzed on a FACS Cantoll (BD Biosciences). Binding curves were analyzed using nonlinear regression (sigmoidal dose response with variable slope) using GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA).

[0643] Figure 1A shows the binding of IgG1λ-huCD3 variants IgG1-huCD3-H1L1 (SEQ ID NOs: 6 and 10, respectively), IgG1-huCD3-H1L2 (SEQ ID NOs: 6 and 11, respectively), IgG1-huCD3-H1L3 (SEQ ID NOs: 6 and 12, respectively), IgG1-huCD3-H3L3 (SEQ ID NOs: 8 and 12, respectively), and IgG1-huCD3-H4L1 (SEQ ID NOs: 9 and 10, respectively) with wild-type Fc regions to Jurkat cells. IgG1-huCLB-T3 / 4, included as a positive control, bound more strongly to Jurkat cells than the IgG1-huCD3 variants, as observed for all variants, and IgG1-CD3-LFLEDA (the parent CD3 antibody described in Example 1, with a non-activating LFLEDA mutation) and IgG1-huCD3-H3L1-LFLEDA with a non-activating LFLEDA mutation. For the negative control antibody IgG1-b12, no binding was observed.

[0644] Figure 6 A shows that IgG1-CD3-LFLEDA with non-activating LFLEDA mutations (parent CD3 antibody described in Example 1, with non-activating LFLEDA mutations), 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 bind to Jurkat cells similar to the binding ability of huCD3 variants with wild-type Fc regions. IgG1-huCLB-T3 / 4, included as a positive control, binds to Jurkat cells more strongly than the IgG1-huCD3 variants at low antibody concentrations, but is similar at higher antibody concentrations. Overall, the humanized CD3 variants retain the ability to bind to CD3 as IgG1-CD3 antibodies. No binding was observed for the negative control antibody IgG1-b12.

[0645] Figure 1B 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 ​​of these bispecific antibodies are higher than those of the monospecific antibodies. The EC50 concentrations of the bispecific antibodies are 6-10 times. Again, no binding was observed for the negative control antibody IgG1-b12.

[0646] Figure 6 B shows that 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 ​​of these bispecific antibodies are higher than those of monospecific antibodies. The EC50 concentration of bispecific antibodies is 4-10 times. Monovalent binding allows more antibodies to aggregate on the cell surface, therefore, allowing higher binding values ​​of bispecific antibodies. Again, no binding was observed for the negative control antibody IgG1-b12.

[0647] Figure 2 A shows that the IgG1-huCD3 variants IgG1-huCD3-H1L1, IgG1-huCD3-H1L2, IgG1-huCD3-H1L3, IgG1-huCD3-H3L3 and IgG1-huCD3-H4L1, and IgG1-CD3-LFLEDA, IgG1-huCD3-H3L1-LFLEDA with wild-type Fc regions bind similarly to the cynomolgus T cell line HSC-F. No binding was observed for the control antibody huCLB-T3 / 4, which does not cross-react with cynomolgus CD3, and the negative control antibody IgG1-b12.

[0648] Figure 7A shows that the binding of the 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 the cynomolgus monkey T cell line HSC-F is similar. No binding was observed for the negative control antibody IgG1-b12.

[0649] 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 ​​of these bispecific antibodies are higher than those of the monospecific anti-CD3 variants. The EC50 concentrations of the bispecific antibodies are 10-12 times that of the monospecific anti-CD3 antibodies. Again, no binding was observed for the negative control antibody IgG1-b12.

[0650] 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 ​​of these bispecific antibodies are higher than those of the monospecific anti-CD3 variants. The EC50 concentration of the bispecific antibodies is 3-6 times that of the monospecific anti-huCD3 antibodies. Again, no binding was observed for the negative control antibody IgG1-b12.

[0651] Example 3 - T cell activation by humanized CD3 antibody variants

[0652] CD69 expression is an early marker of T cell activation. CD3 antibodies can mediate crosslinking of T cells and immune cells through the binding of CD3 expressed by T cells and Fc receptors expressed by immune cells via the Fc region of the antibody, such as IgG1 Fc region. This can lead to T cell activation and induction of CD69. Antibody variants (LFLEDA mutations) containing non-activated Fc regions do not bind to Fc receptors. Therefore, it is expected that non-activated CD3 antibodies do not induce T cell activation and CD69 expression, because non-activated Fc regions do not bind to immune cells expressing Fc receptors, and therefore cannot crosslink T cells and immune cells.

[0653] After incubation with humanized CD3 (huCD3) variants containing and without LFLEDA mutations in the Fc region, CD69 expression on T cells was evaluated by FACS analysis to determine early activation of T cells. In addition to the non-activating mutations, the LFLEDA variants also contained F405L or K409R mutations, as described in Example 1.

[0654] PBMCs were isolated from whole blood or buffy coats by density gradient separation using Leucosep tubes (#227290; Greiner Bio-one, Alphen a / d Rijn, The Netherlands), washed with PBS and resuspended in culture medium.

[0655] A dose response series of huCD3 antibody variants, negative controls (IgG1-b12), and positive controls (IgE-huCD3 and parental IgG1-CD3) were prepared in culture medium (range 0.1-1,000 ng / mL, in 10-fold dilutions) and added to each well of a 96-well round-bottom plate containing human or cynomolgus monkey PBMCs. After 16-24 hours of incubation, cells were pelleted by centrifugation, and supernatants (containing cytokines) were collected and stored at -20°C. 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 markers) and mouse-anti-human CD69-APC antibodies (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. Cells were resuspended in 150 μL / well, and CD69 expression on CD28 positive cells was measured on a FACS Canto II (BD Biosciences).

[0656] Figure 3 It was shown that IgG1-CD3 with a wild-type IgG1 Fc region (as described in Example 1) and humanized IgG1-huCD3 variants were expressed in human ( Figure 3 A) and cynomolgus monkeys ( Figure 3 Similar levels of CD69 expression were induced on T cells derived from IgG1- and IgG1-huCD3-H3L1 (LFLEDA) sources. Non-activated (LFLEDA) IgG1-CD3-LFLEDA and IgG1-huCD3-H3L1 variants induced low levels of CD69 expression in human T cells. CD69 expression was not induced in cynomolgus monkey T cells by non-activated IgG1-huCD3 variants. Control antibody IgG1-b12 also did not induce CD69 expression in human or cynomolgus monkey T cells.

[0657] Figure 8 The 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 were shown to induce low levels of CD69 expression in human T cells. Figure 8 A and 8B are shown in inducing CD69 expression on T cells from cynomolgus monkeys. The less activation observed by non-activated variants may be due to the cross-linking of CD3 molecules caused by the bivalent binding of CD3 antibodies. Such an explanation is supported by the following observations: activation is reduced at the highest concentration, when antibody binding is monovalent. Control antibody IgG1-b12 does not induce the expression of CD69 in people or cynomolgus monkey T cells either.

[0658] Figure 8 C and 8D 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 HER2-related chromatin in vitro and in vivo. Figure 8 C) or cynomolgus monkey ( Figure 8 D) Expression of CD69 in T cells. However, some induction of CD69 expression was observed at higher antibody concentrations.

[0659] Example 4 - T-cell proliferation induced by humanized CD3 antibody variants

[0660] 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) according to the manufacturer's instructions.

[0661] Human or cynomolgus monkey PBMCs isolated from whole blood or buffy coat were incubated with IgG1 huCD3 antibody variants in a dilution series (range 0.1-1,000 ng / mL, diluted 10 times) in 96-well culture plates. IgE-CD3 and IgG1-huCLB-T3 / 4 were included as positive controls and IgG1-b12 as negative controls. After 3 days of incubation with antibodies, BrdU (Roche Applied Science, Mannheim, Germany) was added to the culture medium, and the plates were incubated for 5 hours. The cells were then precipitated by centrifugation, and the supernatant was collected and stored at -20°C. The plates were dried and stored at 4°C until ELISA was performed.

[0662] BrdU incorporation in DNA is measured by ELISA according to the manufacturer's instructions (Roche Applied Science). Cells are fixed to the plate, and then the plate is incubated for 90 minutes at room temperature with an anti-BrdU antibody conjugated to peroxidase. The plate is washed with PBST and combined with ABTS buffer (TMB solution provided in place of the test kit). After 30 min, 2% oxalic acid is added to each well to stop the color development. Then OD405 nm is measured on an EL808 ELISA-reader.

[0663] Figure 4 Incubation of PBMCs with parental IgG1-CD3 and humanized IgG1-huCD3 variants with wild-type IgG1 Fc regions induced human ( Figure 4 A) and cynomolgus monkeys ( Figure 4 B) Strong proliferation of T cells, even at very low antibody concentrations. 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 4Thus, 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), the proliferation of human T cells was not induced by these non-activated IgG1-huCD3 variants.

[0664] Fig. 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 Fig. 9 C) or cynomolgus monkey ( Fig. 9 D) Proliferation of isolated T cells.

[0665] Example 5 - In vitro T cell-mediated cytotoxicity induced by humanized CD3 antibody variants

[0666] Tumor-specific T cell cytotoxicity can be mediated by a bispecific antibody that binds to CD3 with one arm and to a tumor-specific target such as HER2 with the other arm. Bispecific antibodies that bind to both T cells and tumor cells simultaneously will result in T cell activation and tumor cell-specific cytotoxicity. In this example, T cell-mediated cytotoxicity against HER2-positive tumor cells is evaluated using a bispecific antibody against CD3 (humanized variant) and HER2.

[0667] Therefore, AU565 (human breast cancer) cells were cultured in RPMI 1640 supplemented with 10% (vol / vol) 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% (vol / vol) CO2 humidified incubator. AU565 cells were cultured to near confluence, after which the cells were trypsinized, resuspended in culture 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% CO 2 for at least 3 hours to allow attachment to the plate.

[0668] People or cynomolgus monkey PBMC are separated from whole blood or buffy coat. The separated PBMC are washed with PBS, resuspended in culture medium and added to AU565 tumor cells in 96-well plates at a ratio of 1:1. The percentage of T cells present in PBMC 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 PBMC group used is generally 50-60%.

[0669] A dilution series (final concentration range 0.001 to 10,000 ng / mL) of the 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 were prepared in culture medium and added to the plate. IgG1-HER2-LFLEDA and IgG1-b12 were included as controls. In addition to the non-activating mutations, LFLEDA antibody variants contained F405L or K409R mutations for preparation in bispecific format (see Example 1). The plates were incubated at 37°C, 5% CO2 for 3 days. The cells were incubated with 1 µM staurosporine (#S6942-200, Sigma) and used as a reference for 100% tumor cell killing. The plates were washed twice with PBS, and 150 μL of culture medium containing 10% Alamar blue was added to each well. The plates were incubated at 37°C, 5% CO2 for 4 hours. The absorbance at 590 nm was measured (Envision, Perkin Elmer, Waltham, MA).

[0670] The bispecific CD3xHER2-LFLEDA antibody variants (bsIgG1-huCLB-T3 / 4xHER2-LFLEDA and bsIgG1-CD3xHER2-LFLEDA) were expressed at low concentrations using human effector cells ( Figure 5 A) or cynomolgus monkey effector cells ( Figure 5B) Induced killing of AU565 cells. The CD3 bispecific control antibody huCLB-T3 / 4xHER2-LFLEDA, which showed no cross-reactivity with cynomolgus monkey CD3, induced killing of AU565 cells only when human PBMCs were used ( Figure 5 A). Thus, no killing of target cells was observed when cynomolgus monkey effector cells were used in this assay ( Figure 5 B) Incubation with monospecific IgG1-b12 or IgG1-HER2-LFLEDA or bsIgG1-CD3xb12-LFLEDA antibodies did not induce non-specific killing of target cells.

[0671] The 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 were expressed at low concentrations using human effector cells ( Fig.10 A) or cynomolgus monkey effector cells ( Fig.10 B) Induced killing of AU565 cells. Incubation with monospecific IgG1-b12 or IgG1-HER2-LFLEDA antibodies did not induce nonspecific target cell killing ( Fig.10 A and B). Thus, humanized CD3 variants comprising a non-activating Fc region did not induce non-specific target cell killing, suggesting that variants comprising a non-activating Fc region can be used to ensure targeted T cell activation and thus avoid non-targeted T cell activation.

[0672] Example 6 - Rhesus monkey T cell activation by humanized CD3 antibody variants

[0673] After incubation with a humanized CD3 (huCD3) antibody variant with a wild-type IgG1 Fc region, CD69 expression on rhesus macaque T cells was evaluated to determine early activation of T cells. Isolation of rhesus macaque PBMCs and evaluation of CD69 expression by flow cytometry was performed as described in Example 3.

[0674] Fig.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 rhesus monkey-derived T cells to a level similar to that of IgG1-CD3 (as described in Example 1). The negative control antibody IgG1-b12 did not induce expression of CD69 in rhesus monkey T cells. Therefore, the huCD3 variants of the present invention can be used in experiments involving rhesus monkey CD3. The huCD3 variants cross-react with rhesus monkey CD3.

[0675] Example 7 - T cell activation by non-activating variants of huCLB-T3 / 4

[0676] After incubation with IgG1-huCLB-T3 / 4 variants with Fc region mutations (see Example 1), CD69 expression on T cells was evaluated by FACS analysis to determine early activation of T cells.

[0677] PBMCs 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 culture medium.

[0678] A dose response series of IgG1-huCLB-T3 / 4 variants, negative controls (IgG1-huCLB-T3 / 4-Fab), and positive controls (IgE-huCLB-T3 / 4) were prepared in culture medium (range 1-1000 ng / mL, in 3-fold dilutions) and added to each well of a 96-well round-bottom plate containing PBMCs. After 16-24 hours of incubation, cells were pelleted by centrifugation, and supernatants (containing cytokines) were collected and stored at -20°C. 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 markers) and mouse-anti-human CD69-APC antibodies (340560; BD Biosciences, Franklin Lakes, NJ). Unbound antibody was removed by washing twice with PBS / 0.1% BSA / 0.02% Azide. Cells were resuspended in 150 µL / well and CD69-expression on CD28-positive cells was measured on a FACS Canto II (BD Biosciences).

[0679] Fig.12 A shows high CD69 expression 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 induced slightly lower CD69 expression levels compared to wild-type IgG1-huCLB-T3 / 4, and incubation with IgG1-huCLB-T3 / 4-LFLE and IgG1-huCLB-T3 / 4-LFLEPS induced CD69 to a lower extent. Incubation of PBMCs with IgG1-CD3 Fab, IgG1-huCLB-T3 / 4-LFLEDA, IgG1-huCLB-T3 / 4-LFLENQ, IgG1-huCLB-T3 / 4-DANQ, and IgG1-huCLB-T3 / 4-LFLEDANQPS antibodies did not induce any expression of CD69 on T cells.

[0680] Fig.12 B shows high CD69 expression on cells incubated with IgE-huCLB-T3 / 4 and IgG1-huCLB-T3 / 4. Incubation with IgG1-huCLB-T3 / 4-LALA induced slightly lower CD69 expression levels compared to wild-type IgG1-huCLB-T3 / 4, and incubation with IgG1-huCLB-T3 / 4-LFLEDA and IgG1-b12 (negative control) did not induce any expression of CD69 on T cells.

[0681] Example 8 - T-cell proliferation by non-activated variants of huCLB-T3 / 4

[0682] The effect of huCLB-T3 / 4 variants (described in Example 1) on T cell proliferation was evaluated by a cell proliferation ELISA kit from Roche Applied Science (Cell Proliferation ELISA, BrdU Kit, #11647229001; Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions.

[0683] PBMC separated from whole blood or buffy coat were incubated with IgG1-CD3 variants of dilution series (range 0.1-1000ng / mL) in 96-well culture plates. IgE-CD3 and IgG1-CD3 were included as positive controls and IgG1-b12 (with K409R mutation for the production of bispecific antibodies) was used as negative controls. After 3 days of incubation with antibodies, BrdU (Roche Applied Science, Mannheim, Germany) was added to the culture medium, and the plate was incubated for 5 hours. The cells were then precipitated by centrifugation, and the supernatant was collected and stored at -20°C. The plate was dried and stored at 4°C until ELISA was performed.

[0684] BrdU incorporation in 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 plate and then incubated for 90 minutes at room temperature (RT) with an anti-BrdU antibody conjugated to peroxidase. The plate was washed with PBST and the binding was determined using ABTS buffer (instead of the TMB solution provided by the kit). Color development was stopped after 30 min by adding 2% oxalic acid to each well. The OD405 nm was then measured on an EL808 ELISA-reader.

[0685] Fig.13 A shows that incubation of PBMC with IgG1-huCLB-T3 / 4, IgG1-huCLB-T3 / 4-DA and IgG1- huCLB-T3 / 4-DAPS induced strong proliferation of T cells, even at very low concentrations of antibodies. Incubation with IgG1-huCLB-T3 / 4-N297Q induced a dose-dependent proliferation that was comparable to the IgE-huCLB-T3 / 4 positive control. Incubation of PBMC 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 did not induce proliferation of T cells.

[0686] Fig.13B shows that incubation of PBMC with IgG1-huCLB-T3 / 4 induced strong 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.

[0687] Based on the results of Examples 7 and 8, a subset of mutants deemed to be the least activated were further analyzed.

[0688] Example 9 - In vitro T cell-mediated cytotoxicity induced by the non-activating antibody variant huCLB-T3 / 4

[0689] AU565 (human breast cancer) cells were cultured in RPMI 1640 supplemented with 10% (vol / vol) 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% (vol / vol) CO2 humidified incubator. AU565 cells were cultured to near confluence. Cells were trypsinized, resuspended in culture 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.

[0690] 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 PBMCs were washed with PBS, resuspended in culture medium and added to the AU565 tumor cells in 96-well plates at a 1:1 ratio. The percentage of T cells present in PBMCs was measured by FACS-analysis using mouse anti-human CD3-PerCP (BD, #345766) antibodies (for staining T cells). The T cell content in the PBMC group used is generally 50-60%.

[0691] A 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, represented as different Fc-variants, wild-type, N297Q, LFLE, LALA, LFLENQ, LFLEDA, DANQ and LFLEDENQPS, were prepared in culture medium and added to the plates. The plates were incubated at 37ºC, 5% CO2 for 3 days. Cells were incubated with 1 µM staurosporine (#S6942-200, Sigma) as a reference for 100% tumor cell killing. After incubation, the supernatant was removed and stored at -20°C. The plates were washed twice with PBS, and 150 μL of culture medium containing 10% Alamar blue was added to each well. The plates were incubated for 4 h at 37°C, 5% CO2. The absorbance was measured at 590 nm (Envision, Perkin Elmer, Waltham, MA).

[0692] Two experiments were performed using PBMC from different donors. In the first experiment, the Fc-variants N297Q, LFLE, LFLENQ, LFLEDA, DANQ and LFLEDANQPS ( Fig.14 In a second experiment, the Fc-variants LFLEDA and LALA ( Fig.15 AC). Antibodies with wild-type Fc-domains were included as reference in both experiments. Incubation with wild-type monospecific IgG1-huCLB-T3 / 4 or bispecific huCLB-T3 / 4xb12 antibodies induced nonspecific killing of target cells ( Fig.14 AG and 15A-C). Monospecific IgG1-huCLB-T3 / 4 and bsIgG1-huCLB-T3 / 4xb12 variant N297Q ( Fig.14 AG) and LALA ( Fig.15 AC) still induced some non-specific target cell killing, although to a lesser extent than the wild-type antibody tested in the same experiment. Non-specific target cell killing was not induced by any of the other tested IgG1-huCLB-T3 / 4 or bsIgG1-huCLB-T3 / 4xb12 antibodies with non-activating mutations ( Fig.14 AG and 15A-C).

[0693] All bispecific huCLB-T3 / 4xHER2 antibodies induced dose-dependent killing of AU565 cells with at least comparable efficacy to the wild-type bispecific huCLB-T3 / 4xHER2 antibody without the non-activating mutations ( Fig.14 AG and 15A-C). Maximum killing occurred at very low concentrations.

[0694] No cytotoxicity was induced by wild-type or non-activated variants of monospecific b12 or HER2 antibodies ( Fig.14 AG and 15A-C), which is as expected.

[0695] Example 10 - Evaluation of Binding of C1q to Non-Activating Antibody Variants of huCLB-T3 / 4

[0696] The interaction of CIq with antibodies bound to target cells is the first step of the classical pathway of complement activation.Since wild-type IgG1 contains the interaction site for CIq, the interaction of CIq with these non-activated IgG1 variants was evaluated by ELISA.

[0697] A dilution series (range 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 above in Example 1 were coated on 96-well Microlon ELISA plates (Greiner, Germany) overnight at 4° C. The plates were washed and blocked with PBS supplemented with 0.025% Tween 20 and 0.1% gelatin. The plates were sequentially incubated with 3% pooled human serum (Sanquin, product#M0008) at 37˚C for 1 h, 100 μL / well rabbit anti-human C1q (DAKO, product# A0136, 1 / 4,000) at room temperature for 1 h, and 100 μL / well pig anti-rabbit IgG-HRP (DAKO, P0399, 1:10,000) as detection antibody at room temperature for 1 h, with washing between incubations. Detection was performed by adding 1 mg / mL 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany) for approximately 30 min. The reaction was stopped by adding 100 μL 2% oxalic acid. The absorbance at 405 nm was measured in a microplate reader (Biotek, Winooski, VT). Log-transformed data were analyzed by fitting sigmoidal dose-response curves with variable slopes using GraphPad Prism software.

[0698] Fig.16A shows that antibodies IgG1-CD20 and IgG1-huCLB-T3 / 4 with wild-type IgG1 Fc regions show C1q binding. No C1q binding was detected in all evaluated antibody variants with non-activating mutations (N297Q, LFLE, LFLENQ, LFLEDA, DA,DAPS, DANQ, LFLEPS, LFLEDANQPS, LALA).

[0699] Fig.16 B shows that the antibody bsIgG1-huCLB-T3 / 4xHER2 with a wild-type IgG1 Fc region shows C1q binding. No C1q binding was detected in all evaluated antibody variants with non-activating mutations (N297Q, LFLE, LFLENQ, LFLEDA, DA,DAPS, DANQ, LFLEPS, LFLEDANQPS, LALA).

[0700] Fig.16 C and Fig.16 D shows that antibodies IgG1-CD20, IgG1-huCLB-T3 / 4 and bsIgG1-huCLB-T3 / 4xHER2 with wild-type IgG1 Fc regions show C1q binding. No binding of C1q was detected in antibody variants with non-activating mutations (LFLEDA and LALA).

[0701] Example 11 - Pharmacokinetic (PK) analysis of non-activated antibody variants

[0702] The mice in this study were housed in an isolation unit at the Central Laboratory Animal Facility (Utrecht, The Netherlands) and maintained in overhead cleanroom cages with free access to water and food. All experiments were approved by the Animal Ethics Committee of Utrecht University. 7-10 week old CB-17 SCID mice (CB-17 / Icr-Prkdc <scid> / IcrIcoCrl, Charles-River) were injected intravenously with 100 μg of wild-type antibody (IgG1-huCLB-T3 / 4, IgG1-HER2, or bsIgG-huCLB-T3 / 4xHER2) or its non-activated variants (LALA, LFLEDA, LFLENQ, DANQ, or LFLEDANQPS), using 3 mice / group. 50 μL blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days, and 21 days after antibody administration. Blood was collected into heparin-containing vials and lysed at 10,000 x g Centrifuge for 5 minutes and store the plasma at –20°C until the antibody concentration is determined.

[0703] Human IgG concentration was determined using a total hIgG sandwich ELISA. For this assay, mouse mAb anti-human IgG-kappa clone MH16 (#M1268, CLB Sanquin, The Netherlands), coated at a concentration of 2 μg / mL on 96-well Microlon ELISA plates (Greiner, Germany), was used as the capture antibody. After blocking the plates with PBS supplemented with 0.2% bovine serum albumin, samples serially diluted with ELISA buffer (PBS supplemented with 0.05% Tween 20 and 0.2% bovine serum albumin) were added and incubated on a plate shaker at room temperature (RT) for 1 h. The plates were 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 terminated after 30 min by adding 2% oxalic acid to each well. The absorbance at 405 nm was measured in a microplate reader (Biotek, Winooski, VT).

[0704] Plasma clearance (mL / day / kg) was calculated based on the area under the curve (AUC) according to the following equation:

[0705]

[0706] Data analysis was performed using Graphpad Prism software.

[0707] Fig.17 A shows that when compared to the wild-type antibody, for the antibody variants N297Q, DANQ, LFLENQ and LFLEDANQPS, the plasma human IgG concentration is lower, indicating a more rapid clearance. For the antibody variants LFLEDA and LALA, the human IgG concentration in plasma is similar to the wild-type antibody.

[0708] Fig.17 B shows that the plasma clearance of antibody variants N297Q, DANQ and LFLENQ is 2-3 times that of wild-type antibody. The clearance of antibody variant LFLEDANQPS is 3-5 times that of wild-type antibody. The plasma clearance of antibody variants LFLEDA and LALA is similar to that of wild-type antibody.

[0709] Example 12 - In vitro immunogenicity evaluation of IgG1-LFLEDA backbone

[0710] To determine the potential for clinical immunogenicity of the IgG1-LFLEDA-K409R backbone, Antitope's EpiScreen™ platform was applied to IgG1-HER2-LFLEDA. Briefly, PBMCs were isolated from a group of 50 HLA-type healthy donors representing European and North American populations. After CD8+ T cell depletion, the PBMC populations were frozen and stored individually. The thawed PBMCs were then cultured and incubated for 5-8 days with IgG1-HER2-LFLEDA-K409R or one of the control samples (IgG1-HER2 or IgG1-HER2-LFLE-K409R). The ability of the samples to induce CD4+ T cell responses was measured 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.

[0711] EpiScreen™ analysis showed that for IgG1-HER2-LFLEDA, 4 donors (8%) showed positive CD4+ T cell responses, which was comparable to 4 (8%) and 3 (6%) positive responses for IgG1-HER2 and IgG1-HER2-LFLE, respectively ( Fig.18 ). Therefore, IgG1-HER2-LFLEDA-K409R (as well as IgG1-HER2 and IgG1-HER2-LFLE-K409R) showed low immunogenicity potential, with reaction frequencies below 10%. The positive control humanized A33 (e.g.

[84] ) was used as a clinical benchmark control antibody, which showed high levels of immunogenicity in the clinic and typically induced 20-30% T cell responses in the EpiScreen assay.

[0712] Example 13 - Generation of mutants to optimize production of humanized CD3 antibodies

[0713] Generation of huCD3-L1 mutant plasmids

[0714] Several IgG1-huCD3-L1 LC mutants were generated to improve the expression level of IgG1-huCD3-H1L1 in transient transfection assays, see Table 2. The selection of residues was based on comparison with germline sequences or screening for the presence of rare residues in huCD3-L1, and crystal structures from homologous antibodies. The selected sequences were synthesized at GeneArt (Life Technologies, Germany). p33L encodes the constant domain of the human IgLC2 / IgLC3 lambda light chain of SEQ ID NO:31. p33G1f encodes the IgG1m(f) heavy chain constant region of SEQ ID NO:15.

[0715] Table 2

[0716]

[0717] Transient expression in Expi293F cells

[0718] For single antibodies, plasmids encoding heavy chain (HC) and light chain (LC) were transiently transfected into Freestyle Expi293F cells (Life technologies, USA) using ExpiFectamine 293 (Life technologies). A total of 1.5 µg HC encoding plasmid and 1.5 µg LC encoding plasmid (Table 2) were diluted in 150 µL Opti-MEM (Gibco, USA). To prepare the transfection mixture, 8 µL ExpiFectamine 293 was diluted in 150 µL Opti-MEM and incubated at room temperature for 5 minutes. Next, DNA / Opti-MEM and ExpiFectamine 293 / Opti-MEM solutions were mixed, incubated at room temperature for 20 minutes and added to 2.55 mL containing 7.5x10 6 Expi293F cells and 50 U / mL Pen-Strep in Expi293 expression medium. The cells were incubated at 37°C, 8% CO2 and shaking at 200 rpm. To increase expression, 21 hours after transfection, 15 µL of Enhancement Mix 1 and 150 µL of Enhancement Mix 2 were added. The cells were incubated for 4 days and the supernatant was then harvested. The supernatant was centrifuged at 3,000x g The cells were centrifuged and sterilized by filtration through a 0.2 µm filter. IgG expression levels were measured using an anti-human IgG sensor (ForteBio, USA) on an Octet RED (ForteBio, US).

[0719] IgG concentration analysis

[0720] For all mutants listed, IgG expression levels were measured individually or in combination. Fig. 20 The IgG expression levels measured in the supernatant are shown for the expressed antibody panels listed in Table 2. The IgG1-huCD3-H1L1 antibody was expressed to 72 µg / mL. For the IgG1-huCD3-H1L1-LT41K mutant, a 4-fold increase in IgG expression was observed (295 µg / mL). Similar expression levels were observed for the IgG1-huCD3-H1L1-LLKNH mutant, which specifically included the T41K mutation (311 µg / mL). Other mutations in these constructs did not show increased expression when tested alone compared to IgG1-huCD3-H1L1 (IgG1-huCD3-H1L1-LF10L, IgG1-huCD3-H1L1-LK55N, IgG1-huCD3-H1L1-LL97H).

[0721] A second set of mutations that increased expression was observed for the combination of R23A and A35P. Although the IgG1-huCD3-H1L1-LLTGPEAEY version lacking R23A and A35P did not show increased expression (83 µg / mL), the IgG1-huCD3-H1L1-LLAPTGPEAEY containing the additional R23A and A35P mutations did show a 3-fold increase in expression (237 µg / mL). Individually, R23A or A35P did not show increased expression levels (56 and 81 µg / mL, respectively).

[0722] Example 14 - Binding of huCD3-H1L1-LFLEDA variants to Jurkat cells

[0723] The apparent binding affinity of huCD3-H1L1-LFLEDA variants to Jurkat cells was tested. IgG1-CD3-hmAb286 disclosed in US2012038219 was used as a reference antibody.

[0724] Jurkat cells (DSMZ, Brunswick, Germany) were thawed at 37°C and washed once with 30 mL PBS and cultured at 300x g Centrifuge at 4°C for 10 minutes. Resuspend the cell pellet in 25 mL PBS / 0.1% BSA / 0.02% azide to a final concentration of 0.8x10 6 cells / mL. 100 µL of the cell suspension (0.8x10 5 Cells / well) were transferred to polystyrene 96-well round-bottom plates (Greiner Bio-one, Alphen a / d Rijn, The Netherlands) and incubated with serially diluted supernatant preparations (range 3-10,000 ng / mL, in 3-fold dilutions) in PBS / 0.1% BSA / 0.02% azide at 4°C for 30 minutes. After washing three times with PBS / 0.1% BSA / 0.02% azide, Jurkat cells were incubated with secondary antibodies in 50 μL at 4°C for 30 min. As secondary antibodies, R-phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab')2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1 / 200 in PBS / 0.1% BSA / 0.02% azide was used for all experiments. Next, Jurkat cells were washed twice with PBS / 0.1% BSA / 0.02% azide, resuspended in 150 μL PBS / 0.1% BSA / 0.02% azide and analyzed on a FACS Cantoll (BD Biosciences). Binding curves were analyzed using nonlinear regression (sigmoidal dose response with variable slope) using GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA).

[0725] Fig.21 Binding curves are shown, and Table 3 shows the EC of IgG1-huCD3-H1L1-LFLEDA mutants 50 The T41K mutation had no effect on binding affinity, while the K55N and LKNH mutants showed an apparent affinity that was 1 / 5 of that of wild-type IgG1-huCD3-H1L1-LFLEDA. The affinity of the reference antibody IgG1-CD3-hmAb286-LFLEDA was lower than that of IgG1-huCD3-H1L1-LFLEDA and similar to the IgG1-huCD3-H1L1-LFLEDA variant containing the K55N mutation.

[0726] Table 3

[0727] Antibody <![CDATA[EC 50 (μg / mL)]]> IgG1-huCD3-H1L1-LFLEDA 0.096 IgG1-huCD3-H1-LFLEDA-LF10L 0.169 IgG1-huCD3-H1-LFLEDA-LT41K 0.096 IgG1-huCD3-H1-LFLEDA-LK55N 0.531 IgG1-huCD3-H1-LFLEDA-LL97H 0.095 IgG1-huCD3-H1-LFLEDA-LLKNH 0.438 IgG1-CD3-hmAb286-LFLEDA 0.397

[0728] Example 15 - Binding of bispecific huCD3 x HER2 affinity variants to T cells

[0729] Binding of purified affinity variants of bispecific (bs) IgG1-huCD3 x HER2 molecules with or without LFLEDA mutation in the Fc region to human T cells isolated from buffy coats (donor number: N001814268806, purchased from Sanquin, Amsterdam, The Netherlands) was analyzed by FACS analysis. T cells were isolated by StemSep according to the manufacturer's instructions. TM Human T cell enrichment kit was used for isolation (Stem Cell Technologies, Vancouver, Canada). Herceptin-LbH1-mutant was identified from a public database from Bostrom et al. (85, 86) and has a lower affinity for HER2 compared to Herceptin (trastuzumab).

[0730] To evaluate binding, isolated T cells (1x10 5 Cells / well) were incubated in polystyrene 96-well round-bottom plates (Greinerbio-one 650101) with serially diluted antibody preparations (range 6.4-10,000 ng / mL, in 5-fold dilutions) in 100 μL PBS / 0.1% BSA / 0.02% azide at 4°C for 30 minutes. After washing twice with PBS / 0.1% BSA / 0.02% azide, T cells were incubated with secondary antibodies in 50 μL at 4°C for 30 minutes. As secondary antibodies, R-phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab')2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1 / 200 in PBS / 0.1% BSA / 0.02% azide was used for all experiments. Next, T cells were washed twice with PBS / 0.1% BSA / 0.02% Azide, resuspended in 100 μL PBS / 0.1% BSA / 0.02% Azide and analyzed on a FACS Cantoll (BD Biosciences). Binding curves were analyzed using nonlinear regression (sigmoidal dose response with variable slope) using GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA).

[0731] The binding curve results are shown in Fig. 22 , and EC 50 The values ​​are shown in Table 4. As can be seen, the EC 50 The maximum difference between values ​​is 2 times.

[0732] Table 4

[0733] Antibody <![CDATA[EC 50 (μg / mL)]]> bsIgG1-huCD3-H1L1 / Herceptin-LFLEDA ~0.40 bsIgG1-huCD3-H1L1 / Herceptin-LbH1-LFLEDA ~0.28 bsIgG1-huCD3-H1L1 / HER2-LFLEDA 0.26 bsIgG1-huCD3-H1L1-LK55N / Herceptin-LFLEDA ~0.44 bsIgG1-huCD3-H1L1-LK55N / Herceptin-LbH1 -LFLEDA 0.40 bsIgG1-huCD3-H1L1-LK55N / HER2-LFLEDA 0.61 bsIgG1-huCD3-hmAb286 / Herceptin-LFLEDA 0.53 bsIgG1-huCD3-hmAb286 / Herceptin-LbH1 -LFLEDA 0.68 bsIgG1-huCD3-hmAb286 / HER2-LFLEDA 0.46

[0734] Example 16 - In vitro T cell-mediated cytotoxicity induced by huCD3 antibody affinity variants

[0735] In this example, T cell-mediated cytotoxicity for HER2-positive tumor cells was evaluated using bispecific antibodies with different affinities for huCD3 and HER2. The bispecific antibodies were prepared as described above and contained F405L mutations in the CD3 arm and K409R mutations in the HER2 arm. A431 and AU565 (human breast cancer) cells with different HER2 expression levels were used for this assay. The HER2 copy number was ~20,000 for A431 cells and ~1,000,000 for AU565 cells. AU565 cells were cultured in RPMI 1640 (Lonza, Basel Switzerland) supplemented with 10% (vol / vol) iron-containing bovine serum (Life Technologies, Germany), 1.5 g / L sodium bicarbonate (Lonza, Germany), 1 mM sodium pyruvate (Lonza, Germany), 4.5 g / L glucose (Sigma), 50 IU / mL penicillin and 50 μg / mL streptomycin (Lonza, Basel, Switzerland) with HEPES and L-glutamine. A431 (ATCC, epidermoid carcinoma) cells were cultured in RPMI 1640 (Lonza, Basel, Switzerland) supplemented with 10% (vol / vol) iron-containing bovine serum (Life Technologies, Germany), 50 IU / mL penicillin and 50 μg / mL streptomycin (Lonza, Basel, Switzerland) with HEPES and L-glutamine. The cell lines were maintained at 37°C in a 5% (vol / vol) CO2 humidified incubator. AU565 and A431 cells were cultured until near confluence, after which the cells were trypsinized, resuspended in culture medium and passed through a cell strainer to obtain a single cell suspension. AU565 and A431 cells were cultured at 3x10 4 and 1.6x10 4 Cells / well were seeded into 96-well culture plates and incubated at 37°C, 5% CO2 for at least 3 hours to allow attachment to the plates.

[0736] Human T cells were isolated from buffy coats as described in Example 15. Isolated T cells were washed with PBS, resuspended in culture medium (RPMI 1640, 10% serum) and added to AU565 at a 1:3 ratio or to A431 cells at a 1:10 ratio in 96-well plates.

[0737] A dilution series (final concentration range 0.1 to 10,000 ng / mL) of the bispecific antibody variants bsIgG1-huCD3 / Herceptin-LFLEDA, bsIgG1-huCD3 / Herceptin-LbH1—LFLEDA, bsIgG1-huCD3-H1L1 / HER2-LFLEDA, bisIgG1-huCD3-LK55N / Herceptin-LbH1—LFLEDA, bsIgG1-huCD3-LK55N / HER2-LFLEDA, bsIgG1-huCD3-hmAb286 / Herceptin-LFLEDA, bsIgG1-huCD3-hmAb286 / Herceptin-LbH1—LFLEDA, bsIgG1-CD3-hmAb286 / HER2-LFLEDA and bsIgG1-huCD3-H1L1 / b12-LFLEDA were prepared in culture medium and added to the plates. The plates were incubated at 37°C, 5% CO2 for 3 days. The cells were incubated with 1 μM staurosporine (Sigma) as a reference for 100% tumor cell killing. The plates were washed twice with PBS, and 150 μL of culture medium containing 10% Alamarblue (Life Technologies, Germany) was added to each well. The plates were incubated at 37°C, 5% CO2 for 4 hours. The absorbance at 590 nm was measured (Envision, Perkin Elmer, Waltham, MA).

[0738] Figure 23 A431 cells treated with CD3 and HER2 affinity variants are shown ( Figure 23 A) and AU565 cells ( Figure 23 The viability of B).The lower HER2 affinity combined high and low CD3 affinity leads to lower efficacy (shown as open symbols in two figures) in two cell lines.The bispecific antibody (Herceptin and HER2) with similar high affinity to HER2 combined with wild-type huCD3-H1L1, huCD3-LK55N or CD3-hmAb286 CD3 arms show similar efficacy to A431 cells.The affinity change of CD3 arms has no effect on the efficacy of A431 cells.The bispecific antibody (Herceptin and HER2) with similar high affinity to HER2 combined with CD3-hmAb286 CD3 arms, huCD3-LK55N arms and wild-type huCD3-H1L1 arms show similar efficacy to AU565 cells, and for CD3 hmAb286 CD3 arms, relatively higher efficacy is shown.For CD3 arms, lower binding affinity is more favorable in AU565 cells with higher HER2 expression compared with A431 cells.

[0739] Example 17 - In vivo tumor killing of huCD3-H1L1xHER2-LFLEDA and huCD3-H1L1xCD20-LFLEDA in NOD-SCID mice

[0740] The in vivo anti-tumor efficacy of the bispecific CD3xHER2 antibody bsIgG1- huCD3-H1L1xHER2-LFLEDA was evaluated in the subcutaneous NCI-N87 xenograft model. In this model, human unstimulated PBMCs are used as a source of human T cells and co-inoculated with tumor cells, similar to the model described by Brischwein et al. (Mol. Immunol. 43 (2006), 1129–1143). In these experiments, 6-11 week old female NOD-SCID (NOD.CB17-Prkdcscid / NcrCrl) mice were used. Human PBMCs from healthy donors were isolated from buffy coats as described in Example 15. On day 0, 5x10 6 A mixture of PBMCs and NCI-N87 cells of the cells was inoculated subcutaneously into the right flank of each mouse with 200 μL. Within 1 hour of injection, the mice were randomly assigned to 7 different treatment groups (n=4 / donor). Each group was injected intravenously (iv) with a single dose of (bispecific) antibody. The treatment groups are shown in Table 5. Tumor growth was evaluated twice a week using a caliper (PLEXX) until the endpoint tumor volume was 1500 mm 3 , until the tumor showed ulceration or until the end of the study.

[0741] The results are shown in Figure 24 In. Figure 24 As can be seen in A, huCD3-H1L1xHER2-LFLEDA effectively reduced tumor size at all three concentrations tested. Figure 24 B shows tumor size 29 days after tumor inoculation. Tumor formation was significantly inhibited in mice treated with bsIgG1- huCD3-H1L1xHER2-LFLEDA at doses of 0.05 mg / kg, 0.5 mg / kg and 5 mg / kg compared to the PBS control group (p<0.0001, Tukeys multiple comparison test), while treatment with 0.005 mg / kg did not affect tumor formation. Treatment with 5 mg / kg of the control antibody bsIgG1- huCD3-H1L1xb12-LFLEDA did not inhibit tumor formation, while treatment with bsIgG1- b12xHER2-LFLEDA appeared to induce some tumor growth inhibition compared to the PBS control, but this was not significant according to one-way Anova, Tukey's multiple comparison test.

[0742] Table 5

[0743] Group Antibody Dose 1 PBS 2 bsIgG1- huCD3-H1L1xHER2-LFLEDA 0.1 µg (= 0.005 mg / kg) 3 bsIgG1- huCD3-H1L1xHER2-LFLEDA 1 µg (= 0.05 mg / kg) 4 bsIgG1- huCD3-H1L1xHER2-LFLEDA 10 µg (= 0.5 mg / kg) 5 bsIgG1- huCD3-H1L1xHER2-LFLEDA 100 µg (= 5 mg / kg) 6 bsIgG1- huCD3-H1L1xb12-LFLEDA 100 µg (= 5 mg / kg) 7 bsIgG1- b12xHER2-LFLEDA 100 µg (= 5 mg / kg)

[0744] The in vivo anti-tumor efficacy of the bispecific CD20 x CD3 antibody huCD3-H1L1xCD20-LFLEDA was evaluated in a subcutaneous Raji xenograft model. In this model, human unstimulated PBMCs, as a source of human T cells, were co-inoculated with tumor cells, similar to the model described by Brischwein et al. (Mol. Immunol. 43 (2006), 1129–1143). Female NOD-SCID (NOD.CB17-Prkdcscid / NcrCrl) mice aged 6-11 weeks were used. Human PBMCs from healthy donors were isolated from buffy coats, washed and resuspended in PBS-0.1% BSA. On day 0, 5x10 6 A mixture of both PBMCs and Raji cells of the cells was inoculated subcutaneously in the right flank of each mouse in 200 μL. Within 1 hour of injection, mice were sorted into 7 groups (n=4 / donor) and each group was injected intravenously (iv) with a single dose of the (bispecific) antibody. The treatment groups are shown in Table 6. Tumor growth was evaluated twice a week using calipers (PLEXX) until the endpoint tumor volume was 1500 mm 3 , until the tumor showed ulceration or until the end of the study.

[0745] The results are shown in Figure 25 In. Figure 25 As can be seen in A, huCD3-H1L1xCD20-LFLEDA effectively reduced tumor size at the two higher doses tested (0.5 mg / kg and 0.05 mg / kg) of huCD3-H1L1xCD20-LFLEDA.

[0746] exist Figure 25 In B, tumor size of individual mice is indicated on the last day (day 21) when each group was complete. Tumor formation was significantly inhibited in mice treated with huCD3-H1L1xCD20-LFLEDA at doses of 0.05 mg / kg and 0.5 mg / kg compared to the PBS control group (p-values ​​were p<0.05 and p<0.01, respectively, evaluated using one-way Anova, Tukey's multiple comparison test). Treatment with 0.005 mg / kg did not affect tumor formation. Treatment with control antibody b12 x CD20 (bsIgG1-b12xCD20-LFLEDA) at 0.5 mg / kg and 0.05 did not significantly inhibit tumor formation compared to the PBS control (one-way Anova, Tukey's multiple comparison test).

[0747] Table 6

[0748] Group Antibody Dose 1 PBS 2 bsIgG1- huCD3-H1L1xCD20-LFLEDA 0.1 µg (= 0.005 mg / kg) 3 bsIgG1- huCD3-H1L1xCD20-LFLEDA 1 µg (= 0.05 mg / kg) 4 bsIgG1- huCD3-H1L1xCD20-LFLEDA 10 µg (= 0.5 mg / kg) 5 bsIgG1- b12xCD20-LFLEDA 1 µg (= 0.05 mg / kg) 6 bsIgG1- b12xCD20-LFLEDA 10 µg (= 0.5 mg / kg)

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Claims

1. A humanized IgG1 antibody that binds to human CD3, wherein the antibody comprises a binding region, the binding region comprising: a heavy chain variable (VH) region represented by the sequence of SEQ ID NO: 6, and a light chain variable (VL) region represented by the sequence of SEQ ID NO: 10, wherein the amino acid in position T41 of the lambda light chain of SEQ ID NO: 10 is K, or wherein the amino acids in positions F10, T41, K55 and L97 of the lambda light chain of SEQ ID NO: 10 are L, K, N and H, respectively, or wherein the amino acids in positions F10, R23, A35, R47, D71, A82, D83, S86, I87 and F89 of the lambda light chain of SEQ ID NO: 10 are L, A, P, T, G, P, E, A, E and Y, respectively.

2. The antibody of claim 1, wherein the antibody is a full-length antibody.

3. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

4. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that binding of CIq to the antibody is reduced by at least 70% compared to a wild-type antibody, wherein CIq binding is determined by ELISA.

5. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that binding of CIq to the antibody is reduced by at least 80% compared to a wild-type antibody, wherein CIq binding is determined by ELISA.

6. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that binding of CIq to the antibody is reduced by at least 90% compared to a wild-type antibody, wherein CIq binding is determined by ELISA.

7. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that binding of CIq to the antibody is reduced by at least 95% compared to a wild-type antibody, wherein CIq binding is determined by ELISA.

8. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that binding of CIq to the antibody is reduced by at least 97% compared to a wild-type antibody, wherein CIq binding is determined by ELISA.

9. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that binding of CIq to the antibody is reduced by 100% compared to a wild-type antibody, wherein CIq binding is determined by ELISA.

10. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody mediates at least 50% reduction in Fc-mediated T-cell proliferation compared to a wild-type antibody, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC) based functional assay.

11. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody mediates at least 60% reduction in Fc-mediated T-cell proliferation compared to a wild-type antibody, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC) based functional assay.

12. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody mediates at least 70% reduction in Fc-mediated T-cell proliferation compared to a wild-type antibody, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC) based functional assay.

13. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody mediates at least 80% reduction in Fc-mediated T-cell proliferation compared to a wild-type antibody, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC) based functional assay.

14. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody mediates at least 90% reduction in Fc-mediated T-cell proliferation compared to a wild-type antibody, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC) based functional assay.

15. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody mediates at least 99% reduction in Fc-mediated T-cell proliferation compared to a wild-type antibody, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC) based functional assay.

16. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody mediates 100% reduction in Fc-mediated T-cell proliferation compared to a wild-type antibody, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC) based functional assay.

17. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by at least 50% when compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

18. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by at least 60% when compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

19. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by at least 70% when compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

20. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by at least 80% when compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

21. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by at least 90% when compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

22. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by at least 99% when compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

23. The antibody of any one of claims 1-2, wherein the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by 100% when compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

24. The antibody of any one of claims 1-2, wherein the antibody comprises a first and a second immunoglobulin heavy chain, wherein in at least one of the first and second immunoglobulin heavy chains, one or more amino acids at positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are not L, L, D, N and P, respectively.

25. The antibody of claim 24, wherein in at least one of the first and second immunoglobulin heavy chains, the amino acid at position D265 of the human IgGl heavy chain is not D.

26. The antibody of claim 24, wherein in at least one of said first and second immunoglobulin heavy chains, the amino acid at position N297 of the human IgGl heavy chain is not N.

27. The antibody of claim 24, wherein in at least one of said first and second immunoglobulin heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are not L and L, respectively.

28. The antibody of claim 24, wherein in at least one of said first and second immunoglobulin heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are F and E; or A and A, respectively.

29. The antibody of claim 28, wherein in at least one of said first and second immunoglobulin heavy chains, the amino acids at positions L234 and L235 of the human IgGl heavy chain are F and E, respectively.

30. The antibody of claim 28, wherein in at least one of said first and second immunoglobulin heavy chains, the amino acids at positions L234 and L235 of the human IgG1 heavy chain are A and A, respectively.

31. The antibody of claim 24, wherein in at least one of said first and second immunoglobulin heavy chains, the amino acids at positions L234, L235, and D265 of the human IgGl heavy chain are not L, L, and D, respectively.

32. The antibody of claim 24, wherein in at least one of the first and second immunoglobulin heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively.

33. The antibody of claim 32, wherein in at least one of the first and second immunoglobulin heavy chains, the amino acids at positions L234, L235 and D265 of the human IgGl heavy chain are F, E and A, respectively.

34. The antibody of claim 32, wherein in at least one of said first and second immunoglobulin heavy chains, the amino acids at positions L234, L235, and D265 of the human IgG1 heavy chain are A, A, and A, respectively.

35. The antibody of claim 24, wherein in at least one of said first and second immunoglobulin heavy chains, the amino acids at positions L234, L235, D265, N297 and P331 of the human IgGl heavy chain are F, E, A, Q and S, respectively.

36. The antibody of any one of claims 1-2, wherein the antibody comprises a constant heavy chain (HC) and a constant light chain (LC), wherein positions L234, L235 and D265 of the human IgG1 heavy chain of SEQ ID NO: 15 are F, E and A, respectively, and wherein the position of F405 of the human IgG1 heavy chain of SEQ ID NO: 15 is L, and wherein (i) positions F10, T41, K55 and L97 of the lambda light chain of SEQ ID NO: 10 are L, K, N and H, respectively, or (ii) position T41 of the lambda light chain of SEQ ID NO: 10 is K.

37. A bispecific antibody comprising a first binding region of the antibody of any one of claims 1-36, and a second binding region that binds to a different target than the first binding region.

38. The bispecific antibody of claim 37, wherein said antibody comprises a first and a second heavy chain.

39. The bispecific antibody of claim 38, wherein a) the bispecific antibody comprises an Fc region as defined in any one of claims 4 to 23; or b) at least one of the first and second heavy chains comprises one or more amino acids as defined in any one of claims 24-36.

40. The bispecific antibody of any one of claims 37-39, wherein the first and second heavy chains each comprise at least a hinge region, a CH2 region, and a CH3 region, wherein in the first heavy chain at least one amino acid at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain is substituted, and in the second heavy chain at least one amino acid at a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain is substituted, and wherein the first and the second heavy chains are not substituted at the same position.

41. The bispecific antibody of claim 40, wherein the amino acid at position F405 of the human IgG1 heavy chain in the first heavy chain is L, and the amino acid at position K409 of the human IgG1 heavy chain in the second heavy chain is R, or vice versa.

42. The bispecific antibody of claim 37, wherein the second binding region binds human HER2 or human CD20.

43. The bispecific antibody of claim 42, wherein the second binding region binds human CD20.

44. The bispecific antibody of claim 43, wherein the second binding region that binds human CD20 comprises: (i) the VH CDR1 region of SEQ ID NO:34, the VH CDR2 region of SEQ ID NO:35, the VH CDR3 region of SEQ ID NO:36, the VL CDR1 region of SEQ ID NO:37, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:38, (ii) the VH CDR1 region of SEQ ID NO:41, the VH CDR2 region of SEQ ID NO:42, the VH CDR3 region of SEQ ID NO:43, the VL CDR1 region of SEQ ID NO:44, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:45, (iii) the VH CDR1 region of SEQ ID NO:48, the VH CDR2 region of SEQ ID NO:49, the VH CDR3 region of SEQ ID NO:50, the VL CDR1 region of SEQ ID NO:51, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:52, or (iv) the VH CDR1 region of SEQ ID NO:55, the VH CDR2 region of SEQ ID NO:56, the VH CDR3 region of SEQ ID NO:57, the VL CDR1 region of SEQ ID NO:58, the VL CDR2 region of DAS, and the VL CDR3 region of SEQ ID NO:

59.

45. The bispecific antibody of claim 44, wherein the second binding region that binds human CD20 comprises: (i) the VH sequence of SEQ ID NO:29 and the VL sequence of SEQ ID NO:30, (ii) the VH sequence of SEQ ID NO:39 and the VL sequence of SEQ ID NO:40, (iii) the VH sequence of SEQ ID NO:46 and the VL sequence of SEQ ID NO:47, or (iv) the VH sequence of SEQ ID NO:53 and the VL sequence of SEQ ID NO:

54.

46. ​​An expression vector comprising (i) a nucleic acid sequence encoding the heavy chain sequence of the humanized IgG1 antibody of claim 1; and (ii) a nucleic acid sequence encoding the light chain sequence of the humanized IgG1 antibody of claim 1.

47. A host cell comprising the expression vector of claim 46.

48. The host cell of claim 47, wherein the host cell is a recombinant eukaryotic host cell or a recombinant prokaryotic host cell.

49. The host cell of claim 47, wherein the host cell is a recombinant microbial host cell.

50. A composition comprising the antibody of any one of claims 1-36 or the bispecific antibody of any one of claims 37-45.

51. A pharmaceutical composition comprising the antibody of any one of claims 1 to 36 or the bispecific antibody of any one of claims 37 to 45, and a pharmaceutically acceptable carrier.

52. Use of the antibody of any one of claims 1-36, the bispecific antibody of any one of claims 37-45, the composition of claim 50, or the pharmaceutical composition of claim 51 in the preparation of a medicament for treating breast cancer in a subject in need thereof.

53. Use of the antibody of any one of claims 1-36, the bispecific antibody of any one of claims 37-45, the composition of claim 50 or the pharmaceutical composition of claim 51 in the preparation of a kit for diagnosing a disease characterized by involvement or accumulation of cells expressing CD3, optionally wherein the antibody or the bispecific antibody is labeled with a detectable agent.

54. A method for producing an antibody according to any one of claims 1 to 36 or a bispecific antibody according to any one of claims 37 to 45, comprising the following steps a) culturing the host cell according to any one of claims 47 to 49; and b) purifying the antibody from the culture medium.

55. A diagnostic composition comprising the antibody of any one of claims 1-36 or the bispecific antibody of any one of claims 37-45.

56. Use of the antibody of any one of claims 1 to 36 or the bispecific antibody of any one of claims 37 to 45 in the preparation of a kit for detecting the presence of CD3 antigen or cells expressing CD3 in a sample.

57. A kit for detecting the presence of CD3 antigen or cells expressing CD3 in a sample, comprising i) the antibody of any one of claims 1 to 36 or the bispecific antibody of any one of claims 37 to 45; and ii) instructions for use of the kit.

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