EA045070B1 - CD3 BINDING MOLECULES CAPABLE OF BINDING TO HUMAN AND NON-HUMAN CD3 - Google Patents

Description

This application claims priority to U.S. Patent Application No. 61/488,716 (filed May 21, 2011; pending) and U.S. Patent Application No. 61/530,353 (filed Sept. 1

2011; pending), each of which is incorporated herein by reference in its entirety.

Link to sequence list

The application includes one or more sequence listings in accordance with 37 CFR § 1.821 et seq., which are presented in both paper and machine-readable media, and these paper and machine-readable media representations are incorporated herein by reference therein. in full.

Prerequisites for creating the invention

Field of technology

The present invention relates to CD3-binding molecules capable of binding to human CD3 and non-human CD3, and in particular to such molecules that are cross-reactive with CD3 of a non-human mammal (eg, cynomolgus monkey). The present invention also relates to the use of such antibodies and antigen-binding fragments for the treatment of malignancies, autoimmune and/or inflammatory diseases and other conditions.

Description of the technical field

The body's immune system serves as a defense for a number of conditions, including, for example, injury, infection and neoplasia, and is mediated by two separate but interrelated systems: the cellular and humoral immune systems. In general, mediators of the humoral system are soluble products (antibodies or immunoglobulins) that have the ability to combine with products recognized by this system as foreign to the body and neutralize them. In contrast, the cellular immune system involves the activation of specific cells called T cells, which perform a number of therapeutic roles. T cells are lymphocytes that originate from the thymus gland and circulate between tissues, the lymphatic system, and the cardiovascular system. They act against, or in response to, a number of foreign structures (antigens). In many cases, these foreign antigens are presented on host cells as a result of neoplasia or infection. Although T cells themselves do not secrete antibodies, they are usually required for antibody secretion by a second class of lymphocytes, B cells (which are bone marrow-derived). It is important that T cells exhibit exceptional immunological specificity in order to be able to distinguish one antigen from another.

A naïve T cell, for example a T cell that has not yet encountered its specific antigen, is activated when it first encounters a specific peptide:MHC complex on an antigen-presenting cell. The antigen presenting cell may be a B cell, a macrophage, or a dendritic cell. When an unexposed T cell encounters a specific peptide:MHC complex on an antigen-presenting cell, a signal is delivered through the T cell receptor that causes a change in the conformation of lymphocyte function-associated antigen (LFA) molecules and increases their affinity for the molecules intercellular adhesion cell (ICAM) present on the surface of the antigen-presenting cell. The signal generated by the interaction of a T cell with an antigen presenting cell is necessary, but not sufficient, to activate the naïve T cell. A second costimulatory signal is required. An unexposed T cell can only be activated by an antigen-presenting cell that carries both a specific peptide:MHC complex and a co-stimulatory molecule on its surface. Antigen recognition by a naïve T cell in the absence of costimulation results in the T cell becoming anergic. The requirement for two signals to activate T cells and B cells so that they achieve adaptive immune responses may provide a mechanism for avoiding reactions to self-antigens that may be present on the antigen-presenting cell at locations in the system at which it can be recognized by the T cell. If contact of a T cell with an antigen presenting cell results in the generation of only one of the two required signals, the T cell does not become activated and an adaptive immune response does not take place.

The efficiency with which humans and other mammals mount an immunological response to pathogens and foreign substances depends on two characteristics: the high specificity of the immune response for antigen recognition and immunological memory, which allows for faster and stronger responses after reactivation by the same antigen (Portoles, P. et al. (2009) The TCR/CD3 Complex: Opening the Gate to Successful Vaccination, Current Pharmaceutical Design 15: 3290-3300; Guy, C.S. et al. (2009) Organization of Proximal Signal Initiation at the TCR: CD3 Complex, Immunol Rev. 232(1): 7-21). The specificity of the T cell response is mediated by recognition of the antigen (presented on antigen presenting cells (APCs)) by a molecular complex including the T cell receptor (TCR) and the cell surface receptor ligand, CD3. The TCR is a covalently linked heterodimer of α and β chains (TCRae). These

- 1045070 chains are class I membrane polypeptides with a length of 259 (α) and 296 (β) amino acids. The CD3 molecule is a complex containing a CD3 γ chain, a CD3 δ chain, and two CD3 ε chains linked as three dimers (εγ, εδ, ζζ,) (Guy, C.S. et al. (2009) Organization of Proximal Signal Initiation at the TCR:CD3 Complex, Immunol Rev. 232(1): 7-21; Call, M. E. et al. (2007) Common Themes In The Assembly And Architecture Of Activating Immune Receptors, Nat. Rev. Immunol. 7: 841 -850; Weiss, A. (1993) T Cell Antigen Receptor Signal Transduction: A Tale of Tails and Cytoplasmic Protein-Tyrosine Kinases, Cell 73: 209-212). The TCR and CD3 complex, along with the zeta chain, the ζ chain of CD3 (also known as the T3 cell receptor zeta chain or CD247), comprises the TCR complex (van der Merwe, P.A. etc. (epub Dec. 3, 2010) Mechanisms For T Cell Receptor Triggering, Nat. Rev. Immunol. 11: 47-55; Wucherpfennig, K. W. et al. (2010) Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling, Cold Spring Harb Perspect Biol 2:a005140). This complex is particularly important because it contains a large number (ten) of immunoreceptor tyrosine activating motifs (ITAMs).

In the case of mature T cells, activation of TCR/CD3 by foreign antigenic peptides bound to self-MHC molecules is the first step necessary for the expansion of antigen-specific T cells and their differentiation into effector T lymphocytes or memory T cells. These processes involve phosphorylation of the immunoreceptor tyrosine activating motifs (ITAMs) of the TCR complex. Because the TCR complex contains such a large number of ITAMs (10 in total), and these ITAMs are located sequentially one after the other (due to dimerization of the constituent chains), phosphorylation of the corresponding tyrosine residues as a result of TCR ligation creates paired protein attachment sites that contain domains homologous to regions of Src protein 2 (SH2), such as ζ-chain-associated protein with M.m. 70 kDa (ZAP-70), and thereby initiates an amplifying signaling cascade that leads to T cell activation and differentiation (Guy, C.S. et al. (2009) Organization of Proximal Signal Initiation at the TCR:CD3 Complex, Immunol Rev. 232 (1): 7-21).

The outcome of these processes is regulated by the intensity and quality of the antigenic stimulus, as well as the nature of the accompanying signals transmitted by the coreceptor and co-stimulatory surface molecules, or cytokine receptors (Portoles, P. et al. (2009) The TCR/CD3 Complex: Opening the Gate to Successful Vaccination, Current Pharmaceutical Design 15: 3290-3300 Riha, P. et al (2010) CD28 Co-Signaling In The Adaptive Immune Response, Self/Nonself 1(3): 231-240). Although TCR stimulation is a prerequisite for T cell activation, it is generally accepted that engagement with co-stimulatory molecules such as CD28 is necessary for full T cell activation and differentiation (Guy, C.S. et al. (2009) Organization of Proximal Signal Initiation at the TCR:CD3 Complex, Immunol Rev. 232 (1): 7-21).

Because of the fundamentality of CD3 in initiating responses to antigens, monoclonal antibodies against this receptor have been proposed that are capable of blocking or at least modulating the immune process, and thus as agents for the treatment of inflammatory and/or autoimmune disease. Indeed, anti-CD3 antibodies were the first antibody approved for treatment in humans (St. Clair E.W. (2009) Novel Targeted Therapies for Autoimmunity, Curr. Opin. Immunol. 21(6): 648-657). Anti-CD3 antibody (marketed by Janssen-Cilag as ORTHOCLONE™ OKT3™) has been administered to reduce acute rejection in organ transplant patients and as a treatment for lymphoblastic leukemia (Cosimi, A.B. et al. (1981) Use Of Monoclonal Antibodies To T- Cell Subsets For Immunologic Monitoring And Treatment In Recipients Of Renal Allografts, N. Engl. J. Med. 305: 308-314; Rung, P. et al. (1979) Monoclonal antibodies defining distinctive human T cell surface antigens, Science 206: 347-349; Vigeral, P. et al. (1986) Prophylactic Use Of OKT3 Monoclonal Antibody In Cadaver Kidney Recipients. Utilization Of OKT3 As The Sole Immunosuppressive Agent, Transplantation 41: 730-733; Midtvedt, K. et al. (2003 ) Individualized T Cell Monitored Administration Of ATG Versus OKT3 In SteroidResistant Kidney Graft Rejection, Clin. Transplant. 17(1): 69-74; Gramatzki, M. et al. (1995) Therapy With OKT3 Monoclonal Antibody In Refractory T Cell Acute Lymphoblastic Leukemia Induces Interleukin-2 Responsiveness, Leukemia 9(3): 382-390; Herold, K.C. et al. (2002) Anti-CD3 Monoclonal Antibody In New Onset Type I Diabetes Mellitus, N. Engl. J. Med. 346: 1692-1698; Cole, M.S. et al. (1997) Human IgG2 Variants Of Chimeric Anti-CD3 Are Nonmitogenic to T cells, J. Immunol. 159(7): 3613-3621; Cole, M.S. et al. (1999) Hum291, A Humanized Anti-CD3 Antibody, Is Immunosuppressive To T Cells While Exhibiting Reduced Mitogenicity in Vitro, Transplantation 68: 563-571; US Patent Nos. 6491916, 5585097 and 6706265).

However, treatment with such anti-CD3 antibodies has not been found to be sufficiently specific to avoid side effects (Ludvigsson, J. (2009) The Role of Immunomodulation Therapy in Autoimmune Diabetes, J. Diabetes Sci. Technol. 3(2): 320-330) . Repeated daily administration of OKTZ results in potent immunosuppression and provides effective treatment of rejection after kidney transplantation. In vivo, OKTZ administration leads to both T cell activation and suppression of immune responses. However, the use of OKTZ has been hampered by the first toxic dose response syndrome, which is associated with the events of initial T-cell activation and the release of cytoplasmic acid.

- 2,045,070 kin, which occurs before immunosuppression of T-cell responses. Side effects reported in the literature that follow the first and sometimes the second injection of this murine monoclonal antibody include a flu-like syndrome consisting of fever, chills, headache and gastrointestinal symptoms (vomiting and diarrhea), and in severe cases, swelling lungs within hours of treatment (Thistlethwaite, J.R. Jr. et al. (1988) Complications and Monitoring of OCT Therapy, Am. J. Kidney Dis. 11: 112-119). This syndrome is believed to reflect OCTS-mediated cross-linking of the TCR/CD3 complex on the surface of T cells and the resulting release of cytokines (eg, tumor necrosis factor alpha (TNFa), interferon-γ, interleukins IL-2, IL-3, IL- 4, IL-6, IL-10 and granulocyte-macrophage colony-stimulating factor (Masharani, U.B. et al. (2010) Teplizumab Therapy For Type I Diabetes, Expert Opin. Biol. Ther. 10(3): 459465; Abramowicz, D. et al. (1989) Release Of Tumor Necrosis Factor, Interleukin-2, And Gamma-Interferon In Serum After Injection Of OKT3 Monoclonal Antibody In Kidney Transplant Recipients, Transplantation 47: 606-608; Ferran, C. et al. (1990) Cytokine-Related Syndrome Following Injection Of Anti-CD3 Monoclonal Antibody: Further Evidence For Transient In Vivo T Cell Activation, Eur J Immunol 20: 509-515; Hirsch, R. et al (12989) Effects Of In Vivo Administration Of Anti-CD3 Monoclonal Antibody On T Cell Function In Mice II. In Vivo Activation Of T Cells, J. Immunol. 142: 737-743)). The use of anti-CD3 antibodies is described in US Patent Nos. 7883703, 7728114, 7635472, 7575923 and 7381903 and US Patent Application Publications Nos. 2010/0150918, 2010/0209437, 2010/0183554, 2010/0015142 , 2008/0095766, 2007/ 0077246 and PCT Application Publication No. WO 2008/119567.

A particular disadvantage of previous antibodies is their specificity for only human CD3. This deficiency is a significant hindrance in the development of such antibodies as therapeutic agents for the treatment of human diseases. To obtain marketing authorization, any new drug candidate must undergo rigorous testing. This test can be divided into preclinical and clinical phases. While clinical testing, further subdivided into the commonly known clinical phases I, II and III, is performed on human patients, preclinical testing is performed on animals. The purpose of preclinical testing is to confirm that the drug candidate has the desired activity and, most importantly, is safe. Only if preclinical testing has established the safety in animals and the potential efficacy of the drug candidate will the appropriate regulatory authority authorize human clinical testing of the drug candidate. The safety of drug candidates can be tested in animals in the following three ways: (i) on the relevant species, i.e. form in which the drug candidates can recognize orthologous antigens, (ii) in a transgenic animal containing human antigens, and (iii) through the use of a substitute drug candidate that can bind to orthologous antigens present in the animal. The disadvantages of transgenic animals are that this technology is typically confined to rodents. However, rodents and humans have significant differences in physiology that may make it difficult to extrapolate safety data obtained in rodents to predict safety in humans. The disadvantages of a substitute drug candidate are that the chemical compound is different from the actual drug candidate, and the animals often used are rodents, with the disadvantages discussed above. Therefore, preclinical data obtained in rodents have limited predictive power regarding drug candidates. The preferred approach to safety testing is to use the relevant species, preferably a lesser primate. Now, a disadvantage of CD3 binding molecules suitable for therapeutic use in humans described in the art is that the relevant species are great apes, in particular cynomolgus macaques. Accordingly, an anti-CD3 antibody capable of binding to both human and primate CD3 is highly desirable. Such antibodies are described in US Patent Application Publication No. 20100150918 and PCT Application Publication No. WO2008/119567.

Despite these advances, there remains a need for anti-human CD3 antibodies and antigen-binding fragments thereof that are capable of cross-reacting with non-human mammal CD3 (eg, cynomolgus monkey). The present invention addresses this need and the need for improved therapeutics for cancer, autoimmune and inflammatory diseases.

Summary of the invention

The present invention relates to CD3 binding molecules capable of binding to human CD3 and non-human CD3, and in particular to those molecules that are cross-reactive with non-human mammal CD3 (eg, cynomolgus monkey). The present invention also relates to the uses of such antibodies and antigen binding fragments for the treatment of cancer, autoimmune and/or inflammatory diseases and other conditions.

In detail, the present invention provides a CD3 binding molecule comprising an antigen binding fragment of an antibody, wherein the antigen binding fragment includes a CD3 specific VL domain of an antibody and a CD3 specific VH domain of an antibody, wherein the CD3 specific VL domain and the CD3 specific VH domain form an antigen binding domain. a domain capable of immunospecific binding to both a human CD3 epitope and a non-human mammalian CD3 epitope, wherein:

(I) The CD3-specific VL domain is selected from the group consisting of VL-1 h-mab2 (SEQ ID NO: 16), VL-2 h-mab2 (SEQ ID NO: 18), VL-3 h-mab2 ( SEQ ID NO: 20), VL-4 h-mab2 (SEQ ID NO:22), VL-5 hmab2 (SEQ ID NO:24), VL-6 h-mab2 (SEQ ID NO:26), VL-7 h-mab2 (SEQ ID NO: 28), VL-8 h-mab2 (SEQ ID NO: 30), VL-9 h-mab2 (SEQ ID NO: 32) and VL-10 h-mab2 (SEQ ID NO: 34), and said CD3-specific VH domain is selected from the group consisting of VH-1 h-mab2 (SEQ ID NO: 36), VH-2 hmab2 (SEQ ID NO: 38), VH-3 h-mab2 (SEQ ID NO: 40), VH-4 h-mab2 (SEQ ID NO: 42), VH-5 h-mab2 (SEQ ID NO: 44), VH-6 h-mab2 (SEQ ID NO: 46), VH-6L h-mab2 (SEQ ID NO: 54), VH-7 h-mab2 (SEQ ID NO: 48), VH-8 h-mab2 (SEQ ID NO: 50), VH-8L h-mab2 (SEQ ID NO: 55), VH-8 di-1 h-mab2 (SEQ ID NO:56), VH-8 di-2 h-mab2 (SEQ ID NO: 57), VH-6M h-mab2 (SEQ ID NO: 72) , VH-8M h-mab2 (SEQ ID NO: 74), VH-2k h-mab2 (SEQ ID NO: 87) and VH-5k h-mab2 (SEQ ID NO: 88); or (II) the CD3-specific VL domain is selected from the group consisting of VL-1 h-mab1 (SEQ ID NO: 10) and VL-2 h-mab1 (SEQ ID NO: 12), and the CD3-specific VH- domain is VH h-mabl SEQ ID NO: 14).

The present invention particularly relates to an embodiment of the CD3 binding molecule described above in which the CD3-specific VL domain is VL-6 h-mab2 (SEQ ID NO:26).

The present invention further relates to an embodiment of the CD3 binding molecule described above in which the CD3 specific VH domain is VH-8 h-mab2 (SEQ ID NO: 50), VH-6 h-mab2 (SEQ ID NO: 46) or VH-2k h-mab2 (SEQ ID NO: 87).

The present invention particularly relates to an embodiment of the CD3 binding molecule described above, wherein the molecule is an antibody and, in particular, wherein the antibody lacks an Fc region or includes an Fc region that:

(A) lacks effector function or has reduced effector function; or (B) has been modified to impair the ability of the Fc region of the antibody to bind to the Fc receptor; moreover, a decrease in effector function and a weakening of binding ability occurs relative to that of the wild-type Fc region.

The present invention further relates to an embodiment of the CD3 binding molecules described above, wherein the molecule is a CD3 binding diabody that includes a first polypeptide chain and a second polypeptide chain, the chains being covalently linked to each other, wherein:

I. The first polypeptide chain includes the amino terminus and the carboxyl terminus and from the N-terminus to the C-terminus:

(i) domain (A) including a CD3-specific VL domain;

(ii) a domain (B) including the binding region of the second immunoglobulin heavy chain variable domain (VH2); and (iii) domain (C);

wherein domains (A) and (B) do not associate with each other to form an epitope-binding site; and (II) the second polypeptide chain includes the amino-terminus and the carboxyl-terminus and from the N-terminus to the C-terminus:

(iv) a domain (D) including the binding region of the second immunoglobulin light chain variable domain (VL2);

(v) domain (E) including a CD3-specific VH domain; and (vi) domain (F);

wherein domains (D) and (E) do not associate with each other to form an epitope-binding site; and moreover:

(1) domains (A) and (E) associate to form an antigen binding domain that is capable of immunospecific binding to both human and non-human mammalian CD3;

(2) domains (B) and (D) associate to form a binding site that immunospecifically binds to a second epitope, wherein the second epitope is different from the CD3 epitope to which the antigen binding domain formed due to the association of domains (A) and (E) binds ; and (3) domains (C) and (F) are covalently linked together.

The present invention further relates to an embodiment of the CD3 binding molecules described above wherein the second epitope is not a CD3 epitope.

The present invention further relates to an embodiment of the above-described

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CD3-binding molecules, in which the second epitope is a CD3 epitope that is different from the CD3 epitope to which the antigen-binding domain formed due to the association of domains (A) and (E) binds.

The present invention further relates to an embodiment of the CD3 binding molecules or antibodies or diabodies described above, wherein such a molecule is humanized.

The present invention further relates to an embodiment of the CD3-binding molecules or antibodies or diabodies described above, wherein such a molecule is capable of immunospecific binding to CD3 and fluorescein.

The present invention further relates to an embodiment of the CD3-binding molecules or diabodies described above, wherein such molecule is capable of immunospecific binding to both (i) CD3 and (a) a tumor-specific antigen, or (ii) )(b) a cell surface antigen, receptor or cell surface receptor ligand.

The present invention further relates to an embodiment of the CD3-binding molecules or diabody described above, wherein the molecule or diabody is capable of immunospecific binding to CD3 and a tumor-specific antigen presented on a tumor cell, wherein the tumor cell is a cancer cell selected from group consisting of breast cancer, prostate cancer, stomach cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cancer, pharyngeal cancer, esophageal cancer, cancer larynx, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, bladder cancer, kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma and leukemia.

The present invention further relates to an embodiment of the CD3 binding molecules or diabody described above, wherein the molecule or diabody is capable of immunospecific binding to CD3 and a cell surface antigen, receptor or cell surface receptor ligand, wherein the cell surface antigen, receptor or the cell surface receptor ligand is HER2/neu, B7-H3, CD20, PSMA, IGF-1R, Ep-CAM, or is a molecule that is involved in the association of a T cell with a B cell, which leads to activation of the T cell or B -cells during the adaptive immune response.

The present invention further relates to an embodiment of the CD3-binding molecules or diabodies described above, wherein the molecule or diabody is capable of immunospecific binding to CD3 and to a molecule that is involved in T cell-B cell association, and a molecule, which is involved in the association of a T cell with a B cell is selected from the group consisting of CD19, CD20, CD22, CD23, CD27, CD32B, CD38, CD40, CD79a, CD79b, CD80, CD86, LFA-I, LFA-3 and CFA-I.

The present invention further relates to a pharmaceutical composition comprising any of the CD3 binding molecules, antibodies or diabodies described above and a pharmaceutically acceptable carrier, excipient or diluent.

The present invention further relates to the pharmaceutical composition described above for use in the treatment of cancer or an autoimmune or inflammatory disease.

The present invention further relates to the pharmaceutical composition described above for use in the treatment of an autoimmune or inflammatory disease selected from the group consisting of insulin-dependent diabetes type I, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, celiac disease, Gougerot-Sjögren's syndrome, Graves' disease, Crohn's disease, autoimmune hepatitis, psoriasis, psoriatic arthritis, asthma, allergic rhinitis, effects due to organ transplantation or homologous disease (GVHD). The present invention relates in particular to the pharmaceutical composition described above for use in the treatment of insulin-dependent diabetes type I.

Brief description of the figures

In fig. 1A-1B show the results of a capture ELISA in which the ability of the anti-CD3 antibody mAB1 (FIG. 1A) or a chimeric mABl derivative (ch-mAb1) (FIG. 1B) was assessed using soluble human CD3 (shCD3).

In fig. 2A-2B show the results of a capture ELISA in which the ability of anti-CD3 antibody mAB2 (FIG. 2A) or a chimeric derivative of mAB2 (ch-mAb2) (FIG. 2B) was assessed using soluble human CD3 (shCD3) or soluble Javanese CD3 macaque (scCD3).

In fig. 3 shows the results of assays to determine the effect of variations in the Kabat-numbered residues 41-46 of the mAb2 light chain framework regions.

In fig. 4 shows the results of assays to determine the effect of variations in Kabat-numbered residues 36, 38, 44, and 46 of the mAb2 light chain framework regions.

In fig. 5 shows the results of assays to determine the effect of variations in Kabat-numbered residues 36, 38, and 46 of the mAb2 light chain framework regions.

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In fig. 6 shows the results of assays to determine the effect of variations in the Kabat-numbered residues 30, 49, and 93 of the mAb2 heavy chain framework regions.

In fig. 7 shows the results of additional analyzes performed to determine the effect of variations in the Kabat-numbered residues 30, 49, and 93 of the mAb2 heavy chain framework regions.

In fig. 8A-8B show the results of assays performed to determine the ability of the chimeric and humanized mAb2 to bind to non-human CD3.

In fig. 9A-9D show sensorgram recordings of BIACORE™ assays performed to determine the binding kinetics of ch-mAB2 or h-mAb2 to scCD3 or scCD3.

In fig. 10A-10D show the results of capture ELISAs performed on DART™ diabodies containing a CD3-binding first epitope-binding site and a second epitope-binding site that binds either Her2/neu or CD19 or EGFR or B7-H3.

In fig. 11A-11B show the ability of B7H3 x CD3 bispecific DART™ diabodies to mediate redirected killing of tumor cells expressing B7H3.

In fig. 12A-12E show the ability of A33 x CD3 bispecific DART™ diabodies to mediate redirected killing of A33-expressing tumor cells.

In fig. 13A and 13B show the results of a comparison of the ability of the DART™ CD19-hmAb2 diabody and the CD19 x CD3 bispecific diabody to induce redirected T cell-mediated killing. The DART™ CD19-h-mAb2 diabody exhibits specificity for human CD3 as well as non-human CD3; The CD19 x CD3 bispecific diabody DART exhibits specificity for only human CD3. Fig. 13A: redirected killing of Raji human B-cell lymphoma cells; fig. 13B: redirected killing of lymphoma cells from JeKo-1 tissue cells.

In fig. 14A and 14B show that the DART™ CD19-h-mAb2 diabody of the present invention was capable of mediating cytolysis in the presence of either human or cynomolgus effector T cells.

In fig. 15A and 15B show the ability of the DART™ ERBITUX™-h-mAb2 diabody of the present invention or the (ERBITUX™-T cell receptor)-bispecific DART™ diabody to mediate an increase in CD69 MFI after incubation with CD4+ or CD8+ T cells. The control diabody ERBITUX™-CD3 FN18 DART™ (capable of binding to EGFR and cynomolgus CD3) did not cause an increase in CD69 MFI.

In fig. 16A-16D show the results of binding studies of either DART™ ERBITUX™-h-mAb2 diabody, DART™ ERBITUX™-m-mAb2 diabody, or DART™ 4420-h-mAb2 diabody (as a negative control) or control secondary antibody with A498 or A431 cells (FIGS. 16A and 16C, respectively) and in mediating redirected killing of such cells (FIGS. 16B and 16D, respectively).

Detailed Description of the Present Invention

The present invention relates to antibodies against human CD3 and antigen-binding fragments thereof, and in particular to such antibodies that are cross-reactive with CD3 of a non-human mammal (eg, cynomolgus monkey). The present invention also relates to the uses of such antibodies and antigen binding fragments for the treatment of cancer, autoimmune and/or inflammatory diseases and other conditions.

I. Definitions

As used herein, the term CD3-binding molecule means a molecule capable of immunospecific binding to both human CD3 and non-human mammalian CD3 due to at least one antigen recognition site (eg, an antibody antigen binding domain) located in the variable region of the molecule. As used herein, such ability to immunospecifically bind to both human and non-human mammalian CD3 is not intended to imply the ability of one antigen binding domain to simultaneously bind both such CD3 molecules, but rather to mean that such antigen binding domain exhibits cross-reactive such that it will immunospecifically bind to human CD3 when incubated in the presence of human CD3 and will immunospecifically bind to non-human mammalian CD3 when incubated in the presence of such non-human mammalian CD3.

As used herein, the term CD3-binding molecule covers not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') ₂ Fv), single chain (ScFv), mutants thereof, naturally occurring variants fusion proteins comprising a portion of a protein with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, BiTE®, DART™ diabody molecules and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site of the required specificity. The term BiTE (bispecific T cell recruiters) refers to a single chain polypeptide molecule that has two antigen binding domains, one of which binds to a T cell antigen and the second of which binds to an antigen present on the surface of the target (WO 05/061547; Baeuerle, P et al. (2008) BiTE®: A New Class Of Antibodies That Recruit T Cells, Drugs of the Future 33: 137-147; Bargou, et al. 2008) Tumor Regression in Cancer Patients by Very Low Doses of a T Cell -Engaging Antibody Science 321:974-977).

The term DART™ (Dual Affinity Retargeting Reagent) diabody refers to an immunoglobulin molecule that includes at least two polypeptide chains that associate (primarily through covalent linkage) to form at least two epitope-binding sites that can recognize the same epitope or distinct epitopes. Each of the polypeptide chains of the DART™ diabody includes an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope-binding site. More specifically, the immunoglobulin heavy chain variable region of one (eg, the first) of the polypeptide chains of a DART™ diabody interacts with the immunoglobulin light chain variable region of a different (eg, second) DART™ polypeptide chain to form an epitope-binding site. Likewise, the immunoglobulin light chain variable region of one (eg, the first) of the polypeptide chains of a DART™ diabody interacts with the immunoglobulin heavy chain variable region of a different (eg, second) DART™ polypeptide chain to form an epitope-binding site. DART™ diabodies can be monospecific, bispecific, trispecific, etc., thus having the ability to simultaneously bind one, two, three or more different epitopes (which may be from the same or different antigens). DART™ diabodies can further be monovalent, divalent, trivalent, tetravalent, pentavalent, hexavalent, etc., thus having the ability to bind one, two, three, four, five, six or more molecules simultaneously. These two characteristics of DART™ diabodies (i.e. degree of specificity and valency) can be combined, for example, to produce bispecific antibodies (i.e. capable of binding two epitopes) that are tetravalent (i.e. capable of binding four series of epitopes), etc. DART™ diabody molecules are described in PCT Application Publications Nos. WO 2006/113665, WO 2008/157379 and WO 2010/080538.

The bispecific (or trispecific or polyspecific) molecules of the present invention will be capable of binding to both human and non-human mammal CD3 (eg, cynomolgus monkey), as well as second (or additional) and distinct antigen(s). ) and epitope(s). The second antigen or epitope is preferably a tumor-specific antigen presented on the tumor cell. Such tumor cells may be from malignant neoplasms, for example, breast cancer, prostate cancer, stomach cancer, lung cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cancer, pharynx cancer, esophageal cancer, laryngeal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, bladder cancer, kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma or leukemia. Additional antigens or epitopes are preferably tumor-specific antigens or epitopes on the cell surface (such as 17-1A, A33, major antigen I of endodermal origin on adult erythrocytes, alpha-fetoprotein, tumor RNA virus envelope antigen, oncoembryonic antigen, urinary tract tumor-specific bladder, B7-H1, B7-H2, B7-H3, B7-H4, B7H5, B7-H6, Burkitt's lymphoma-specific antigen-38.13, CA125, CD18, CD19, human B-cell lymphoma-specific antigen-CD20, CD22 , CD33, CD44, CD52, CEA, CO17-1A, STA-1, CTLA-4, epidermal growth factor receptor, Ep-CAM, EphA2, antigen I on newborn erythrocytes, fibrosarcoma antigen, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, GICA 19-9, gp IIIb/IIIa, gp72, HER1, HER-2/neu, HER3, HER4, melanoma-specific antigen with large molecular weight, HLA-DR antigen, human leukemia-specific T-cell antigen- Gp37, human lung carcinoma-specific antigen L20, human lung carcinoma-specific antigen L6, human milk fat globular antigen, IgE, carcinoma-specific KS 1/4 pan antigen, LEA, adenocarcinoma-specific antigen F3, malignant-specific antigen human lymphocyte antigen-APO-1, melanoma-specific antigen gp75, melanoma-associated antigen p97, neoglycoprotein, nuC242, polymorphic epithelium-specific antigen mucin type, prostate-specific antigen, prostate-specific membrane antigen, prostate-specific acidic phosphate, SK-1 antigen, TAG-72, T antigen, tumor-specific antigen CA125, tumor-specific antigen MUC1, tumor-specific cell surface transplantation antigen, vascular endothelial growth factor, vascular endothelial growth factor receptor and ανβ3). Alternatively, such additional antigens or epitopes may be associated with a pathogen such as hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, veterinary virus

- 7 045070 smallpox, adenovirus, herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), causative agent of rinderpest, rhinovirus, ECHO virus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackievirus, mumps virus, measles virus, rubella virus, poliovirus, variola virus, Epstein-Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II ( HIV-II), the causative agent of viral meningitis, the causative agent of viral encephalitis, the causative agent of dengue, the causative agent of smallpox; mycobacteria, rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia burgdorferi, Bacillus anthracis, Streptococcus, Staphylococcus, Mycobacterium, tetanus, whooping cough, cholera, plague, diphtheria, chlamydia and legionella; leishmania, the causative agent of coccidiosis, trypanosome or the causative agent of malaria; chlamydia and rickettsia.

The term monoclonal antibody refers to a homogeneous collection of antibodies, where the monoclonal antibody is composed of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of antigen. Monoclonal antibodies are highly specific, being directed against one region of the determinant. The term monoclonal antibody covers not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2 Fv), single chain (ScFv), mutants thereof, fusion proteins comprising part of the antibody , humanized monoclonal antibodies, chimeric monoclonal antibodies and any other modified configuration of an immunoglobulin molecule that includes an antigen recognition site of the required specificity and with the ability to bind to the antigen. It is not intended to be limited by the source of the antibody or the method in which it is produced (eg, using hybridoma, phage selection, expression of recombinant molecules, transgenic animals, etc.). The term includes complete immunoglobulins, as well as fragments, etc., described above in the definition of antibody.

The term humanized antibody refers to a chimeric molecule, typically prepared using recombinant DNA techniques, having an antigen binding site derived from an immunoglobulin of a non-human species and the remaining structure of the immunoglobulin molecule being based on the structure and/or sequence of the human immunoglobulin. The antigen binding site may include either entire variable domains inserted into constant domains, or only complementarity determining regions (CDRs) transplanted into appropriate framework regions of the variable domains. Antigen binding sites may be wild type or modified by one or more amino acid substitutions. This precludes the constant region from functioning as an immunogen in human subjects, but the possibility of an immune response to a foreign variable region remains possible (LoBuglio, A.F. et al. (1989) Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response, Proc. Natl. Acad. Sci. (U.S.A.) 86: 4220–4224). Another approach focuses not only on providing human-derived constant regions, but also on modifying the variable regions to give them a new form as close as possible to the human form. The variable regions of both the heavy and light chains are known to contain three complementarity determining regions (CDRs) that vary in response to the corresponding antigens and determine binding ability, flanked by four framework regions (FRs) that are relatively conserved in a particular species and which presumably provide the framework for CDR. When preparing non-human antibodies to a particular antigen, the variable regions can be reshaped or humanized by grafting CDRs derived from the non-human antibody into FRs present in the human antibody that are modified. The application of this approach to various antibodies was reported in Sato, K. et al. (1993) Cancer Res 53: 851-856; Riechmann, L. et al. (1988) Reshaping Human Antibodies for Therapy, Nature 332: 323-327; Verhoeyen, M. et al. (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239: 1534-1536; Kettleborough, C.A. et al. (1991) Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation, Protein Engineering 4: 773-3783; Maeda, H. et al. (1991) Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity, Human Antibodies Hybridoma 2: 124-134; Gorman, S.D. et al. (1991) Reshaping A Therapeutic CD4 Antibody, Proc. Natl. Acad. Sci. (U.S.A.) 88: 41814185; Tempest, P.R. et al. (1991) Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in Vivo, Bio/Technology 9: 266-271; Co, M.S. et al. (1991) Humanized Antibodies For Antiviral Therapy, Proc. Natl. Acad. Sci. (U.S.A.) 88: 2869-2873; Carter, P. et al. (1992) Humanization Of An Anti-pl85her2 Antibody For Human Cancer Therapy, Proc. Natl. Acad. Sci. (U.S.A.) 89: 4285-4289; and So, M.S. et al. (1992) Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen, J. Immunol. 148: 1149-1154.

In some embodiments, all CDR sequences are retained in the humanized antibodies (eg, in a humanized mouse antibody that contains all six CDRs from the mouse antibodies). In other embodiments, humanized antibodies contain one or more CDRs (one, two, three, four, five, six) that are modified from the parent antibody,

- 8 045070 which are also referred to as one or more CDRs derived from one or more CDRs of the parent antibody. As described below, preferred antibodies of the present invention contain specific identified CDRs. The present invention, however, provides equivalent antibodies containing altered CDRs.

An antibody or polypeptide, as used herein, is said to bind immunospecifically or, equivalently, specifically to a site of another molecule (i.e., epitope) if it reacts or associates more frequently, more quickly, with greater duration, and/or with greater affinity with this epitope than with alternative epitopes. For example, an antibody that specifically binds to a CD3 epitope is an antibody that binds to that CD3 epitope with greater affinity, avidity, faster, and/or longer duration than it binds to other CD3 epitopes or non-CD3 epitopes. It is also intended by this definition that, for example, an antibody (or moiety or epitope) that immunospecifically binds to a first target may or may not bind specifically or preferentially to a second target. As such, immunospecific binding does not necessarily imply (although it may include) exclusive binding. Typically, but not necessarily, reference to binding means immunospecific binding.

As used herein, the term immunologically active, in relation to an epitope that is or remains immunologically active, refers to the ability of an antibody (eg, anti-CD3 antibody) to bind to the epitope under various conditions, for example, after exposing the epitope to reducing and denaturing conditions. For example, if an antibody is no longer able to bind to a denatured epitope, the epitope is said to have become immunologically inactive.

Various biological functions are associated with the anti-CD3 antibodies of the present invention, and such antibodies may exhibit any or all of the following characteristic properties, or may lack one, two, three or more of such characteristic properties: the ability to specifically bind to human CD3, which is endogenously present on the surface of a normal human T cell; the ability to specifically bind to human CD3, which is endogenously present on the surface of human leukemia T cells; the ability to specifically bind to CD3 of a non-human mammal (eg, cynomolgus monkey), which is endogenously present on the surface of a normal non-human mammal T cell; the ability to specifically bind to non-human CD3, which is endogenously present on the surface of normal non-human T cells; the ability to specifically bind to non-human CD3, which is endogenously present on the surface of non-human leukemia T cells; the ability to neutralize (ie, block or interfere with binding) complex formation with CD3; the ability to neutralize the formation of a complex with the TCR; the ability to modulate (either antagonistically or agonistically) signal transmission by the TCR complex; ability to bind to the Fc receptor; the ability to competitively inhibit the preferential binding of a known anti-CD3 antibody to CD3, including the ability to preferentially bind to the same CD3 epitope to which the parent antibody preferentially binds; the ability to bind to a portion of CD3 that is present on the surface of a living cell in vitro or in vivo; the ability to bind to the part of CD3 that is present on the surface of a living cancer cell; the ability to deliver a chemotherapeutic agent to a cancer T cell; and/or the ability to deliver a therapeutic agent, toxin or detectable marker to a T cell. As discussed here, the polypeptides (including antibodies) of the present invention may have any one or more of these characteristic properties.

As used herein, the term agent refers to a biological, pharmaceutical or chemical compound. Non-limiting examples include a simple or complex organic or inorganic molecule, peptide, protein, oligonucleotide, antibody, antibody derivative, antibody fragment, vitamin derivative, carbohydrate, toxin, or chemotherapeutic compound. Various compounds can be synthesized, such as small molecules and oligomers (eg, oligopeptides and oligonucleotides), and synthetic organic compounds based on various basic structures. In addition, various natural sources such as plant or animal extracts and the like can provide compounds for selection. The agents used in the methods of this invention may be randomly selected or rationally selected or designed. An agent, as used herein, is said to be randomly selected if the agent is selected without prior consideration or knowledge of the specific amino acid or other chemical moieties involved in the association of the molecule with its natural binding partner(s) or known antibodies. An example of a randomly selected agent is an agent that is identified through the use of or screening of a chemical library or peptide combinatorial library. An agent, as used herein, is said to be rationally selected or designed if the agent is selected on a non-random basis that takes into account the sequence of the target site and/or its conformation in relation to the action of that agent. The agent can be selected rationally or designed

- 9 045070 rationally through the use of peptide sequences that form contact sites in the receptor/ligand and/or CD3/anti-CD3 antibody complex. For example, a rationally selected peptide agent may be a peptide whose amino acid sequence is identical to an epitope that appears on CD3 when exposed on the surface of a living cell in its natural environment. Such an agent will reduce or block the association of the anti-CD3 antibody with CD3, or the association of CD3 with its natural ligand, as desired, upon binding to the anti-CD3 antibody or its natural ligand.

As used herein, the term labeled, with respect to an antibody, is intended to encompass direct labeling of the antibody by coupling (i.e., physical association) with a detectable substance, such as a radioactive agent or fluorophore (e.g., phycoerythrin (PE) or fluorescein isothiocyanate (also known as fluoroisothiocyanate or FITC) with an antibody, as well as indirect labeling of a probe or antibody resulting from reactivity with the detectable substance.

As used herein, the term association, with respect to an antibody, includes the covalent or non-covalent attachment or binding of an agent (eg, a chemotherapeutic agent) to the antibody(ies). Association of an antibody with an agent (eg, a chemotherapeutic agent) can be accomplished by direct binding or indirect binding through attachment to a common platform such that the antibody localizes the agent to the cancer cell to which the antibody binds without the antibody and agent being substantially dissociated under physiological conditions, so that the agent is not directed to the same cancer cell to which the antibody binds, or so that the effectiveness of the agent is not reduced.

The term biological sample covers many types of samples that are obtained from an individual and may be used in research for diagnostic or monitoring purposes. The definition covers saliva, blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen, or tissue cultures or cells derived from them and their progeny, such as cells obtained from a tissue sample taken from an individual suspected of having the presence of cancer, in preferred embodiments, tissue of the ovary, lung, prostate, pancreas, colon and breast. The definition also includes samples that have been manipulated after their receipt in any way, such as by treatment with reagents, solubilization or enrichment for specific components such as proteins or polynucleotides, or embedding in a semi-solid or solid matrix for the purpose of sectioning. The term biological specimen covers a clinical specimen and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, body fluid, and tissue samples.

The term host cell includes a single cell or cell culture that can be or has been the recipient of the vector(s) for incorporating polynucleotide inserts. Host cells include the progeny of a single host cell, and these progeny may not necessarily be completely identical (in morphology or genomic DNA) to the original parent cell due to natural, random or non-random mutation. The host cell includes cells transfected in vivo with the polynucleotide(s) of this invention.

As used herein, an effective amount of a pharmaceutical composition, in one embodiment, is an amount sufficient to provide beneficial or desired results, including, but not limited to, clinical results such as reduction in tumor size or rate of growth, delay or attenuation of an inflammatory response, improvement quality of life of those suffering from a disease, reducing the dose of other drugs needed to treat such a disease, enhancing the effect of another drug, for example, through targeted delivery and/or internalization, delaying the progression of the disease and/or increasing the life expectancy of individuals. Such an effective amount may be administered in one or more administrations. For the purposes of this invention, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to improve the clinical observed condition.

In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an effective amount may be considered in terms of the administration of one or more additional agents, and one agent may be considered to be administered in an effective amount if, together with one or more other agents, the desired result can be or is achieved. Although individual needs vary, determination of optimal effective amount ranges for each compound is a matter of skill in the art. A typical dose administered to a patient is usually from 0.0001 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dose administered to the patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg. kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.7 5 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg and 0.25 mg/ kg, 0.0001 and 0.15 mg/kg, 0.0001 and 0.10 mg/kg, 0.001 and 0.5 mg/kg, 0.01 and 0.25 mg/kg or 0.01 and 0, 10 mg/kg body weight

- 10 045070 patient's body. The dose and frequency of administration of the molecules of the present invention can be reduced or modified by increasing the absorption and tissue penetration of the molecules of the present invention through modifications such as, for example, lipidation.

A nucleic acid molecule or agent, antibody, composition or cell, etc. as used herein is said to be isolated if the nucleic acid molecule, agent, antibody, composition or cell, etc. separated substantially from contaminant molecules of nucleic acids, antibodies, agents, compositions or cells, etc., which are naturally present at its original source.

The term individual refers to a vertebrate animal, preferably a mammal. Mammals include, but are not limited to, humans, farm animals, sporting animals, pets, primates, mice and rats. In the most preferred embodiment, the term individual means a human being.

The terms polypeptide, oligopeptide, peptide and protein are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be straight or branched, it may include modified amino acids, and it may be interrupted by non-amino acids. The terms also cover a polymer of amino acids that has been modified naturally or as a result of interference, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation to a tag moiety. Also included in this definition are, for example, polypeptides containing one or more amino acid analogues (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It will be understood that, since the polypeptides of this invention are antibody based, the polypeptides may occur as single chains or as linked chains.

As used herein, the term substantially pure refers to a material that is at least 50% pure (i.e., free of impurities), more preferably at least 90% pure, more preferably at least 95% pure, more preferably at least 98% pure, more preferably at least 99% pure, and most preferably greater than 99% pure.

As used herein, the term toxin refers to any substance that produces a negative reaction in a cell. For example, a toxin directed at a cancer cell will have a negative, sometimes harmful effect on the cancer cell. Examples of toxins include, but are not limited to, taxane, maytansinoid, auristatin (e.g., monomethyl auristatin (MMAE), monomethyl auristatin F (MMAF), auristatin E (AE), etc.) (e.g., those described in U.S. Pat. Nos. 5208020, 5416064, 6333410, 6340701, 6372738, 6436931, 6441163, 6596757, 7276497, 7585857 or 7851432), calicheamicin, anthracycline (e.g. doxorubicin), CC analogue -1065, docetaxel, cathepsin B or E, ricin, gelonin, Pseudomonas exotoxin , diphtheria toxin and RNase; radiolabeled antibodies (for example, conjugated with thiuxetane or labeled with a toxic radioisotope (for example, ⁹⁰ Y; ¹³ I, ¹⁷⁷ Lu, ^{1 6} Re, ¹⁸⁸ Re, ²¹¹ At, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac, etc.) ).

As used herein, the term treatment means a method of obtaining a beneficial or desired result, including preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reduction of inflammation or autoimmune response, reduction of proliferation (or destruction) of cancer cells or other unhealthy cells, reduction of metastasis of cancer cells found in malignancies, reduction in tumor size , reducing symptoms resulting from a disease, improving the quality of life of those suffering from the disease, reducing the dosage of other medications needed to treat the disease, delaying the progression of the disease, and/or increasing the life expectancy of individuals.

II. Methods for creating antibodies and polypeptides of the present invention

Methods for creating monoclonal antibodies are known in the art. One method that can be used is the method of Kohler, G. et al. ((1975) Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity, Nature 256: 495-497) or a modification thereof. Typically, monoclonal antibodies are created in non-human species such as mice. Typically a mouse or rat is used for immunization, but other animals can also be used. Antibodies are produced when mice are immunized with immunogenic amounts of cells, cell extracts, or protein preparations that contain human CD3. The immunogen may be, but is not limited to, fetal cells, cultured cell lines, cancer cells, nucleic acids, or tissue.

In one embodiment, monoclonal antibodies that bind to CD3 are produced using host cells that overexpress CD3 as an immunogen. Such cells include, by way of example and not limitation, human T cells.

To monitor antibody production, a small biological sample (eg blood) can be obtained from the animal and tested for antibody titer against the immunogen. Spleen

- 11 045070 and/or several large lymph nodes can be removed and dissociated into individual cells. If desired, spleen cells can be screened (after removing nonspecifically attached cells) by adding a cell suspension to an antigen-coated plate or well. B cells expressing membrane-bound immunoglobulin specific for the antigen will bind to the plate and will not be washed away with the rest of the suspension. The resulting B cells, or all dissociated spleen cells, can then be fused with myeloma cells (eg, X63-Ag8.653 and cells from the Salk Institute, Cell Distribution Center, San Diego, CA). Polyethylene glycol (PEG) can be used to fuse spleen cells or lymphocytes with myeloma cells to form a hybridoma. The hybridoma is then cultured in a selective medium (eg, hypoxanthine, aminopterin, thymidine medium, otherwise known as HAT medium). The resulting hybridomas are then plated by serial dilution and screened for the production of antibodies that specifically bind to the immunogen using, for example, FACS (fluorescence-excited cell sorting) or immunohistochemical (IHC) selection. Selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (eg, in tissue culture mats or hollow fiber reactors) or in vivo (eg, as ascitic fluids in mice).

As another alternative to the cell fusion method, B cells immortalized by Epstein-Barr virus (EBV) can be used to produce monoclonal antibodies of the subject invention. Hybridomas are expanded and subcloned, if desired, and supernatants are tested for anti-immunogenic activity using conventional assay procedures (eg, FACS, IHC, radioimmunoassay, enzyme-linked immunosorbent assay, fluorescence immunoassay, etc.).

As another alternative, the anti-CD3 monoclonal antibody or any other equivalent antibodies can be sequenced and produced recombinantly using any method known in the art (eg, humanization, use of transgenic mice to produce fully human antibodies, phage display technology, etc. .). In one embodiment, the anti-CD3 monoclonal antibody is sequenced and then the polynucleotide sequence is cloned into an expression or extension vector. The sequence encoding the antibody of interest can be maintained in the vector in a host cell, and the host cell can then be expanded and frozen for later use.

The polynucleotide sequence of an anti-CD3 monoclonal antibody and any other equivalent antibodies can be used for genetic manipulation to create a humanized antibody, increase affinity, or improve other characteristics of the antibody. The general principle in humanizing an antibody involves maintaining the core sequence of the antigen-binding portion of the antibody while replacing the non-human remainder of the antibody with human antibody sequences. There are four general steps for the purpose of humanizing a monoclonal antibody. These steps are: (1) determination of the nucleotide and predicted amino acid sequence of the variable domains of the light and heavy chains of the parent antibody;

(2) construction of a humanized antibody, i.e. deciding which antibody framework to use during the humanization process; (3) methodologies/techniques for actual humanization; and (4) transfection and expression of the humanized antibody. See, for example, US Patent Nos. 4816567, 5807715, 5866692 and 6331415.

A number of humanized antibody molecules have been described incorporating an antigen-binding site derived from a non-human immunoglobulin, including chimeric antibodies containing rodent-derived or modified rodent-derived V regions and associated complementarity determining regions (CDRs) fused to constant domains humans (see, for example, Winter et al. (1991) Man-made Antibodies, Nature 349: 293-299; Lobuglio et al. (1989) Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response, Proc. Natl. Acad. Sci. (U.S.A.) 86: 4220-4224 (1989), Shaw et al (1987) Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor-Associated Antigen, J. Immunol. 138: 4534-4538, and Brown et al (1987) Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody, Cancer Res. 47: 3577-3583). Other references describe rodent-derived CDRs transplanted into a human supporting framework region (FR) prior to fusion with the corresponding human antibody constant domain (see, for example, Riechmann, L. et al. (1988) Reshaping Human Antibodies for Therapy, Nature 332 : 323-327; Verhoeyen, M. et al. (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239: 1534-1536; and Jones et al. (1986) Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse, Nature 321: 522525). Another reference document describes rodent-derived CDRs supported by recombinantly venated rodent-derived framework regions. See, for example, European Patent Publication No. 519596. These humanized molecules are designed to minimize adverse immunological reactions to anti-human molecules

- 12 045070 rodent antibodies, which limits the duration and effectiveness of therapeutic applications of these constituents in human recipients. Other methods for humanizing antibodies that can also be used are described in Daugherty et al. (1991) Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins, Nucl. Acids Res. 19: 2471-2476 and US Patent Nos. 6180377, 6054297, 5997867 and 5866692.

Also covered by the present invention are the single chain Fv fragments (scFv) of the antibodies of this invention, such as mouse anti-CD3. Single chain Fv fragments are created by linking light and/or heavy chain variable regions using a short linker peptide. Bird et al. ((1988) Single-Chain Antigen-Binding Proteins, Science 242: 423-426) provide an example of linker peptides that form an approximately 3.5 nm bridge between the carboxyl terminus of one variable region and the amino terminus of another variable region. areas. Linkers with other sequences have been developed and used (Bird et al. (1988) Single-Chain AntigenBinding Proteins, Science 242: 423-426). Linkers can in turn be modified for additional functions, such as attaching drugs or attaching to solid supports. Single chain variants can be produced either recombinantly or synthetically. An automated synthesizer can be used to synthesize scFv. For recombinant production of scFv, a suitable plasmid containing a polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic such as yeast, plant, insect or mammalian cells, or prokaryotic such as E. coli. Polynucleotides encoding the scFv of interest can be generated using conventional manipulations such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification methods known in the art.

The present invention includes modifications to anti-CD3 antibodies and binding fragments thereof. Modification of polypeptides is a common practice in the art and is not required to be described in detail here. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not result in significantly deleterious changes in functional activity, or the use of chemical analogues. Amino acid residues that may be conservatively substituted for one another include, but are not limited to: glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine; lysine/arginine and phenylalanine/tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation using various Sugars, acetylation and phosphorylation. Preferably, the amino acid substitutions would be conservative, i.e. the replaced amino acid would have chemical properties similar to those of the original amino acid. Such conservative substitutions are known in the art, and examples are provided above. Amino acid modifications can range from changing or modifying one or more amino acids to completely remodeling a region, such as a variable region. Changes in the variable region may result in changes in affinity and/or specificity. Other modification methods include the use of coupling methods known in the art, including, but not limited to, enzymatic methods, oxidative displacement and chelation. Modifications can be used, for example, to attach labels in the case of an immunoassay, for example, to attach radioactive moieties in the case of a radioimmunoassay. Modified polypeptides are generated using procedures well known in the art and can be screened using standard assays known in the art.

The fact that a change in one amino acid residue in a CDR can lead to loss of functional binding (Rudikoff, S. etc. (1982) Single Amino Acid Substitution Altering AntigenBinding Specificity, Proc. Natl. Acad. Sci. (USA) 79(6): 1979-1983), provides a method for methodically identifying alternative functional CDR sequences. In one preferred method of producing such CDR variants, the polynucleotide encoding the CDR is mutated (e.g., by nonspecific mutagenesis or a site-directed mutagenesis method (e.g., polymerase chain reaction amplification using primers that encode the mutated locus)) to produce the CDR, containing a substituted amino acid residue. By comparing the identifier of the corresponding residue in the original (functional) CDR sequence with the identifier of the CDR sequence variant with a (non-functional) substitution, a score for this substitution can be determined BLOSUM62.iij. The BLOSUM system provides a matrix of amino acid substitutions created by analyzing a sequence database for the sake of reliable alignments (Eddy, S.R. (2004) Where Did The BLOSUM62 Alignment Score Matrix Come From?, Nature Biotech. 22(8): 1035-1036; Henikoff, J.G. (1992) Amino acid substitution matrices from protein blocks, Proc. Natl. Acad Sci (USA) 89: 10915-10919 Karlin, S. et al (1990) Methods For Assessing

- 13 045070

The Statistical Significance Of Molecular Sequence Features By Using General Scoring Schemes, Proc. Natl. Acad. Sci. (USA) 87: 2264-2268; Altschul, S.F. (1991) Amino Acid Substitution Matrices From An Information Theoretical Perspective, J. Mol. Biol. 219, 555-565). Currently, the most developed BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table 1 presents the scores for the BLOSUM62.iij substitutions (the higher the score, the more conservative the substitution is, and, accordingly, the more likely it is that the substitution will not affect function). If the antigen binding fragment comprising the resulting CDR does not bind to CD3, then the BLOSUM62.iij substitution score is considered to be insufficiently conservative and a new candidate substitution having a higher score is selected and implemented. Thus, for example, if the original residue was glutamate (E) and the nonfunctional replacement residue was histidine (H), then the BLOSUM62.iij substitution score would be 0, and more conservative substitutions (e.g., aspartate, asparagine, glutamine) would be preferred or lysine).

Table 1 A R N D WITH Q E G AND I L To m G R S T W Y V A B4th -1 _9 _9 0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 +1 0 -3 -2 0 R -1 0 _9 -3 +1 0 -2 0 -3 -2 +2 -1 -3 -2 -1 -1 -3 -2 -3 N _2 0 +6 +1 -3 0 0 0 +1 -3 -3 0 -2 -3 -2 +1 0 -4 -2 -3 D _2 _2 +1 +6 -3 0 +2 -1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3 WITH 0 -3 -3 -3 +9 -3 -4 -3 -3 -1 -1 -3 -1 _9 -3 -1 -1 _9 _9 -1 ABOUT -1 +1 0 0 -3 +5 +2 0 -3 -2 +1 0 -3 -1 0 -1 -2 -1 -2 E -1 0 0 +2 -4 +2 +5 0 -3 -3 +1 -2 -3 -1 0 -1 -3 -2 -2 G 0 -2 0 -1 -3 _2 +6 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3 N -2 0 +1 -1 -3 0 0 _9 +8 -3 -3 -1 -2 -1 -2 -1 -2 -2 +2 -3 I -1 -3 -3 -3 -1 -3 -3 -4 -3 +2 -3 +1 0 -3 -2 -1 -3 -1 +3 L -1 -2 -3 -4 -1 -2 -3 -4 -3 +2 +2 0 -3 -2 -1 -2 -1 +1 TO -1 +2 0 -1 -3 +1 +1 -2 -1 -3 NTS® -1 -3 -1 0 -1 -3 -2 -2 M -1 -1 -2 -3 -1 0 -2 -3 -2 +1 +2 -1 0 _9 -1 -1 -1 -1 +1 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 +6 -4 _9 -2 +1 +3 -1 R -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 _9 -4 -1 -1 -4 -3 -2 S +1 -1 +1 0 -1 0 0 0 -1 _9 _9 0 -1 _9 -1 th+4th +1 -3 _9 -2 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 +1 _9 _9 0 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 +1 -4 -3 _9 Bit® +2 -3 Y -2 -2 -2 -3 -2 -1 -2 -3 +2 -1 -1 -2 -1 +3 -3 -2 _9 +2 -1 V 0 -3 -3 -3 -1 -2 -2 -3 -3 +3 +1 -2 +1 -1 -2 -2 0 -3 -1 (T42

Thus, the present invention provides for the use of non-specific mutagenesis to identify improved CDRs. Alternatively, phage display technology can be used to increase (or decrease) the affinity of CDRs. This technology, called affinity maturation, uses mutagenesis or CDR walking, and re-selection uses the target antigen or antigenic fragment to identify antibodies containing CDRs that bind with greater (or less) affinity to the antigen than the original or parent antibody (see, for example, Glaser et al. (1992) J. Immunology 149: 3903). Mutating entire codons rather than individual nucleotides results in a semi-random set of amino acid mutations. Libraries can be created consisting of a collection of variant clones, each of which differs in one amino acid change in one CDR, and containing variants that reflect every possible amino acid change for each CDR residue. Mutants with increased (or decreased) affinity for an antigen can be selected by bringing the immobilized mutants into contact with a labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased or decreased affinity for an antigen (eg, ELISA) (see Wu et al. 1998, Proc Natl. Acad Sci. USA 95: 6037; Yelton et al. ., 1995, J. Immunology 155: 1994). A CDR walk can be used, which imparts randomness to the light chain (see Schier et al., 1996, J. Mol. Bio. 263: 551).

Methods for performing such affinity maturation are described, for example, in

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Krause, J.C. et al. (2011)An

Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function Of A Human Antibody, MBio. 2(1) pii: e00345-10. doi: 10.1128/mBio.00345-10; Kuan, C.T. et al.

(2010) Affinity-Matured Anti-Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas And Melanomas, Int. J Cancer 10.1002/ijc.25645; Hackel, B.J. et al. (2010) Stability And CDR Composition Biases Enrich Binder Functionality Landscapes, J. Mol. Biol. 401(1): 84-96; Montgomery, D. L. et al. (2009) Affinity Maturation And Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41, MAbs 1(5): 462-474; Gustchina, E. et al. (2009) Affinity Maturation By Targeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab Derived From A Synthetic Naive Human Antibody Library And Directed Against The Internal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With Improved HIV-1 Neutralization Potency And Breadth, Virology 393(1): 112-119; Finlay, W.J. et al. (2009) Affinity Maturation Of A Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The Complementarity-Determining Regions, J. Mol. Biol. 388(3): 541-558; Bostrom, J. et al. (2009) Improving Antibody Binding Affinity And Specificity For Therapeutic Development, Methods Mol. Biol. 525: 353-376; Steidl, S. et al. (2008) In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR-Diversification, Mol. Immunol. 46(1): 135-144; and Barderas, R. et al. (2008) Affinity maturation of antibodies assisted by in silico modeling, Proc. Natl. Acad. Sci. (USA)

105(26): 9029-9034.

In a preferred embodiment, multiwell plates can be coated with the selected anti-CD3 antibody (e.g., 100 ng/well in carbonate buffer at room temperature for 2 hours) and subsequently incubated with soluble CD3 added at a 1/10 dilution and incubated at room temperature in for 16 hours, or diluted to 50 ng/ml in PBS-TBSA (0.05 ml added to each well and incubated for at least 2 hours at room temperature). The plate is then washed and dilutions of recombinant antibodies starting at 0.5 μg/ml in PBS-T-BSA are then added and incubated for 1 h at room temperature. The binding of the recombinant antibodies to the captured antigen is then measured using, for example, an anti-human IgG-HRP conjugate and a TMB substrate. After color development has been stopped, using dilute sulfuric acid, the plate is read at 450 nm and higher affinity antibodies are identified (see, for example, US Pat. No. 7,351,803).

The present invention includes polypeptides comprising the amino acid sequence of the antibodies of this invention. The polypeptides of this invention can be created using procedures known in the art. Polypeptides can be produced by proteolytic or other cleavage of antibodies, by methods using recombinant molecules (ie, single or fused polypeptides) as described above, or by chemical synthesis. Antibody polypeptides, especially shorter polypeptides up to approximately 50 amino acids, are readily generated by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, an anti-CD3 polypeptide could be produced by an automated polypeptide synthesizer using a solid phase method.

The present invention also includes fusion proteins comprising one or more fragments or regions from the polypeptides and antibodies of this invention. In one embodiment, a fusion polypeptide is provided comprising at least 10 contiguous light chain variable region amino acids and at least 10 contiguous heavy chain variable region amino acids. In another embodiment, the fusion polypeptide comprises a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide comprises a light chain variable region and a heavy chain variable region of an antibody produced by an openly deposited hybridoma. For purposes of this invention, an antibody fusion protein contains one or more polypeptide domains that specifically

- 15 045070 binds to CD3, and another amino acid sequence to which it is not attached in a natural molecule, for example, a heterologous sequence or a homologous sequence from another region.

Ahtu-CD3 polypeptides and other CD3 agonists, antagonists and modulators can be created using methods known in the art, for example, synthetically or recombinantly. One method for producing such molecules involves chemical synthesis of the polypeptide followed by treatment under oxidative conditions suitable to obtain the natural conformation, i.e. corresponding compounds using disulfide bonds. This can be accomplished using methodologies well known to those skilled in the art (see, for example, Kelley, R.F. et al. (1990) In: GENETIC ENGINEERING PRINCIPLES AND METHODS, Setlow, J.K. Ed., Plenum Press, N.Y., vol. 12, pp. 1-19; Stewart, J. M. et al. (1984) SOLID PHASE PEPTIDE SYNTHESIS, Pierce Chemical Co., Rockford, IL; see also US Patent Nos. 4105603, 3972859, 3842067 and 3862925).

The polypeptides of the present invention can be readily prepared using solid-phase peptide synthesis (Merrifield, B. (1986) Solid-Phase Synthesis, Science 232(4748): 341-347; Houghten, R. A. (1985) General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen-Antibody Interaction At The Level Of Individual Amino Acids, Proc. Natl. Acad. Sci. (U.S.A.) 82(15): 5131-5135; Ganesan, A. (2006) Solid-Phase Synthesis In The Twenty-First Century, Mini Rev Med Chem 6(1): 3-10).

However, in another embodiment, fully human antibodies can be produced by using commercially available mice that have been engineered to express human specific immunoglobulin proteins. Transgenic animals, which are created to produce a more desirable (eg, fully human antibody) or stronger immune response, can also be used to produce humanized or human antibodies. Examples of such technology are XENOMOUSE™ (Abgenix, Inc., Fremont, CA) and HUMAB-MOUSE (R) and TC MOUSE™ (both from Medarex, Inc., Princeton, NJ).

In an embodiment, antibodies can be generated recombinantly and expressed using any method known in the art. Antibodies can be generated recombinantly by first isolating the generated antibodies from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (eg, CHO cells). Another method that can be used is to express the antibody sequence in plants (eg tobacco) or transgenic milk. Suitable methods for expressing antibodies recombinantly in plants or milk have been described (see, for example, Peeters et al. (2001) Production Of Antibodies And Antibody Fragments In Plants, Vaccine 19: 2756; Lonberg, N. et al. (1995) Human Antibodies From Transgenic Mice, Int. Rev. Immunol 13: 65-93; and Pollock et al. (1999) Transgenic Milk As A Method For The Production Of Recombinant Antibodies, J. Immunol Methods 231: 147-157). Suitable methods for preparing antibody derivatives, eg humanized, single chain, etc., are known in the art. Alternatively, antibodies can be created recombinantly using phage display technology (see, for example, US Patent Nos. 5565332, 5580717, 5733743, 6265150, and Winter, G. et al. (1994) Making Antibodies By Phage Display Technology, Annu. Rev. Immunol 12: 433-455).

The antibody or protein of interest can be sequenced using Edman digestion of proteins, which is well known to those skilled in the art. Peptide information obtained from mass spectrometry or Edman digestion of proteins can be used to design probes or primers that are used to clone the protein of interest.

An alternative method for cloning a protein of interest is by panning, using purified CD3 or portions thereof of cells expressing the antibody or protein of interest. The panning procedure can be performed by generating a cDNA library from tissues or cells that express CD3, overexpressing the cDNA in a second cell type, and screening the transfected cells of the second cell type for specific binding to CD3. Detailed descriptions of methods used in cloning mammalian genes encoding cell surface proteins by panning can be found in the art (see, for example, Aruffo, A. et al. (1987) Molecular Cloning Of A CD28 cDNA By A High Efficiency COS Cell Expression System, Proc. Natl. Acad. Sci. (U.S.A.) 84: 8573-8577 and Stephan, J. et al. (1999) Selective Cloning Of Cell Surface Proteins Involved In Organ Development: Epithelial Glycoprotein Is Involved In Normal Epithelial Differentiation , Endocrinol 140: 5841–5854).

cDNAs encoding anti-CD3 antibodies and other CD3 peptide agonists, antagonists and modulators can be obtained by reverse transcribing mRNA from a specific cell type according to standard methods in the art. In particular, mRNA can be isolated using various lytic enzymes or chemical solutions according to procedures outlined, for example, in MOLECULAR CLONING: A LABORATORY MANUAL, Third Edition

- 16 045070 (Sambrook et al. Eds., 2001) Cold Spring Harbor Press, Cold Spring Harbor, NY), or extract using commercially available nucleic acid binding resins following the accompanying instructions provided by the manufacturers (e.g., Qiagen, Invitrogen, Promega). The synthesized cDNAs can then be inserted into an expression vector to produce the antibody or protein of interest in a second cell type. The expression vector is expected to be replicable in host cells either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and cosmids.

Vectors containing polynucleotides of interest can be introduced into a host cell by any of a number of appropriate methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microparticle bombardment; lipofection and infection (for example, if the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on the characteristics of the host cell.

Any host cells capable of overexpressing heterologous DNA can be used to isolate genes encoding an antibody, polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. Preferably, the host cells express the cDNA at a level that is about 5 times higher, more preferably 10 times higher, even more preferably 20 times higher than that of the corresponding endogenous antibody or protein of interest, if present, in the cells - the owners. Screening host cells for specific CD3 binding is performed using an immunoassay or FACS. A cell overexpressing an antibody or protein of interest can be identified.

III. Methods for screening polypeptides and monoclonal antibodies

Several methods can be used to screen for polypeptides and monoclonal antibodies that bind to CD3. It is understood that binding refers to biologically or immunologically relevant specific binding and does not refer to non-specific binding, which may occur, for example, when using immunoglobulin in a very high concentration against a non-specific target. In one embodiment, the monoclonal antibodies are screened for binding to CD3 using standard screening methods. In this way, a monoclonal antibody against CD3 can be obtained. Preferred hybridomas of the present invention are those that produce antibodies mAb1 and mAb2, or chimeric or humanized derivatives thereof. However, additional monoclonal antibodies can be identified that bind to CD3. To this end, monoclonal antibodies are screened for their differential ability to bind to human CD3 as well as primate CD3.

Any of several different detection systems can be used to detect antibody binding to a tissue section. Typically, an immunohistochemical study involves binding a first antibody to the tissue, followed by a second antibody directed against the species from which the first antibody was derived and conjugated to a detectable marker (eg, horseradish peroxidase (HRP) or diaminobenzidine (DAB)). One alternative method that can be used is polyclonal Mirror Image Complementary Antibodies or polyMICA™ (The Binding Site Limited, Birmingham, UK; Mangham, D.C. et al. (1999) A Novel Immunohistochemical Detection System Using Mirror Image Complementary Antibodies (MICA), Histopathology 35(2): 129-33). The PolyMICA™ method can be used to test the binding of first antibodies (eg, anti-CD3 antibodies) to normal and cancer tissue. There are several types of polyMICA™ detection kits commercially available: Product No. HK004.D is a polyMICA™ detection kit that uses the DAB chromogen; Product No. HK004.A is a polyMICA™ detection kit that uses the AEC chromogen. Alternatively, the first antibody can be directly labeled with a detectable marker.

IV. Methods for characterizing anti-CD3 antibodies

Any of several methods can be used to characterize anti-CD3 antibodies. One way is to identify the epitope to which it binds. Epitope mapping is available on the market from various suppliers, such as Pepscan Systems (Lelystad, The Netherlands). Epitope mapping can be used to determine the sequence to which an anti-CD3 antibody binds. The epitope may be a linear epitope, i.e. contained in a single fragment of amino acids, or a conformational epitope formed as a result of the spatial interaction of amino acids that may not necessarily be contained in a single fragment.

Peptides of various lengths (e.g., preferably at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used for binding assays

- 17 045070 with anti-CD3 antibody. The epitope to which the anti-CD3 antibody binds can be determined by methodical screening using overlapping peptides derived from the extracellular sequence and detecting binding by the anti-CD3 antibody.

However, another method that can be used to characterize an anti-CD3 antibody is the use of competition assays with other antibodies that are known to bind to the same antigen, i.e. CD3, to determine whether anti-CD3 antibodies bind to the same epitope as other antibodies. Examples of commercially available anti-CD3 antibodies may be available and can be identified using the binding assays set forth herein. Competition analyzes are well known to those skilled in the art, and such procedures and explanatory data are detailed below in the Examples section. Antibodies against CD3 can further be characterized according to the tissue, type of cancer or type of tumor to which they bind.

V. Preferred Compositions of the Present Invention

The present invention covers compositions, including pharmaceutical compositions, comprising anti-CD3 antibodies, polypeptides derived from anti-CD3 antibodies, polynucleotides comprising a sequence encoding anti-CD3 antibodies, and other agents described herein. The present invention further provides conjugates of any CD3 peptide agonist, antagonist or modulator and additional chemical structures that implement the intended function or functions of the particular CD3 peptide agonist, antagonist or modulator. These conjugates include a CD3 peptide agonist, antagonist or modulator covalently linked to a macromolecule, such as any insoluble, solid support matrix used in the diagnostic, screening or purification methods discussed herein. Suitable matrix materials include any substance that is chemically inert, has a high degree of porosity, and has a large number of functional groups capable of forming covalent bonds with peptide ligands. Examples of matrix materials and methods for preparing matrix-ligand conjugates are given in Dean et al. (Eds) AFFINITY CHROMATOGRAPHY: A PRACTICAL APPROACH, IRL Press (1985); Lowe, An Introduction to Affinity Chromatography, in Work et al. (eds) LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, Vol. 7, Part II, NorthHolland (1979); Porath et al., Biospecific Affinity Chromatography, in Neurath, H. et al. (eds), THE PROTEINS, 3rd ed., Vol. 1, pp. 95-178 (1975); and Schott, H. AFFINITY CHROMATOGRAPHY, Macel Dekker, Inc. N.Y. (1984).

Also provided herein are conjugates of a CD3 agonist, antagonist or modulator peptide and any reporter moiety used in the diagnostic procedures described herein. The CD3 peptide agonist, antagonist or modulator agents, polypeptides and proteins of this invention, including anti-CD3 antibodies, are further identified and characterized according to any one or more of the following criteria:

(1) the ability to specifically bind to human CD3, which is endogenously present on the surface of normal human T cells;

(2) the ability to specifically bind to human CD3, which is endogenously present on the surface of human leukemia T cells;

(3) the ability to specifically bind to non-human CD3 (eg, cynomolgus CD3) that is endogenously present on the surface of normal non-human T cells;

(4) the ability to specifically bind to non-human CD3, which is endogenously present on the surface of non-human leukemia T cells;

(5) the ability to neutralize (ie, block or interfere with binding) complex formation with CD3; the ability to neutralize the formation of a complex with the TCR;

(6) the ability to modulate (either antagonistically or agonistically) T CR complex signaling;

(7) ability to bind to the Fc receptor;

(8) the ability to competitively inhibit the preferential binding of a known anti-CD3 antibody to CD3, including the ability to preferentially bind to the same CD3 epitope to which the parent antibody preferentially binds;

(9) the ability to bind to the portion of CD3 that is present on the surface of a living cell in vitro or in vivo; the ability to bind to the part of CD3 that is present on the surface of a living cancer cell;

(10) the ability to deliver a chemotherapeutic agent to a cancer T cell; and/or (11) the ability to deliver a therapeutic agent, toxin or detectable marker to a T Cell.

A preferred antibody of the present invention will exhibit differential IHC staining of tumor tissue relative to normal, non-cancerous tissue and will further be capable of testing in primate (and especially cynomolgus) models the effectiveness of the antibody.

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Preferred antibodies of the present invention will also exhibit desired levels of affinity and antigenic specificity. Preferred antibodies of the present invention will further exhibit desired levels of immunomodulatory activity and cellular internalization.

In some embodiments, an antibody of the present invention is an antibody that produces a mAb1 hybridoma or an mAb2 hybridoma, which respectively express a mouse mAb1 antibody and a mouse mAb2 antibody, or its progeny. Also covered by the present invention are various preparations of antibodies produced by these hybridomas and equivalent antibodies or polypeptide fragments (e.g. Fab, Fab', F(ab') ₂ Fv, Fc, etc.), chimeric antibodies, single chain (ScFv) , mutants thereof, fusion proteins comprising a portion of an antibody, humanized antibodies, and any other modified configuration of any of these or equivalent antibodies that includes an antigen recognition site (CD3) of the required specificity. The present invention also provides human antibodies exhibiting one or more biological characteristics of a member of the anti-CD3 family. Equivalent anti-CD3 family antibodies (including humanized and human antibodies), polypeptide fragments, and polypeptides comprising any of these fragments are identified and characterized in accordance with any one or more of the criteria described above.

Accordingly, the present invention provides any of the following (or compositions, including pharmaceutical compositions, comprising any of the following): (a) an antibody produced by a host cell or its progeny; (b) a humanized form of such antibody; (c) an antibody comprising one or more light chain and/or heavy chain variable regions of such an antibody; (d) a chimeric antibody comprising variable regions homologous to or derived from the variable regions of the heavy chain and light chain of such an antibody, and constant regions homologous to or derived from the constant regions of the heavy chain and light chain of a human antibody; (e) an antibody comprising one or more CDRs (at least one, two, three, four, five or six) of the light chain and/or heavy chain of such antibody; (f) an antibody comprising a heavy and/or light chain of such an antibody; (g) a human antibody that is equivalent to such an antibody. The humanized form of the antibody may or may not contain CDRs identical to those of the parent antibody or the host cell-produced antibody defined above. The determination of CDR regions is entirely part of the technical knowledge of the art. Other embodiments include antibodies that contain at least two, three, four, five or six CDRs that are substantially homologous to at least two, three, four, five or six CDRs of an antibody produced by a deposited hybridoma as defined herein, or come from such an antibody. It is understood that in the context of this invention, the binding specificity and/or overall activity is generally maintained, although the degree of activity may differ compared to the antibody produced by the deposited hybridoma (may be higher or lower). The present invention also provides methods for creating any of these antibodies. Methods for generating antibodies are known in the art and are described herein.

The present invention also provides polypeptides comprising the amino acid sequence of the antibodies of the present invention. In some embodiments, the polypeptides include one or more light chain and/or heavy chain variable regions of an antibody. In some embodiments, the polypeptides include one or more CDRs of an antibody light chain and/or heavy chain. In some embodiments, the polypeptides include three CDRs of an antibody light chain and/or heavy chain. In some embodiments, the polypeptide includes an antibody amino acid sequence that contains any of the following: at least 5 consecutive amino acids of the parent antibody sequence, at least 8 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids, wherein at least 3 of amino acids come from the variable region of the antibody. In one embodiment, the variable region is derived from the light chain of the parent antibody. In another embodiment, the variable region is derived from the heavy chain of an antibody. In another embodiment, 5 (or more) consecutive amino acids are derived from the complementarity determining region (CDR) of the antibody.

In some embodiments of this invention, cells of this invention that express CD3, a portion of CD3, anti-CD3 antibodies, and other CD3-binding polypeptides of this invention are administered directly to an individual to modulate the in vivo biological activity of CD3.

Preferred anti-CD3 antibodies of the present invention are mAb1 and mAb2, and humanized or chimeric derivatives and antigen-binding fragments thereof, which react with the human and cynomolgus CD3 molecule. Below are the amino acid sequences of the variable region of the light chain and the variable region of the heavy chain of the murine antibodies mAbl and mAb2 and the polynucleotide sequences encoding them. The CDR sequences of the exemplary antibodies (mAb1 and mAb2) are shown in bold and underlined.

A. Sequences of the variable regions of the mouse monoclonal antibody mAb1

Amino acid sequence of the murine monoclonal antibody light chain variable region mAb1 (SEQ ID NO: 1):

QWLTQSPAI MSAFPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDS SKLASGVPAR FSGSGSGTSY SLTISSMETE DAATYYCQQW SRNPPTFGGG TKLQITR

Polynucleotide sequence encoding the light chain variable region of the mouse monoclonal antibody mAb1 (SEQ ID NO: 2):

caggtggtgc tgacccagtc ccccgccatc atgtccgcct tccccggcga gaaagtgaca atgacctgct ccgcctcctc ctccgtgtcc tacatgaact ggtatcagca gaagtccggc acctccccca agcggtggat ctacgactcc tccaagctgg cctccggcgt gcccgccaga ttct ctggct ccggctccgg caccagctac tccctgacca tctcctccat ggaaaccgag gacgccgcca cctactactg ccagcagtgg tcccggaacc cccctacctt cggcggaggc accaagctgc agatcaccag a

Amino acid sequence of the murine monoclonal antibody mAb1 heavy chain variable region (SEQ ID NO: 3):

QVQLQQSGAE LARPGASVKM SCKASGYTFT RSTMHWVKQR PGQGLEWIGY INPSSAYTNY NQKFKDKATL TADKSSSTAY MQLSSLTSED SAVYYCASPQ VHYDYNGFPY WGQGTLVTVS S

Polynucleotide sequence encoding the heavy chain variable region of the murine monoclonal antibody mAb1 (SEQ ID NO: 4):

caggtgcagc tgcagcagtc tggcgccgag ctggccagac ctggcgcctc cgtgaagatg tcctgcaagg cctccggcta caccttcacc cggtccacca tgcactgggt gaaacagcgg cctggacagg gcctggaatg gatcggctac atcaacccct ccagcgccta caccaactac aaccagaagt t caaggacaa ggccaccctg accgccgaca agtcctccag caccgcctac atgcagctgt cctccctgac ctccgaggac tccgccgtgt actactgcgc ctccccccag gtgcactacg actacaacgg cttcccctac tggggccagg gcaccctggt gacagtgtcc tcc

B. Sequences of the variable regions of the mouse monoclonal antibody mAb2

Amino acid sequence of the murine monoclonal antibody light chain variable region mAb2 (SEQ ID NO: 5):

IQAVVTQESAL TTSPGETVTL TCRSSTGAVT TSNYANWVQE KPDHLFTGLI GGTNKRAPGV PARFSGSLIG DKAALTITGA QTEDEAIYFC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding the light chain variable region of the murine monoclonal antibody mAb2 (SEQ ID NO: 6):

caggccgtgg tgacacagga gtcagctctg accacatccc caggcgaaac agtgactctg acctgcagat ccagcactgg agcagtgact acctctaact acgctaattg ggtgcaggag aagcccgacc acctgttcac tgggctgatc ggcggaacca acaaaagggc acccggtgtg cctgc ccggt tttctggcag tctgatcgga gacaaggccg ctctgacaat tactggcgcc cagacagagg atgaagctat ttacttctgt gcactgtggt atagcaatct gtgggtgttt gggggtggca ccaaactgac agtgctggga

Amino acid sequence of the murine monoclonal antibody mAb2 heavy chain variable region (SEQ ID NO: 7):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYADSVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Polynucleotide sequence encoding the heavy chain variable region of the murine monoclonal antibody mAb2 (SEQ ID NO: 8): gaggtgaagc tgctggaaag cggcggagga ctggtgcagc caaagggatc actgaaactg tcctgcgccg cctccggctt cacctttaac acatacgcta tgaattgggt gcgacaggca cctggcaagg gcct ggagtg ggtggcaagg atcaggtcca agtacaacaa ttatgcaacc tactatgccg actctgtgaa ggatagattc acaatcagtc gcgacgattc ccagagcatt ctgtatctgc agatgaacaa tctgaaaact gaagacaccg ccatgtacta ttgtgtgcgg cacggtaact tcggcaattc ttacgtgtct tggtttgctt attggggaca ggggacactg gtgactgtgt cttcc

Position 40 of the heavy chain is the position of the anchor residue of the MHC class II high affinity binding peptide. Positions 44, 48, 54, 94, 99 and 108 of the heavy chain are the anchor residue positions of the MHC class II intermediate affinity binding peptide. Position 69 of the light chain is the position of the anchor residue of the high affinity binding peptide

- 20 045070 class II MHC. Position 59 of the light chain is the position of the anchor residue of the MHC class II intermediate affinity binding peptide. These residues can be replaced, using standard molecular biology techniques, with any residue to weaken or eliminate the recognition site

MHC class II.

C. Anti-CD3 antibodies with engineered Fc

During normal functioning of the immune system, the interaction of antibody-antigen complexes with cells of the immune system results in a wide range of responses ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation and phagocytosis to immunomodulatory signals such as proliferation of regulatory lymphocytes and antibody secretion. Any of these interactions are initiated by the binding of the Fc domain of antibodies or immune complexes to specialized cell surface receptors of hematopoietic cells. The diversity of cellular responses triggered by antibodies and immune complexes is a consequence of the structural heterogeneity of three Fc receptors: FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16). FcyRI (CD64), FcyRIIA (CD32A) and FcyRI11 (CD16) are activating (ie, enhancing the immune system) receptors; FcyRIIB (CD32B) is an inhibitory (ie, calms the immune system) receptor. The amino acid sequence of the Fc region of IgG1 is presented below (as SEQ ID NO:9, numbered according to Kabat et al. (SEQUENCE OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, NIH, MD (1991), which is included in the present description in verbal form by reference, below referred to as Kabat EU) Residues 230-341 are the CH2 region of the Fc. Residues 342-447 are the CH3 region of the Fc.

SEQ ID NO:9

PAPELLGGPS 230 VFLFPPKPKD 240 TLMISRTPEV 250 TCVVVDVSHE 260 DPEVKFNWYV 270 DGVEVHNAKT 280 KPREEQYNST 290 YRVVSVLTVL 300 HQDWLNGKEY 310 KCKVSNKALP 320 APIEKTISKA 330 KGQPREPQVY 340 TLPPSREEMT 350 KNQVSLTCLV 360 KGFYPSDIAV 370 EWESNGQPEN 380 NYKTTPPPVLD 390 SDGSFFLYSK 400 LTVDKSRWQQ 410 GNVFSCSVMH 420

EALHNHYTQK SLSLSPGK 430 440

Because non-Fc receptor (FcR) CD3-specific antibodies are minimally depleting, it has been hypothesized that they may alter TCR signaling such that immunological tolerance could be induced (St. Clair E.W. (2009) Novel Targeted Therapies for Autoimmunity, Curr Opin Immunol 21(6): 648–657. Thus, such therapy has potential application in the treatment of autoimmune disease and rejection due to host-versus-graft disease. Non-FcR binding CD3-specific antibodies have also been postulated to induce tolerance in the form of remission of type 1 diabetes mellitus (St. Clair E.W. (2009) Novel Targeted Therapies for Autoimmunity, Curr. Opin. Immunol. 21(6): 648- 657; Masharani, U. B. et al. (2010) Teplizumab Therapy For Type 1 Diabetes, Expert Opin. Biol. Ther. 10(3): 459-465).

Thus, the present invention includes CD3-specific binding antibodies that include variant Fc regions having Fc regions that are modified (eg, contain substitutions, deletions, insertions in one or more portions) to be unable or less capable of binding with the Fc receptor (compared to an antibody containing the same CDRs, but the wild-type Fc region).

In one embodiment, such antibodies will be incapable of binding to any Fc receptor. Alternatively, the Fc region of the antibody will be modified to enable it to bind to Fc receptors such as FcyRIIB, which are inhibitory, but not to Fc receptors such as FcyRIIA, FcyRIIIA or FcyRIIIB, which stimulate immune system activation.

Preferably, the binding abilities of the molecules of the present invention are determined using in vitro functional assays to determine one or more FcyR-mediated functions of effector cells. The affinities and binding abilities of molecules, such as antibodies, of the present invention to (c) FcyR can be determined using in vitro assays (biochemical or immunological assays) known in the art to determine antibody-antigen or Fc-FcyR interactions, i.e. e. specific antigen-antibody binding or Fc region-specific binding to FcyR, respectively, including, but not limited to, ELISA assay, surface plasmon resonance assay, immunoprecipitation assay. In most preferred embodiments, the molecules of the present invention have binding abilities in in vivo models (such as those described and disclosed herein) similar to those in in vitro assays. However, the present invention does not exclude molecules of the present invention that do not exhibit the desired phenotype in in vitro assays but exhibit the desired phenotype in vivo.

In some embodiments, molecules of the present invention including an Fc region variant include at least one amino acid modification (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications) in the CH3 -Fc region domain, which is defined as the domain located from amino acid 342 to amino acid 447. In other embodiments, molecules of the present invention comprising an Fc region variant include at least one amino acid modification (e.g., having 1, 2, 3 , 4, 5, 6, 7, 8, 9 or more amino acid modifications) in the CH2 domain of the Fc region, which is defined as the domain located from amino acid 231 to amino acid 341. In some embodiments, the molecules of the present invention include at least two amino acid modifications (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications), at least one such modification is in the CH3 region, and at least one such modification is in CH2 regions. The present invention further covers modification of an amino acid at the hinge region. In a specific embodiment, the present invention covers amino acid modification in the CH1 domain of the Fc region, which is defined as the domain located from amino acid 216 to amino acid 230.

In particularly preferred embodiments, the present invention covers molecules comprising an Fc region variant, wherein the variant imparts or possesses reduced ADCC activity and/or reduced binding to FcyRIIA (CD32A), as determined using methods known to one skilled in the art. specialist and are given here as an example. ADCC assays used in accordance with the methods of the present invention can be NK-dependent or macrophage-dependent.

In particularly preferred embodiments, the present invention covers molecules comprising an Fc region variant, wherein the variant imparts or possesses reduced ADCC activity and/or reduced binding to FcyRIIIA (CD16A), as determined using methods known to one skilled in the art. specialist and are given here as an example. ADCC assays used in accordance with the methods of the present invention can be NK-dependent or macrophage-dependent.

The Fc variants of the present invention may be in combination with other Fc modifications, such as those described in US Patent Nos. 7632497, 7521542, 7425619, 7416727, 7371826, 7355008, 7335742, 7332581, 7183387, 712263 7 and 6737056; in publications of PCT applications with No. WO 2008/105886; WO 2008/002933; WO 2007/021841; WO 2007/106707; WO 06/088494; WO 05/115452; WO 05/110474; WO 04/1032269 and WO 04/063351; and in Presta, L.G. et al. (2002) Engineering therapeutic antibodies for improved function, Biochem. Soc. Trans. 30(4): 487-490; Shields, R.L. et al. (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcgammaRIII and antibody-dependent cellular toxicity, J. Biol. Chem. 26; 277(30): 26733-26740, and Shields, R.L. et al. (2001) High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgGl variants with improved binding to the Fc gamma R, J. Biol. Chem. 276(9): 6591-6604). The present invention covers the combination of an Fc variant of the present invention with other Fc modifications to impart additive, synergistic or novel properties to the modified antibody.

In other embodiments, the present invention covers the use of any Fc variant known in the art, such as those described in

- 22 045070

Jefferis, V.J. et al. (2002) Interaction Sites On Human IgG-Fc For FcgammaR: Current Models, Immunol. Lett. 82: 57-65; Presta, L.G. et al. (2002) Engineering Therapeutic Antibodies For Improved Function, Biochem. Soc. Trans. 30: 487-90; Idusogie, E.E. et al. (2001) Engineered Antibodies With Increased Activity To Recruit Complement, J. Immunol. 166: 2571-75; Shields, R.L. et al. (2001) High Resolution Mapping Of The Binding Site On Human IgGl For Fc Gamma RI, Fc Gamma RI I, Fc Gamma RIII, And FcRn And Design Of IgGl Variants With Improved Binding To The Fc gamma R, J. Biol. Chem. 27 6: 6591-6604; Idusogie, E.E. et al. (2000) Mapping Of The Clq Binding Site On Rituxan, A Chimeric Antibody With A Human IgG Fc, J. Immunol. 164: 4178-84; Reddy, M.P. et al. (2000) Elimination Of Fc Receptor-Dependent Effector Functions Of A Modified IgG4 Monoclonal Antibody To Human CD4, J. Immunol. 164: 1925-1933; Xu, D. et al. (2000) In Vitro Characterization of Five Humanized OKT3 Effector Function Variant Antibodies, Cell. Immunol. 200: 16-26; Armour, K.L. et al. (1999)

Recombinant human IgG Molecules Lacking Fcgamma Receptor I Binding And Monocyte Triggering Activities, Eur. J. Immunol. 29: 2613-24; Jefferis, R. et al. (1996) Modulation Of

Fc(Gamma)R And Human Complement Activation By IgG3-Core Oligosaccharide Interactions, Immunol. Lett. 54: 101-04; Lund, J. et al. (1996) Multiple Interactions Of IgG With Its Core Oligosaccharide Can Modulate Recognition By Complement And Human Fc Gamma Receptor I And Influence The Synthesis Of Its Oligosaccharide Chains, J. Immunol. 157: 4963-4969; Hutchins et al. (1995) Improved Biodistribution, Tumor Targeting, And Reduced Immunogenicity In Mice With A Gamma 4 Variant Of Campath-1H, Proc. Natl. Acad. Sci. (U.S.A.) 92: 11980-84;

Jefferis, R. et al. (1995) Recognition Sites On Human IgG For Fc Gamma Receptors: The Role Of Glycosylation, Immunol. Lett. 44: 111-17; Lund, J. et al. (1995) Oligosaccharide-Protein

Interactions In IgG Can Modulate Recognition By Fc Gamma Receptors, FASEB J. 9: 115-19; Alegre, M. L. et al. (1994) A Non-Activating Humanized Anti-CD3 Monoclonal Antibody Retains Immunosuppressive Properties In Vivo, Transplantation 57: 1537-1543; Lund et al. (1992) Multiple Binding Sites On The CH2 Domain Of IgG For Mouse Fc Gamma RII, Mol. Immunol. 29: 53-59; Lund et al. (1991) Human Fc Gamma RI And Fc Gamma RII Interact With Distinct But Overlapping Sites On Human IgG, J. Immunol. 147: 2657-2662; Duncan, A.R. et al. (1988)

Localization Of The Binding Site For The Human High-Affinity Fc Receptor On IgG, Nature 332: 563-564;

in US patent Nos. 5624821, 5885573, 6194551, 7276586 and 7317091; and publications of PCT applications No. WO 00/42072 and WO 99/58572.

In some embodiments, an antibody of the present invention includes a variant Fc region (including an Fc derived from any type (e.g., IgG, IgE, IgM, IgD, IgA, and Y) Ig or class (e.g., IgGp IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ ) or a subclass of human immunoglobulins), wherein the Fc region variant includes at least one amino acid modification compared to the wild type Fc region, which (the Fc region variant) exhibits a reduced or

- 23 045070 abrogated binding to one or more effector ligands, as determined by standard assays known in the art and described herein, against a comparable molecule comprising a wild-type Fc region. In some embodiments, the Fc domain variant of an antibody of the present invention includes an amino acid modification (i.e., insertion, substitution, deletion) at one or more of residue positions 233, 234, 235, 236, 237, 238, 265, 270, 2-97, 2 98, 299. In a specific embodiment, the one or more amino acid modifications that reduce or abrogate binding to one or more effector ligands are a phenylalanine or proline substitution at position 233; substitution with alanine at position 234; substitution with alanine or glutamic acid at position 235; alanine substitution at position 236, alanine substitution at position 237, arginine substitution at position 238; substitution with alanine or glutamic acid at position 265; substitution with alanine or asparagine at position 270; substitution with alanine or glutamine at position 297; substitution with phenylalanine, asparagine or proline at position 298; a substitution with any amino acid other than serine or threonine at position 299, or a combination of two or more of the above substitutions. In some embodiments, an antibody of the present invention includes an Fc domain containing an alanine substitution at position 265 and at position 297; substitution with alanine at position 265 and glutamine at position 297; substitution with glutamic acid at position 265 and alanine at position 297; or a substitution with glutamic acid at position 265 and glutamine at position 297. In preferred embodiments, an antibody of the present invention includes an Fc domain containing a modification (eg, substitution, insertion, deletion) at position 234 and at position 235 of the Fc region. In a specific example according to this embodiment, the antibody of the present invention includes an Fc domain containing an alanine substitution at position 234 and a glutamic acid substitution at position 235. However, in a more preferred embodiment, the antibody of the present invention includes an Fc substitution containing alanine at position 234 and substitution with alanine at position 235.

In other embodiments, an antibody of the present invention includes an F region variant that exhibits reduced or abolished binding to one or more effector ligands, as determined by standard assays known in the art and described herein, relative to a comparable control molecule. In some embodiments, an antibody of the present invention has an Fc region exhibiting reduced or abolished binding to one or more effector ligands, which includes phenylalanine or proline at position 233; alanine at position 234; alanine or glutamic acid at position 235; alanine at position 236, alanine at position 237, arginine at position 238; alanine or glutamic acid at position 265; alanine or asparagine at position 270; alanine or glutamine at position 297; phenylalanine, asparagine or proline at position 298; any amino acid other than serine or threonine at position 299 or a combination of two or more of the above substitutions. In some embodiments, an antibody of the present invention includes an Fc domain containing an alanine at position 265 and at position 297; alanine at position 265 and glutamine at position 297; glutamic acid at position 265 and alanine at position 297; or glutamic acid at position 265 and glutamine at position 297. In some embodiments, an antibody of the present invention includes an Fc domain containing an alanine at position 234 and a glutamic acid at position 235. In preferred embodiments, an antibody of the present invention includes an Fc domain containing an alanine at position 234 and alanine at position 235.

Antibodies of the present invention that include an Fc domain containing alanine at positions corresponding to 234 and 235 according to the Kabat numbering system are known as ala-ala antibodies. In some embodiments, the use of ala-ala Fc domains and/or other combinations of the amino acid combinations described herein (including combinations including ala-ala Fc domains) can abrogate the binding of the Fc domain to all FcyRs. The binding of an Fc domain to one or more FcyRs can be determined using any method described herein and/or known in the art.

In some embodiments, one or more amino acid modifications that abolish binding to all FcyRs or reduce or abrogate binding to one or more effector ligands include combinations of modifications listed herein, or combinations of modifications listed herein with any of those that may provide zero-order binding to any FcR (eg, FcyRIIIA, FcyRIIIB, FcyRIIA) as determined by methods described herein or known to one skilled in the art. As will be readily appreciated by one skilled in the art, such antibodies of the present invention may find particular use in the treatment of an autoimmune disease in which anti-CD3 antibodies and antigen binding fragments serve to modulate immune system function without the associated first dose response characteristic of for antibodies against immunocytes.

In some embodiments, anti-CD3 antibodies and antigen-binding fragments

- 24 045070 of the present invention, or antigen binding fragments thereof, are characterized by a reduced (for example, but not limited to, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% compared to binding to a protein comprising a control Fc domain) or, more preferably, undetectable binding to one or more of any FcyRs (eg, FcyRI, FcyRII or FcyRIII) due to its Fc domain, as determined by assays conventional in the art. Additionally or alternatively, the anti-CD3 antibodies and antigen binding fragments of the present invention, or antigen binding fragments thereof, may have reduced (e.g., but not limited to, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less 5% or less than 1% compared to binding to a control protein comprising a control Fc domain) or, more preferably, no detectable binding to any complement receptors such as Clq, as determined in commonly used assays. In certain embodiments, the antibody is non-glycosylated. In other embodiments, the antibody lacks an Fc domain (eg, it is a Fab fragment, F(ab') ₂ or a single chain antibody).

Thus, the antibodies of the present invention are particularly useful because they have reduced or no in vivo toxicity due to lymphokine production or cytokine release. Methods for determining lymphokine production and cytokine release are known and routine in the art and are included herein. For example, cytokine release can be determined by measuring the secretion of cytokines including, but not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-b (IL-6), interleukin-12 (IL- 12), interleukin-16 (IL-16), PDGF, TGF-α, TGF-β, TNF-α, TNF-β, GCSF, GM-CSF, MCSF, IFN-α, IFN-β, TFN-y, IGF-I, IGF-II. For example, see Isaacs et al., 2001, Rheumatology, 40: 724738; Soubrane et al., 1993, Blood, 81(1): 15-19; all of which are incorporated herein by reference in their entirety.

D. CD3-specific DART™ diabodies

As discussed above, the present invention further covers bispecific, trispecific and multispecific antibodies. A particularly preferred example of such antibodies includes DART™ diabody molecules that comprise at least two polypeptide chains that form at least two epitope binding sites, at least one of which specifically binds to CD3. Exemplary DART™ diabody molecules are presented in US 20100174053, US 20090060910, US 20070004909, EP 2158221, EP 1868650, WO 2010080538, WO 2008157379 and WO 20061136 65.

In preferred embodiments, the first polypeptide chain of the DART™ diabody includes:

(i) a domain (A) comprising an epitope-specific first immunoglobulin light chain variable domain (VL1) binding region (1);

(ii) a domain (B) comprising an epitope-specific second immunoglobulin heavy chain variable domain (VH2) binding region (2); and (iii) domain (C).

The second polypeptide chain of the DART™ diabody includes:

(iv) a domain (D) comprising an epitope-specific second immunoglobulin light chain variable domain (VL2) binding region (2);

(v) a domain (E) comprising an epitope-specific first immunoglobulin heavy chain variable domain (VH1) binding region (1); and (vi) domain (F).

Domains (A) and (B) of the DART™ diabody do not associate with each other to form an epitope-binding site. Also, domains (D) and (E) of the DART™ diabody do not associate with each other to form an epitope-binding site. More specifically, domains (A) and (E) of the DART™ diabody associate to form a binding site that binds epitope (1); said domains (B) and (D) of the DART™ diabody associate to form a binding site that binds said epitope (2). Domains (C) and (F) are covalently linked together.

Each polypeptide chain of a DART™ diabody molecule includes a VL domain and a VH domain, which are covalently linked so that self-assembly of the domains is difficult. The interaction of two of the polypeptide chains will result in two VL-VH pairings, forming two epitope-binding sites, i.e. divalent molecule. Neither the VH domain nor the VL domain is limited to any position in the polypeptide chain, i.e. is not limited to the amino (N) terminus or the carboxyl (C) terminus, nor are the domains limited to their positions relative to each other, i.e. The VL domain may be N-terminal to the VH domain and vice versa. The only limitation is that a complementary polypeptide chain must be available to form functional DART™ diabodies. If the VL and VH domains are derived from the same antibody, the two complementary polypeptide chains may be identical. For example, if the binding domains are derived from an antibody, specifically

- 25 045070 with respect to epitope A (ie the binding domain is formed based on the VLA-VHA interaction), each polypeptide will include VHA and VLA. Homodimerization of the two polypeptide chains of an antibody will result in the formation of two VLA-VHA binding sites, resulting in a bivalent monospecific antibody. If the VL and VH domains are derived from antibodies specific for different antigens, the formation of a functional DART™ bispecific diabody requires the interaction of two different polypeptide chains, i.e. heterodimer formation. For example, in the case of a DART™ bispecific diabody, one polypeptide chain will include VLA and VLB; homodimerization of said chain will result in the formation of two VL _A -VH _B binding sites, either non-binding or unpredictably binding. In contrast, if two different polypeptide chains can interact, for example in a recombinant expression system, one comprising VL _A and VH _B and the other comprising VL _B and VH _A , two different binding sites will be formed: VLA-VHA and VLB-VHB. In the case of all pairs of polypeptide chains of DART™ diabody, there is a possibility of misalignment or incorrect binding of the two chains, i.e. VL-VL or VH-VH domain interactions; however, it is easy to purify functional antibodies based on the immunospecificity of properly dimerized binding sites using any affinity method known in the art or exemplified herein, such as affinity chromatography.

One or more of the polypeptide chains of a DART™ diabody may optionally include an Fc domain or a portion thereof (eg, a CH2 domain or a CH3 domain). The Fc domain or portion thereof may be derived from any isotype or allotype of immunoglobulins, including, but not limited to, IgA, IgD, IgG, IgE, and IgM. In preferred embodiments, the Fc domain (or part thereof) is derived from IgG. In specific embodiments, the IgG isotype is IgG1, IgG2, IgG3, or IgG4, or an allotype thereof. In one embodiment, the diabody molecule includes an Fc domain including a CH2 domain and a CH3 domain that are independently selected from any immunoglobulin isotype (i.e., an Fc domain including a CH2 domain derived from IgG and a CH3 domain derived from IgE, or CH2 domain derived from IgG1 and CH3 domain derived from IgG2, etc.). The Fc domain may be designed in a polypeptide chain comprising a diabody molecule of the present invention at any position relative to other domains or portions of said polypeptide chain (e.g., the Fc domain, or a portion thereof, may be C-terminal to both VL and VH domains of a polypeptide chain; may be N-terminal to both VL and VH domains; or may be N-terminal to one domain and C-terminal to the other (i.e., located between two domains of a polypeptide chain )).

The Fc domains in the polypeptide chains of DART™ diabody molecules preferentially undergo dimerization, resulting in a DART™ molecule that exhibits properties similar to those of immunoglobulins, such as Fc-Fcy interactions. Fc-containing diabodies can be dimers, for example, consisting of two polypeptide chains, each of which includes a VH domain, a VL domain and an Fc domain. Dimerization of these polypeptide chains results in the formation of a divalent diabody DART™, including an Fc domain, albeit with a structure different from that of the unmodified divalent antibody. Such DART™ diabody molecules will exhibit altered phenotypes compared to wild-type immunoglobulin, such as altered serum half-life, binding abilities, etc. In other embodiments, DART™ diabody molecules comprising Fc domains may be tetramers. Such tetramers include two heavier polypeptide chains, i.e. a polypeptide chain including VL, VH and Fc domain, and two lighter polypeptide chains, i.e. polypeptide chain including VL and VH. Lighter and heavier chains interact to form a monomer, and these monomers interact through their unpaired Fc domains to form an Ig-like molecule. This DART™ Ig-like diabody is tetravalent and may be monospecific, bispecific or tetraspecific.

VI. Therapeutic Uses of Anti-CD3 Antibodies of the Present Invention

The anti-CD3 antibodies of the present invention and antigen-binding fragments thereof are particularly useful for the treatment of cancers associated with CD3 expression and for the treatment of autoimmune disease and other inflammatory disorders.

These applications may result in the formation of a complex between CD3 and an antibody that specifically binds to CD3. Examples of such antibodies include, but are not limited to, the anti-CD3 monoclonal antibodies mAb1 and mAb2 or, more preferably, humanized derivatives thereof. The formation of such a complex can occur in vitro or in vivo. Without being limited by theory, an anti-CD3 antibody may bind to CD3 through the extracellular domain of CD3 and may then be internalized into a living normal or cancer cell.

A. Cancer treatment

The antibodies and antigen binding fragments of the present invention bind to CD3 present on the surface of T cells. Antigen binding fragments of the present invention

- 26 045070 can be used in the case of a bispecific (or trispecific or polyspecific) molecule, such as a DART or BiTE molecule, to redirect T cells to a tumor cell. The cell can then destroy the tumor cell. The bispecific (or trispecific or polyspecific) molecules of the present invention are capable of binding to both human and non-human mammal CD3 (e.g., cynomolgus monkey), as well as second (or additional) and distinct antigen(s) and epitope(s). The second antigen or epitope is preferably a tumor-specific antigen presented on the tumor cell. Such tumor cells may be from malignant neoplasms, for example, breast cancer, prostate cancer, stomach cancer, lung cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cancer, pharynx cancer, esophageal cancer, laryngeal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, bladder cancer, kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma and leukemia. Such additional antigens or epitopes are preferably tumor-specific antigens or epitopes on the cell surface (such as 17-1A, A33, major endodermal antigen I on adult erythrocytes, alpha-fetoprotein, tumor RNA virus envelope antigen, tumor-specific oncoembryonic antigen bladder, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, Burkitt's lymphoma-specific antigen-38.13, CA125, CD18, CD19, human B-cell lymphoma-specific antigen- CD20, CD22, CD33, CD44, CD52, CEA, CO17-1A, CTA-1, CTLA-4, epidermal growth factor receptor, EpCAM, EphA2, antigen I on newborn erythrocytes, fibrosarcoma antigen, ganglioside GD2, ganglioside Gd3, ganglioside GM2 , ganglioside GM3, GICA 19-9, gp IIIb/IIIa, gp72, HER1, HER-2/neu, HER3, HER4, melanoma-specific high molecular weight antigen, HLA-DR antigen, human leukemia-specific T-cell antigen -Gp37, human lung carcinoma-specific antigen L20, human lung carcinoma-specific antigen L6, human milk fat globular antigen, IgE, carcinoma-specific KS 1/4 pan antigen, LEA, adenocarcinoma-specific antigen F3, specific for human malignant lymphocyte antigen-APO-1, melanoma-specific antigen gp75, melanoma-associated antigen p97, neoglycoprotein, nuC242, polymorphic epithelium-specific antigen mucin type, prostate-specific antigen, prostate-specific membrane antigen, prostate-specific acid phosphate, SK-1 antigen, TAG-72, Tantigen, tumor-specific antigen CA125, tumor-specific antigen MUC1, tumor-specific cell surface transplantation antigen, vascular endothelial growth factor, vascular endothelial growth factor receptor and ανβ3). Alternatively, such additional antigens or epitopes may be associated with a pathogen (such as: hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, varicella zoster virus, adenovirus, herpes simplex virus type I (HSV-I) , herpes simplex virus type II (HSV-II), rinderpest pathogen, rhinovirus, ECHOvirus, rotavirus, respiratory syncytial virus, papillomavirus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackievirus, mumps virus, measles virus , rubella virus, poliovirus, the causative agent of smallpox, Epstein-Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), the causative agent of viral meningitis, the causative agent of viral encephalitis, the causative agent of dengue, the causative agent of natural smallpox, mycobacteria, rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia burgdorferi, Bacillus anthracis, Streptococcus, Staphylococcus, Mycobacterium, tetanus, whooping cough, cholera, plague, diphtheria, chlamydia and legionella; leishmania, the causative agent of coccidiosis, trypanosome or the causative agent of malaria; chlamydia and rickettsia).

The antibodies and antigen binding fragments of the present invention bind to CD3 present on the surface of T cells. Using conventional methods, such antibodies can be labeled with fluorescein as described above. When such labeled molecules are incubated in the presence of a bispecific molecule (such as, for example, a UDART™ diabody containing an epitope-binding domain that binds to the T-cell receptor and an epitope-binding domain that binds to fluorescein (TCR-UDART™)), they can bind labeled with β-fluorescein - and thus localize themselves on the surface of cells that express CD3, and lead to redirected destruction of such cells.

In an alternative embodiment, CD19 can be used as a second epitope such that a bispecific antibody, or more preferably a DART™ diabody, recognizing CD3 and CD19 is used to kill B cell lymphoma by contacting a B cell specific antigen ( CD19), and with the T-cell receptor/CD3 complex on effector T cells. As discussed in Moore, P.A. et al. (2011) Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma, Blood 2011 blood-2010-09306449, CD3/CD19-specific DART™ diabody was used to kill B-cell lymphoma through contact and with a B cell-specific antigen (CD19), and with the T cell receptor/CD3 complex on effector T cells. In a side-by-side comparison with a single chain bispecific antibody containing anti-CD 19 and anti-CD3 Fv sequences, DART was found to be more effective in inducing B cell lysis. Increased activity with CD19χCD3 bispecific DART was observed against all CD19-expressing target B cells when assessed using resting and prestimulated human PBMC or purified effector T cell populations. Characterization of the CD19χTCR bispecific DART revealed efficacy equivalent to that of the CD19xCD3 DART, indicating the flexibility of the DART structure in maintaining T cell/B cell associations for redirected T cell-mediated killing applications. It is important to note that the increased level of killing mediated by DART molecules was not accompanied by any increase in the activation of nonspecific T cells or the lysis of CD19-negative cells. Cell-cell association studies indicate that the DART structure is well suited to maintain cell-cell contacts that undoubtedly contribute to high levels of target cell killing. Finally, the ability of CD19xTCR-bispecific DART to inhibit B-cell lymphoma in NOD/SCID mice when co-administered with human PBMC further demonstrates the value of DART molecules for the treatment of B-cell malignancies. The cross-reactive anti-CD3 antibodies of the present invention could be used in the same manner as the anti-CD3 antibodies of Moore, P. A., et al. Thus, the present invention provides therapy for cancers (especially lymphomas and leukemias) involving CD3-expressing cancer cells.

The bispecific (or trispecific or polyspecific) molecules of the present invention are preferably administered to a patient in one or more unit dosages, typically ranging from 0.0001 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dose administered to the patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg /kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.7 5 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg and 0, 25 mg/kg, 0.0001 and 0.15 mg/kg, 0.0001 and 0.10 mg/kg, 0.001 and 0.5 mg/kg, 0.01 and 0.25 mg/kg or 0.01 and 0.10 mg/kg of the patient's body weight.

B. Treatment of autoimmune disease and inflammation

The present invention also provides methods for treating, preventing, slowing the progression and/or reducing the severity of symptoms of T cell-mediated diseases or disorders, including transplant rejection, homologous disease, delayed-type adverse hypersensitivity reactions (such as delayed-type allergic reactions) mediated by T cells lung diseases and autoimmune diseases. T cell-mediated lung diseases include sarcoidosis, allergic alveolitis, acute interstitial pneumonitis, alveolitis, pulmonary fibrosis, idiopathic pulmonary fibrosis and other diseases characterized by inflammatory lung disease. T cell-mediated autoimmune diseases include multiple sclerosis, neuritis, polymyositis, psoriasis, vitiligo, Gougerot-Sjögren syndrome, rheumatoid arthritis, type I diabetes, autoimmune pancreatitis, inflammatory bowel diseases (eg, Crohn's disease and ulcerative colitis), celiac disease, glomerulonephritis, scleroderma, sarcoidosis, autoimmune thyroid diseases (for example, Hashimoto's goiter and Graves' disease), myasthenia gravis, Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia and systemic lupus erythematosus (in particular the cutaneous form), effects due to organ transplantation, homologous disease (GVHD), etc. In particular, the methods of the present invention are useful for subjects with early stage disease to slow or reduce impairment due to autoimmunity and maintain a high level of functioning and/or reduce the need for other therapy (for example, in the treatment or prevention of type I diabetes mellitus, the methods of the present invention may reduce the need to administer exogenous insulin to the subject). In addition, the methods of the present invention may advantageously reduce or eliminate the incidence of cytokine release syndrome previously associated with the administration of therapeutic antibodies and, in particular, anti-T cell antibodies (eg, anti-CD3) or antigen binding fragments.

In some embodiments, treatment with anti-CD3 antibody or antigen-binding fragments in accordance with the methods of the present invention is repeated at intervals of 2 months, 4 months, 6 months, 8 months, 9 months, 10 months, 12 months, 15 months, 18 months, 24 months, 30 months or 36 months. In specific embodiments, the effectiveness of treatment with the anti-CD3 antibody or antigen-binding fragments of the present invention is determined as described herein or as known in the art at 2 months, 4 months, 6 months, 9 months, 12 months, 15 months, 18 months, 24 months, 30 months or 36 months after previous treatment.

In another embodiment, the subject is administered one or more unit doses of about 0.5-50 μg/kg, about 0.5-40 μg/kg, about 0.5-30 μg/kg, about 0.5-20 μg /kg, approximately 0.5-15 μg/kg, approximately 0.5-10 μg/kg, approximately 0.5-5

- 28 045070 µg/kg, approximately 1-5 µg/kg, approximately 1-10 µg/kg, approximately 20-40 µg/kg, approximately 20-30 µg/kg, approximately 22-28 µg/kg or approximately 25- 26 μg/kg of one or more anti-CD3 antibodies or antigen-binding fragments for the prevention, treatment or amelioration of one or more symptoms of an autoimmune disorder or T-cell malignancy. In another embodiment, the subject is administered one or more unit doses of 200 μg/kg, 178 μg/kg, 180 μg/kg, 128 μg/kg, 100 μg/kg, 95 μg/kg, 90 μg/kg, 85 μg /kg, 80 mcg/kg, 75 mcg/kg, 70 mcg/kg, 65 mcg/kg, 60 mcg/kg, 55 mcg/kg, 50 mcg/kg, 45 mcg/kg, 40 mcg/kg, 35 mcg /kg, 30 mcg/kg, 26 mcg/kg, 25 mcg/kg, 20 mcg/kg, 15 mcg/kg, 13 mcg/kg, 10 mcg/kg, 6.5 mcg/kg, 5 mcg/kg, 3.2 µg/kg, 3 µg/kg, 2.5 µg/kg, 2 µg/kg, 1.6 µg/kg, 1.5 µg/kg, 1 µg/kg, 0.5 µg/kg, 0.25 mcg/kg, 0.1 mcg/kg, or 0.05 mcg/kg of one or more anti-CD3 antibodies or antigen-binding fragments for the prevention, treatment, or amelioration of one or more symptoms of an autoimmune disorder or T-cell malignancy.

In one embodiment, the subject is administered one or more doses of 200 μg/kg or less, 175 μg/kg or less, 150 μg/kg or less, 128 μg/kg or less, 100 μg/kg or less, 95 μg/kg kg or less, 90 mcg/kg or less, 85 mcg/kg or less, 80 mcg/kg or less, 75 mcg/kg or less, 70 mcg/kg or less, 65 mcg/kg or less, 60 mcg/kg or less, 55 mcg/kg or less, 50 mcg/kg or less, 45 mcg/kg or less, 40 mcg/kg or less, 35 mcg/kg or less, 30 mcg/kg or less, 25 mcg/kg or less, 20 mcg/kg or less, 15 mcg/kg or less, 10 mcg/kg or less, 5 mcg/kg or less, 2.5 mcg/kg or less, 2 mcg/kg or less, 1.5 mcg /kg or less, 1 μg/kg or less, 0.5 μg/kg or less, 0.25 μg/kg or less, 0.1 μg/kg or less, or 0.05 μg/kg or less one or more of anti-CD3 antibodies or antigen-binding fragments for the prevention, treatment or amelioration of one or more symptoms of an autoimmune disorder or T-cell malignancy.

In certain embodiments, the subject is administered one or more doses of approximately 5-1200 μg/m ² , preferably 51-826 μg/m ² . In another embodiment, the subject is administered one or more unit doses of 1200 μg/m ² , 1150 μg/m ² , 1100 μg/m ² , 1050 μg/m ² , 1000 μg/m ² , 950 μg/m ² , 900 µg/ ^m2 , 850 µg/ ^m2 , 800 µg/ ^m2 , 750 µg/ ^m2 , 700 µg/m2, 650 µg/ ^m2 , 600 µg/ ^m2 , 550 µg/ ^{m2 ,} 500 µg/ ^m2 , 450 μg/ ^m2 , 400 μg/ ^m2 , 350 μg/ ^m2 , 300 μg/ ^m2 , 250 μg/ ^m2 , 200 μg/ ^m2 , 150 μg/ ^m2 , 100 μg/ ^m2 , 50 µg/ ^m2 , 40 µg/ ^m2 , 30 µg/ ^m2 , 20 µg/ ^m2 , 15 µg/ ^m2 , 10 µg/ ^m2 or 5 µg/ ^m2 of one or more anti-CD3 antibodies or antigen-binding fragments to prevent, treat, slow the progression, delay the onset or reduce the intensity of one or more symptoms of an autoimmune disorder or T-cell malignancy.

In another embodiment, the subject is prescribed a treatment regimen comprising one or more doses of a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen binding fragments, wherein the treatment is administered for 2 days, 3 days, 4 days, 5 days, 6 days , 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days. In one embodiment, the treatment regimen includes dosing a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen binding fragments every day, every other day, every third day, or every fourth day. In some embodiments, the treatment regimen includes dosing a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen-binding fragments on Monday, Tuesday, Wednesday, Thursday of a particular week and not dosing a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies. or antigen-binding fragments on Friday, Saturday, or Sunday of the same week until 14 doses, 13 doses, 12 doses, 11 doses, 10 doses, 9 doses, or 8 doses have been administered. In some embodiments, the dose administered is the same on each day of the regimen. In some embodiments, the subject is prescribed a treatment regimen comprising one or more doses of a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen binding fragments, wherein the prophylactically or therapeutically effective amount is 200 μg/kg/day, 175 μg/kg/day , 150 mcg/kg/day, 125 mcg/kg/day, 100 mcg/kg/day, 95 mcg/kg/day, 90 mcg/kg/day, 85 mcg/kg/day, 80 mcg/kg/day, 75 mcg/kg/day, 70 mcg/kg/day, 65 mcg/kg/day, 60 mcg/kg/day, 55 mcg/kg/day, 50 mcg/kg/day, 45 mcg/kg/day, 40 mcg/kg/day, 35 mcg/kg/day, 30 mcg/kg/day, 26 mcg/kg/day, 25 mcg/kg/day, 20 mcg/kg/day, 15 mcg/kg/day, 13 mcg /kg/day, 10 mcg/kg/day, 6.5 mcg/kg/day, 5 mcg/kg/day, 3.2 mcg/kg/day, 3 mcg/kg/day, 2.5 mcg/kg /day, 2 mcg/kg/day, 1.6 mcg/kg/day, 1.5 mcg/kg/day, 1 mcg/kg/day, 0.5 mcg/kg/day, 0.25 mcg/kg /day, 0.1 mcg/kg/day or 0.05 mcg/kg/day; and/or wherein the prophylactically or therapeutically effective amount is 1200 μg/m ² /day, 1150 μg/m ² /day, 1100 μg/m ² /day, 1050 μg/m ² /day, 1000 μg/m ² /day, 950 µg/ ^m2 /day, 900 µg/ ^m2 /day, 850 µg/ ^m2 /day, 800 µg/ ^m2 /day, 750 µg/ ^m2 /day, 700 µg/ ^m2 /day, 650 µg /m ² /day, 600 μg/m ² /day, 550 μg/m ² /day, 500 μg/m ² /day, 450 μg/m ² /day, 400 μg/m ² /day, 350 μg/m ² /day, 300 µg/m ² /day, 250 µg/m ² /day, 200 µg/m ² /day, 150 µg/m ² /day, 100 µg/m ² /day, 50 µg/m ² / day, 40 µg/m ² /day, 30 µg/m ² /day, 20 µg/m ² /day, 15 µg/m ² /day, 10 µg/m ² /day or 5 µg/m ² /day. In another embodiment, an intravenous dose of 1200 μg/ ^m2 or less, 1150 μg/ ^m2 or less, 1100 μg/ ^m2 or less, 1050 μg/ ^m2

- 29 045070 or less, 1000 μg/ ^m2 or less, 950 μg/ ^m2 or less, 900 μg/m2 or less, 8 50 μg/ ^m2 or less, 800 μg/ ^m2 or less, ⁷⁵⁰ μg/ m ² or less, 700 μg/m ² or less, 650 μg/m ² or less, 600 μg/m ² or less, 550 μg/m ² or less, 50 0 μg/m ² or less, 450 μg/m ² or less, 400 µg/ ^m2 or less, 350 µg/ ^m2 or less, 300 µg/m2 or less, 250 µg/ ^m2 or less, 200 ^µg / ^m2 or less, 150 µg/ ^m2 or less, 100 µg/ ^m2 or less, 50 µg/ ^m2 or less, 40 µg/ ^m2 or less, 30 µg/m2 or less, 20 ^{µg/m2 or} less, 15 µg/ ^m2 or less , 10 μg/m ² or less, or 5 μg/m ² or less, of one or more anti-CD3 antibodies or antigen binding fragments, administered over about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, approximately 14 hours, approximately 12 hours, approximately 10 hours, approximately 8 hours, approximately 6 hours, approximately 4 hours, approximately 2 hours, approximately 1.5 hours, approximately 1 hour, approximately 50 minutes, approximately 40 minutes, approximately 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, about 2 minutes, about 1 minute, about 30 seconds, or about 10 seconds to prevent, treat, or reduce the intensity of one or more symptoms of an autoimmune disorder or T-cell malignancy. The total dose during the implementation of the regimen is preferably less than 9000 µg/ ^m2 , 8000 µg/ ^m2 , 7000 µg/ ^m2 , 6000 µg/ ^m2 and may be less than 5000 µg/ ^m2 , 4000 µg/ ^m2 , 3000 µg/ ^m2 , 2000 µg/ ^m2 or 1000 µg/ ^m2 . In specific embodiments, the total dose administered in the regimen is 100 μg/m ² -200 μg/m ² , 100 μg/m ² -500 μg/m ² , 100 μg/m ² -1000 μg/m ² , or 500 μg /m ² -1000 μg/m ² .

In preferred embodiments, the dose is increased over the first four, first half, and first 2/3 doses (e.g., during the first 2, 3, 4, 5, or 6 days of the dosing regimen for 10, 12, 14, 16, 18, or 20 days of one dose per day) of the treatment regimen until a daily prophylactically or therapeutically effective amount of one or more of the anti-CD3 antibodies or antigen-binding fragments is achieved. In some embodiments, the subject is prescribed a treatment regimen comprising one or more doses of a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen binding fragments, wherein the prophylactically or therapeutically effective amount is increased, for example, by 0.01 μg/kg, 0. 02 mcg/kg, 0.04 mcg/kg, 0.05 mcg/kg, 0.06 mcg/kg, 0.08 mcg/kg, 0.1 mcg/kg, 0.2 mcg/kg, 0.25 µg/kg, 0.5 µg/kg, 0.75 µg/kg, 1 µg/kg, 1.5 µg/kg, 2 µg/kg, 4 µg/kg, 5 µg/kg, 10 µg/kg, 15 mcg/kg, 20 mcg/kg, 25 mcg/kg, 30 mcg/kg, 35 mcg/kg, 40 mcg/kg, 45 mcg/kg, 50 mcg/kg, 55 mcg/kg, 60 mcg/kg, 65 mcg/kg, 70 mcg/kg, 75 mcg/kg, 80 mcg/kg, 85 mcg/kg, 90 mcg/kg, 95 mcg/kg, 100 mcg/kg, or 125 mcg/kg every day; or increase, for example, by 1 μg/ ^m2 , 5 μg/ ^m2 , 10 μg/ ^m2 , 15 μg/ ^{m2, 20 μg/m2} , 30 μg/ ^m2 , 40 μg/ ^m2 , 50 ^μg / ^m2 , 60 μg/ ^m2 , 70 μg/ ^m2 , 80 μg/ ^m2 , 90 μg/m2, 100 μg ^{/ m2} , 150 μg/ ^m2 , 200 μg/ ^m2 , 250 μg/m ² , 300 µg/ ^m2 , 350 µg/ ^m2 , 400 µg/ ^m2 , 450 µg/ ^m2 , 500 µg/ ^m2 , 550 µg/ ^m2 , 600 µg/ ^m2 or 650 µg/ ^m2 , every day during treatment. In some embodiments, the subject is prescribed a treatment regimen comprising one or more doses of a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen binding fragments, wherein the prophylactically or therapeutically effective amount is increased by 1.25 times, 1.5 times, 2 times, 2.25 times, 2.5 times or 5 times until the daily prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen-binding fragments is achieved.

In a specific embodiment, the subject is administered intramuscularly with one or more doses of 200 μg/kg or less, preferably 175 μg/kg or less, 150 μg/kg or less, 125 μg/kg or less, 100 μg/kg or less, 95 mcg/kg or less, 90 mcg/kg or less, 85 mcg/kg or less, 80 mcg/kg or less, 75 mcg/kg or less, 70 mcg/kg or less, 65 mcg/kg or less, 60 mcg /kg or less, 55 mcg/kg or less, 50 mcg/kg or less, 45 mcg/kg or less, 40 mcg/kg or less, 35 mcg/kg or less, 30 mcg/kg or less, 25 mcg/ kg or less, 20 mcg/kg or less, 15 mcg/kg or less, 10 mcg/kg or less, 5 mcg/kg or less, 2.5 mcg/kg or less, 2 mcg/kg or less, 1, 5 mcg/kg or less, 1 mcg/kg or less, 0.5 mcg/kg or less, or 0.5 mcg/kg or less of one or more anti-CD3 antibodies or antigen-binding fragments for the prevention, treatment, or reduction of one or more more symptoms of an autoimmune disorder or T-cell malignancy.

In another embodiment, the subject is administered subcutaneously one or more doses of 200 μg/kg or less, preferably 175 μg/kg or less, 150 μg/kg or less, 125 μg/kg or less, 100 μg/kg or less, 95 mcg/kg or less, 90 mcg/kg or less, 85 mcg/kg or less, 80 mcg/kg or less, 75 mcg/kg or less, 70 mcg/kg or less, 65 mcg/kg or less, 60 mcg /kg or less, 55 mcg/kg or less, 50 mcg/kg or less, 45 mcg/kg or less, 40 mcg/kg or less, 35 mcg/kg or less, 30 mcg/kg or less, 25 mcg/ kg or less, 20 mcg/kg or less, 15 mcg/kg or less, 10 mcg/kg or less, 5 mcg/kg or less, 2.5 mcg/kg or less, 2 mcg/kg or less, 1, 5 mcg/kg or less, 1 mcg/kg or less, 0.5 mcg/kg or less, or 0.5 mcg/kg or less of one or more anti-CD3 antibodies or antigen-binding fragments to prevent, treat, or reduce the intensity of one or more more symptoms of an autoimmune disorder.

In another embodiment, the subject is administered intravenously one or more doses of 100 μg/kg or less, preferably 95 μg/kg or less, 90 μg/kg or less, 85 μg/kg, or

- 30 045070 less, 80 mcg/kg or less, 75 mcg/kg or less, 70 mcg/kg or less, 65 mcg/kg or less, 60 mcg/kg or less, 55 mcg/kg or less, 50 mcg/ kg or less, 45 mcg/kg or less, 40 mcg/kg or less, 35 mcg/kg or less, 30 mcg/kg or less, 25 mcg/kg or less, 20 mcg/kg or less, 15 mcg/kg or less, 10 mcg/kg or less, 5 mcg/kg or less, 2.5 mcg/kg or less, 2 mcg/kg or less, 1.5 mcg/kg or less, 1 mcg/kg or less, 0 .5 μg/kg or less, or 0.5 μg/kg or less, of one or more anti-CD3 antibodies or antigen-binding fragments for the prevention, treatment, or amelioration of one or more symptoms of an autoimmune disorder or T-cell malignancy. In another embodiment, an intravenous dose of 100 mcg/kg or less, 95 mcg/kg or less, 90 mcg/kg or less, 85 mcg/kg or less, 80 mcg/kg or less, 75 mcg/kg or less, 70 mcg/kg or less, 65 mcg/kg or less, 60 mcg/kg or less, 55 mcg/kg or less, 50 mcg/kg or less, 45 mcg/kg or less, 40 mcg/kg or less, 35 µg/kg or less, 30 µg/kg or less, 25 µg/kg or less, 20 µg/kg or less, 15 µg/kg or less, 10 µg/kg or less, 5 µg/kg or less, 2, 5 mcg/kg or less, 2 mcg/kg or less, 1.5 mcg/kg or less, 1 mcg/kg or less, 0.5 mcg/kg or less, or 0.5 mcg/kg or less one or more of anti-CD3 antibodies or antigen-binding fragments, administered over approximately 6 hours, approximately 4 hours, approximately 2 hours, approximately 1.5 hours, approximately 1 hour, approximately 50 minutes, approximately 40 minutes, approximately 30 minutes, approximately 20 minutes, approximately 10 minutes, approximately 5 minutes, approximately 2 minutes, approximately 1 minute, approximately 30 seconds, or approximately 10 seconds to prevent, treat, or reduce the severity of one or more symptoms of an autoimmune disorder or T-cell malignancy.

In particular embodiments in which escalating doses are administered during the first days of a dosing regimen, the dose on day 1 of the regimen is 5-100 mcg/m ² /day and increases to the daily dose set forth immediately above by days 3, 4, 5. 6 or 7. For example, on day 1 the subject is administered a dose of approximately 51 μg/m ² /day, on day 2 approximately 103 μg/m ² /day, on day 3 approximately 207 μg/m ² /day, day 4 - approximately 413 mcg/m ² /day and on subsequent days of this regimen (for example, days 5-14) 826 mcg/m ² /day.

In other embodiments, the initial dose is 1/4, to 1/2, to equal the daily dose at the end of the regimen, but is administered in portions at 6, 8, 10, or 12 hour intervals. For example, a 13 mcg/m ² /day dose administered in four doses equal to 3-4 μg/kg, at intervals of 6 hours, to reduce the level of cytokine release caused by the administration of the antibody.

In specific embodiments, to reduce the potential for cytokine release and other adverse effects, the first 1, 2, 3, or 4 doses or all doses of the regimen are administered more slowly via intravenous administration. For example, a dose of 51 μg/m ² /day may be administered over about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, and about 22 hours. In some embodiments, the dose is administered by slow infusion over a period of time of, for example, 20-24 hours. In certain embodiments, the dose is pumped, preferably increasing the concentration of antibody administered during the infusion.

In other embodiments, a given portion of the regimen may be administered in increasing doses. For example, in the case of the 51 μg/m ² /day-826 μg/m ² /day dosing regimen described above, the portion may be 1/10, 1/4, 1/3, 1/2, 2/3, or ³ / ₄ from daily doses. Accordingly, when the part is equal to 1/10, the daily doses will be 5.1 μg/ ^m2 on day 1, 10.3 μg/ ^m2 on day 2, 20.7 μg/ ^m2 on day 3, 41.3 µg/ ^m2 on day 4 and 82.6 µg/ ^m2 on days 5-14. When the portion is 1/4, the doses will be 12.75 µg/ ^m2 on day 1, 25.5 µg/ ^m2 on day 2, 51 µg/ ^m2 on day 3, 103 µg/ ^m2 on day 4 and 207 mcg/ ^m2 on days 5-14. When the portion is 1/3, the doses will be 17 µg/ ^m2 on day 1, 34.3 µg/ ^m2 on day 2, 69 µg/ ^m2 on day 3, 137.6 µg/ ^m2 on day 4 and 275.3 μg/m ² on days 5-14. When the portion is 1/2, the doses will be 25.5 mcg/ ^m2 on day 1, 51 mcg/ ^m2 on day 2, 103 mcg/ ^m2 on day 3, 207 mcg/ ^m2 on day 4, and 413 µg/ ^m2 on days 5-14. When the portion is 2/3, the doses will be 34 µg/ ^m2 on day 1, 69 µg/ ^m2 on day 2, 137.6 µg/ ^m2 on day 3, 275.3 µg/ ^m2 on day 4 and 550.1 μg/m ² on days 5-14. When the portion is 3/4, the doses will be 38.3 µg/ ^m2 on day 1, 77.3 µg/ ^m2 on day 2, 155.3 µg/ ^m2 on day 3, 309.8 µg/m ² on day 4 and 620 mcg/ ^m2 on days 5-14.

In specific embodiments, the anti-CD3 antibody or antigen binding fragments are not administered in daily doses over a number of days, but rather are administered by infusion continuously over 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours. , 20 hours, 24 hours, 30 hours or 36 hours. The infusion may be unchanged or may begin with a lower dose administered during, for example, the first 1, 2, 3, 5, 6 and 8 hours of the infusion, and subsequently increase to higher dose. During the infusion, the patient receives a dose equal to the amount administered in the exemplary regimens set forth above, for example, a dose of approximately 150 μg/m ² , 200 μg/m ² , 250 μg/m ² , 500 μg/m ² , 750 µg/ ^m2 , 1000 µg/ ^m2 , 1500 µg/m2, 2000 µg/ ^m2 , 3000 µg/ ^{m2 ,} 4000 µg/ ^m2 , 5000 µg/ ^m2 , 6000 µg/ ^m2 , 7000 µg/ ^m2 , 8000 µg/ ^m2 or 9000 µg/ ^m2 . In particular, the rate and duration of infusion are planned according to

- 31 045070 designed to minimize the level of free anti-CD3 antibody or antigen-binding fragments in the subject after administration. In some embodiments, the level of free anti-antibody

CD3 or antigen binding fragments should not exceed 200 ng/ml of free antibody. In addition, the infusion is planned to achieve a combined coverage and modulation of T Cell Receptors of at least 50%, 60%, 70%, 80%, 90%, 95% or 100%.

In some embodiments, anti-CD3 antibody or antigen-binding fragments are administered to achieve a certain level of combination coverage and modulation of T cell receptor complexes on T cells, determined using methods well known in the art, see, for example, Example 11 in the application publication to US Patent No. US 2003/0108548, which is hereby incorporated by reference in its entirety. In certain embodiments, the dosing schedule achieves a combined coverage and T cell receptor modulation of at least 50%, 60%, 70%, 80%, 90%, 95%, or 100%, in certain embodiments , low or no detectable free anti-CD3 antibody or antigen-binding fragments (for example, less than 200 ng/ml of drug is detected in the patient's blood).

In preferred embodiments, the anti-CD3 antibody or antigen binding fragments are administered parenterally, for example intravenously, intramuscularly or subcutaneously, or alternatively administered orally. Anti-CD3 antibody or antigen binding fragments can also be administered as a sustained release formulation.

In a particular embodiment, administration of one or more doses or dosage schedule of a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen-binding fragments does not cause or reduces, relative to other immunosuppressive agents, one or more of the following undesirable or adverse effects: baseline abnormalities body conditions (fever, tachycardia, bradycardia, hypertension, hypotension), hematological manifestations (anemia, lymphopenia, leukopenia, thrombocytopenia), headache, chills, dizziness, nausea, asthenia, lower back pain, chest pain (chest tightness), diarrhea, myalgia, pain, itching, psoriasis, rhinitis, sweating, injection site reaction, dilation of blood vessels, increased risk of opportunistic pathogen infection, activation of Epstein-Barr virus, T-cell apoptosis, and increased risk of developing certain types of cancer. In another specific embodiment, administration of one or more doses of a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen-binding fragments does not cause or reduces, relative to other immunosuppressive agents, one or more of the following undesirable or adverse effects: abnormalities in vital signs ( fever, tachycardia, bradycardia, hypertension, hypotension), hematological manifestations (anemia, lymphopenia, leukopenia, thrombocytopenia), headache, chills, dizziness, nausea, asthenia, lower back pain, chest pain (chest tightness), diarrhea, myalgia , pain, itching, psoriasis, rhinitis, sweating, injection site reaction, dilation of blood vessels, increased risk of opportunistic pathogen infection, Epstein-Barr virus activation, T-cell apoptosis, and increased risk of developing certain types of cancer.

In accordance with the present invention, a dose or dosage regimen comprising a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen binding fragments for treating an autoimmune disorder may be repeated after 1 month, 2 months, 4 months, 6 months, 8 months, 12 months, 15 months, 18 months, or 24 months or longer after an initial or previous dose or dosage regimen comprising a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen-binding fragments. A repeat dose or dosing schedule may be prescribed, typically when symptoms associated with the autoimmune disorder recur after subsiding from the initial or previous dose or dosing schedule, or when symptoms associated with the autoimmune disorder do not improve after the initial dose. or dosing regimens of anti-CD3 antibody or antigen-binding fragments in accordance with the methods of the present invention. For example, with respect to diabetes, a repeat dose or dosing regimen comprising a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen binding fragments may be administered to a subject when, for example, the subject's average daily insulin administration is 1 month, 2 months, 4 months, 6 months, 8 months, 12 months, 15 months, 18 months, or 24 months or longer after initial or previous treatment with anti-CD3 antibody or antigen-binding fragments does not decrease by at least 5%, at least 10%, at least at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to levels before treatment. Alternatively, with respect to diabetes, a repeat dose or dosing regimen comprising a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen binding fragments may be administered to a subject when, for example, the subject's HA 1 or HA 1 C levels after 1 month , 2 months, 4 months, 6 months, 8 months, 12

- 32 045070 months, 15 months, 18 months or 24 months or longer after initial or previous treatment with anti-CD3 antibody or antigen binding fragments do not decrease by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to pre-treatment levels. In another embodiment, with respect to diabetes, a repeat dose or dosing regimen comprising a prophylactically or therapeutically effective amount of one or more anti-CD3 antibodies or antigen-binding fragments may be administered to a subject when, for example, a response of an increase in C-peptide after 1 month, 2 months, 4 months, 6 months, 8 months, 12 months, 15 months, 18 months or 24 months or longer after initial or previous treatment with anti-CD3 antibody or antigen binding fragments decreases by more than 5%, more than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% compared to pre-treatment levels.

Autoimmune diseases are non-infectious immunological diseases caused by immune responses directed against normal components of human cells, tissues and organs. Autoimmune diseases are often chronic diseases that little by little destroy the target tissues and organs. Common diseases now classified as autoimmune diseases due to the presence of inappropriate autoimmune responses include insulin-dependent diabetes type I, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), malignant myasthenia gravis, celiac disease, Gougerot-Sjögren's syndrome, Graves' disease, Crohn's disease, autoimmune hepatitis, psoriasis, psoriatic arthritis, asthma, allergic rhinitis, effects due to organ transplantation or homologous disease (GVHD) and numerous other diseases in which the inflammatory immune response is involved .

Because autoimmune diseases are typically chronic, they typically require lifelong treatment and monitoring. For this reason, traditional therapies for autoimmune disease mainly aim to combat the effects of inflammation caused by the disease, and few autoimmune diseases can be cured or made to disappear with such treatment. For some autoimmune diseases, administration of one of a limited number of immunosuppressive drugs can lead to periods of remission or disappearance of active disease. Immunosuppressive agents used for adjunctive therapy include substances that suppress cytokine production, reduce or suppress the expression of autoantigens, or mask major histocompatibility complex (MHC) antigens. Immunosuppressives include anti-inflammatory drugs (eg, nonsteroidal anti-inflammatory drug (NSAID), cyclophosphamide, bromocriptine, cyclosporine A, methotrexate, steroids such as glucocorticoids, and cytokines or cytokine receptor antagonists. Patients can rarely stop taking these immunosuppressives because the autoimmune disease They usually recur when the drug is stopped.The autoimmune disease may become untreatable with continued use of immunosuppressive drugs over the long term and may even require increasing doses of immunosuppressive drugs.

Therapeutic antibodies directed against CD3 are believed to result in fewer long-term side effects than many of the immunosuppressive chemotherapies currently available for the treatment of autoimmune diseases (WO 2007/117600). However, previous antibody-based therapies have been found to be problematic, especially when multiple administrations are used. Antilymphocyte therapies, such as antilymphocyte globular protein (ALG), and monoclonal antibodies directed against B cells, such as rituximab (Rituxin®) and alemtuzumab (CAMPATH®), reduce circulating and tissue B cell populations in treated subjects. However, these therapies also lead to severe immunosuppression, which is undesirable when treating a chronic autoimmune disease for a long time. The main complication of severe immunosuppressive therapy is infection. Systemic immunosuppression may also be associated with unwanted toxic effects and decreased levels of hematopoietic stem cells. In addition, patients receiving antibody-based therapies often develop significant levels of human anti-mouse antibodies (HAMA), human anti-chimeric antibodies (HACA), and anti-idiotypic responses, which may limit repeat treatments when remission ends.

As discussed above, antibodies directed against T cell antigens such as the T cell receptor (TCR) complex have been proposed as possible therapeutic agents for the immunosuppression of autoimmune disease. Antibodies against CD3 are believed to cause this immunosuppression by reducing pathogenic T cells and inducing regulatory T cells (WO 2007/117600; St. Clair E.W. (2009) Novel Targeted Therapies for Autoimmunity, Curr. Opin. Immunol. 21( 6): 648-657; Ludvigsson, J. (2009) The Role of Immunomodulation Therapy in Autoimmune Diabetes, J. Diabetes Sci. Technol. 3(2): 320-330). For this reason, antibodies against T cells, including

- 33 045070

CD3 have been used to influence the immunological status of a subject by suppressing, enhancing or redirecting T cell responses to an antigen. Specifically, Teplizumab, also known as hOKT3y1 (Ala-Ala) (containing alanine at positions 234 and 235) (MacroGenics, Inc.), is an anti-CD3 antibody that has been engineered to alter the function of T cells that mediate the destruction of insulin- beta-producing pancreatic islets. Teplizumab binds to and acts on the CD3 ε chain epitope present on mature T cells.

Due in part to its cross-reactivity with non-human CD3 (which allows for more precise and flexible dosing), the anti-CD3 antibodies of the present invention are considered to be particularly useful for the treatment of autoimmune disease, despite the obvious failures of the prior art.

Such antibodies and their antigen-binding fragments can be used alone or together with other pharmacological agents. In particular, the present invention provides therapies comprising the administration of such antibodies or antigen binding fragments together with anti-B cell antibodies (or antigen binding fragments thereof). Anti-B cell antibodies are known in the art (see WO 2007/117600; WO 2005/000901; WO 2004/035607; US Patent Nos. 5,500,362 and 5,736,137, US Patent Application Publications Nos. 2003/0219433, 2005/0271658, 2005/0271658, 2005/0281817, 2006/024295, 2006/024300 and 2006/034835 Clark, E. A. et al (1985) Role Of The Bp35 Cell Surface Polypeptide In Human B-Cell Activation, Proc. Natl. Acad. Sci (USA) 82(6): 17661770; Press, O. W. et al (1987) Monoclonal Antibody 1F5 (Anti-CD20) Serotherapy of Human B Cell Lymphomas, Blood 69: 584-591). Such co-administration can be accomplished using co-administration of individual antibodies or antigen-binding fragments thereof, or through the formation of bispecific antibodies, or more preferably, through the formation of DART™ diabodies described above having the ability to bind to both CD3 and B cell antigen .

Preferably, the anti-B cell antibody or antigen binding fragment used will be directed against a B cell surface marker, such as a marker selected from CD19, CD20, CD22, CD23, CD32B, CD40, B7-1 (CD80), B7-2 (CD86) , CD79a, CD79b, CD38, CD27, lymphocyte function-associated antigen (LFA) such as LFA-I or LFA-3, CFA-I, or other accessory molecule involved in the association of T cells, B cells, which leads to to the activation of T cells and B cells during the adaptive immune response. In a further preferred embodiment, the anti-B cell antibody may be a B cell depleting antibody, such as an antibody directed against a marker selected from CD19, CD20, CD22, CD23, CD32B, CD40, B7-1 (CD80), B7-2 (CD86), lymphocyte function-associated antigen (LFA), such as LFA-I or LFA-3, CFA-I, or other accessory molecule involved in T-cell, B-cell association.

Alternatively, such combination therapy may include administration of an anti-CD3 antibody or antigen-binding fragment thereof in combination with an antibody (or antigen-binding fragment thereof) that recognizes an antigen present on an antigen-presenting cell (eg, B7-H3). However, in a further preferred embodiment, the combination therapy includes administration of an anti-CD3 antibody (or an antigen binding fragment thereof) in combination with an antibody (or an antigen binding fragment thereof) that recognizes a polypeptide involved in B cell activation (either directly or indirectly) , or an immunomodulator such as a member of the TNF family of cytokines or an interferon (eg, α, β, or γ-interferon). As will be appreciated by those skilled in the art, such interferons are involved in the regulation of proteins that work together in the processing and presentation of antigens. These cytokines stimulate cells to increase their expression of HLA class I heavy chains. In one preferred embodiment, the combination therapy comprises administering to a subject having an active autoimmune disease an anti-T cell antigen antibody in combination with an anti-interferon-β antibody. In a further preferred embodiment, the combination therapy comprises administering to the subject an antibody directed against a T-cell antigen in combination with an antibody selected from the anti-interferon-β antibodies AVONEX®, BETASERON® and REBIF®. In a further preferred embodiment, the combination therapy comprises administering to a subject an antibody directed against a T-cell antigen in combination with an antibody directed against interferon-β to treat a subject suffering from multiple sclerosis.

In a further embodiment, the anti-CD3 antibody may be a non-mitogenic antibody or a reduced-mitogenic antibody that inhibits or prevents T cell activation when the T cell comes into contact with a specific antigen on an antigen presenting cell, in particular an antigen presenting B cell. As used herein, the term non-T cell mitogenic antibody means an antibody that is constructed by altering the Fc receptor of the antibody so that it does not trigger the initial activation events and subsequent release of cytokines that are noted upon T cell activation. Antibody with reduced mitogen

- 34 045070 T cell antigen is an antibody specific for a T cell antigen that attenuates the initial activation events and subsequent release of cytokines that occur upon T cell activation. A non-mitogenic antibody or an antibody with reduced mitogenicity may be used to prevent the initial first-dose side effects observed when an antilymphocyte antibody is administered to a patient. A non-mitogenic antibody or an antibody with reduced mitogenicity may be an engineered antibody containing a modified Fc moiety that prevents or inhibits binding by effector cells.

C. Methods of administration

In one aspect, embodiments of the present invention provide human subjects treated to achieve and maintain clinical remission for longer periods of time than remissions achieved in patients treated using conventional therapy. For example, where traditional therapy achieves remission of symptoms of an autoimmune disease within three months, the compositions of the present invention can provide complete remission of symptoms for up to six months, up to 12 months, and in some cases up to one to two years or longer. It is envisaged that in the case of some autoimmune diseases, it is possible to achieve complete remission, which does not cease again, especially if therapy is started soon after diagnosis of the autoimmune disease.

Clinical remission achieved with combination therapy may be complete remission, or it may be partial remission, in which significant reductions in disease symptoms are maintained over an extended period. For example, a subject receiving therapy of the present invention may experience decreased autoimmune responses, as determined by decreased levels of detectable autoantibodies in body fluids and tissues, such as cerebrospinal fluid (CSF), serum, urine, or body tissues. A subject receiving combination therapy of the present invention may also experience reduced T cell responses to autoantigens, as determined by in vitro proliferation or cytokine production assays using peripheral blood mononuclear cells (PBMCs) or purified T cells, compared to subjects receiving the combination therapy of the present invention. treated using traditional therapy.

The compositions of the present invention can be administered by any suitable route, including parenteral, topical, subcutaneous, intraperitoneal, intrapulmonary, intranasal and/or intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody can suitably be administered by infusion in an intermittent manner, for example using increasing doses of the antibody. Preferably, dosing is administered intravenously or subcutaneously, and more preferably by intravenous infusion(s). Each exposure can be achieved using the same or different routes of administration. In one embodiment, each exposure is administered via intravenous administration. In another embodiment, each exposure is administered via subcutaneous administration. However, in another embodiment, exposure is accomplished through both intravenous and subcutaneous administration.

In one embodiment, the therapeutic antibodies are administered as a slow intravenous infusion, which can be started at an hourly rate to deliver the molecules of the present invention in approximately 15 minutes to 4 hours. However, if the subject exhibits an infusion reaction, the infusion rate is preferably reduced, for example, to half flow speed. Treated subjects may receive prophylactic treatment with acetaminophen/paracetamol (eg, approximately 1 g) and diphenhydramine HCl (eg, approximately 50 mg or an equivalent dose of similar agent) administered orally approximately 30 to 60 minutes prior to the start of the infusion.

The treatment provided by the combination compositions of the present invention (including DART™ diabodies) can be administered to a subject using an initial dose of the first antibody that is less than the amount of such antibody required to achieve clinical efficacy of the therapy for the autoimmune disease when administered as a single antibody therapy . A dose of a therapeutic anti-T cell antibody that is less than the dose required to achieve depletion of T cells capable of recognizing and responding to self-antigens in a single antibody therapy may be sufficient to achieve the desired clinical efficacy. Methods for determining the dose of a therapeutic antibody required to achieve clinical efficacy are known to those skilled in the art. For example, clinical effectiveness in a subject may be defined as the length of time until disease progression, reduction in clinical symptoms, reduction in laboratory marker levels, reduction in the need for re-treatment, or any other clinical measure recognized as a useful indicator of improvement in an autoimmune disease.

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The second antibody of the combination therapy may also be administered to the subject in need of treatment at an initial dose that is less than the dose effective to achieve clinical efficacy when administered alone with the antibody. For example, doses of an anti-B cell depleting antibody that achieve less than 100% B cell depletion, less than 50% B cell depletion, less than 30% B cell depletion, or even no B cell depletion. cells may be administered together with the first anti-T cell antibody to achieve clinical efficacy of suppressing the immune response against a self-antigen that is equal to the clinical efficacy achieved by administering an amount of B cell depleting antibody that causes 100% depletion of B cells in a subject at administration of only this antibody, or exceeds it.

In some cases, clinical effectiveness is effectiveness that is not achieved by administering either the first antibody alone or the second antibody alone. In other cases, clinical efficacy may be equivalent to that achieved with single antibody therapy, with the combination therapy causing less immunosuppression of the treated subject's immune system than single antibody therapy. In one preferred embodiment, the synergistic response produced by the combination therapy reduces or abolishes anti-self antigen responses in the subject while providing lower levels of immunosuppression. General immunosuppression is a significant problem with previously available antibody-based therapies.

D. Pharmaceuticals

Antibody therapeutic preparations used in embodiments of the present invention are prepared for storage, delivery and administration by mixing the composition of the present invention at the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers generally recognized in the pharmaceutical field, in the form of lyophilized preparations or aqueous solutions .

The term pharmaceutically acceptable means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeias for use in animals and more particularly in humans. The term carrier refers to the diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient or carrier with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water or oils, including petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially in the case of injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol and the like. . The composition, if desired, may also contain minor amounts of wetting agents or emulsifiers or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release preparations, and the like.

The compositions of the present invention include bulk drug compositions useful for the manufacture of pharmaceutical compositions (e.g., admixed or non-sterile compositions) and pharmaceutical compositions (i.e., compositions suitable for administration to a subject or patient) that can be used to prepare standard dosage forms. Such compositions include a prophylactically or therapeutically effective amount of a prophylactic and/or therapeutic agent described herein, or combinations thereof, and a pharmaceutically acceptable carrier. Preferably, the compositions of the present invention include a prophylactically or therapeutically effective amount of the humanized antibodies of the present invention and a pharmaceutically acceptable carrier.

Typically, the ingredients of the compositions of the present invention are supplied either separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or an anhydrous concentrate in a sealed container, such as an ampoule or sachet, indicating the amount of active agent. If the composition is to be administered by infusion, its distribution is accomplished using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, a vial of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration. Pharmaceutical compositions suitable for injection include sterile aqueous solutions in which the active agents are water soluble, or dispersions or sterile powders for the preparation of sterile injectable solutions for immediate administration. Compositions for use in combination therapy can be prepared by including the active antagonist or antibody in the required amount together with suitable

- 36 045070 carriers, for example water, ethanol, polyol (for example glycerin, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Isotonicity agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride may be included in the composition.

The compositions of the present invention can be prepared in neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed by anions, such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed by cations, such as those are obtained from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2ethylaminoethanol, histidine, procaine, etc.

E. Sets

The present invention provides a pharmaceutical kit or kit comprising one or more containers filled with the humanized antibodies of the present invention. In addition, one or more other prophylactic or therapeutic agents useful for treating the disease may also be included in the pharmaceutical kit or kit. The present invention also provides a pharmaceutical kit or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the present invention. Such container(s) may optionally accompany a message in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products reflecting that agency's approval of manufacture, use, or sale for administration to humans.

The present invention provides kits that can be used in the above methods. In one embodiment, the kit includes one or more humanized antibodies of the present invention. In another embodiment, the kit further includes one or more other prophylactic or therapeutic agents useful for treating cancer in one or more containers. In another embodiment, the kit further includes one or more cytotoxic antibodies that bind to one or more cancer antigens associated with cancer. In some embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.

In one embodiment, the present invention provides a finished product containing antibodies that are used in combination therapy for the treatment of an autoimmune disease. The finished product includes a container including a first antibody that binds to an antigen present on a T cell, and a pharmaceutically acceptable carrier, excipient or diluent within the container. The finished product further includes a second container including a second antibody directed against a B cell surface marker and a pharmaceutically acceptable carrier, excipient or diluent and instructions for administering the composition to a subject in need of treatment for an autoimmune disease. If the first and second antibodies are determined to be complementary and do not have an adverse effect on each other, the first and second antibodies may be provided in a single container containing the first and second antibodies at appropriate concentrations for administration, along with a package insert and instructions in regarding introduction.

The finished product containers may be made of any suitable material that will not react with or otherwise affect the drug. The finished product may further include a second or third container including a buffer as a pharmaceutically acceptable diluent, such as bacteriostatic water for injection, phosphate buffered saline, Ringer's solution, and dextrose solution. The finished product may also include other material that may be desirable from a commercial and consumer point of view, including other buffers, diluents, filters, needles and syringes.

VII. Diagnostic methods using anti-CD3 antibodies of the present invention

Anti-CD3 antibodies generated using the methods described herein can also be used to determine the presence or absence of cancer cells or their levels by detecting antigens that are circulating in the blood after they have been dissociated from the cell surface (eg, soluble CD3). Such circulating antigen may be an intact CD3 antigen or a fragment thereof that remains detectable according to the methods set forth herein. Such detection can be accomplished, for example, by analysis and using FACS, using standard methods commonly used in the art.

In a preferred embodiment of the diagnostic methods of this invention, the antibody carries a detectable label. Examples of tags that may be used include a radioactive agent (e.g. scandium-47, technetium-99m, indium-111, iodine-131, rhenium-186, rhenium-188,

- 37 045070 samarium-153, holmium-166, lutetium-177, copper-64, scandium-47, yttrium-900), enzyme or fluorophore such as phycoerythrin or fluorescein isothiocyanate (also known as fluoroisothiocyanate or

FITC).

One way to use antibodies for diagnosis is to visualize a tumor in vivo by binding the antibody to a radioactive or radiopaque agent, administering the antibody to the individual, and using an X-ray machine or other imaging apparatus to visualize the location of the labeled antibody on the surface of cancer cells expressing the antigen. The antibody is administered at a concentration that supports binding under physiological conditions.

In vitro methods for detecting CD3 are common in the art and include enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, immunofluorescence, enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA) and Western blot analysis.

The present invention also provides methods for assisting in diagnosing a cancer characterized by cancer cells that express CD3 in an individual using an antibody that binds to CD3 and any other methods that can be used to determine the level of CD3 expression. As used herein, methods of assisting diagnosis mean that these methods facilitate clinical determination of the classification, or nature, of the cancer and may or may not be determinative as to the exact diagnosis. Accordingly, a method of assisting in diagnosing cancer may include the step of detecting the level of CD3 in a biological sample from an individual and/or determining the level of CD3 expression in the sample. Antibodies that recognize an antigen or a portion thereof may also be used to create diagnostic immunoassays for the detection of antigen released from or secreted by living or dying cancer cells in body fluids including, but not limited to, blood, saliva, urine, alveolar fluid, or ascitic fluid. liquid. Antibodies against CD3 generated using the methods described herein can be used to determine whether an individual diagnosed with cancer can be considered a candidate for immunotherapy using antibodies directed against CD3. In one embodiment, the biopsy sample can be tested for CD3 expression using antibodies directed against CD3. Individuals with cancer cells that express CD3 are suitable candidates for immunotherapy using antibodies directed against CD3. Anti-CD3 staining can also be used to distinguish cancerous tissues from normal tissues.

Methods for using anti-CD3 antibodies for diagnostic purposes are applicable both before and after any form of cancer treatment, such as chemotherapy or radiation therapy, to determine which tumors are most likely to respond to a particular treatment, the prognosis for an individual with a cancer, and the tumor subtype. or the source of metastatic disease, and disease progression or response to treatment.

The compositions of this invention are particularly suitable for diagnosing disease states other than cancer using the methods described generally above for use with other unhealthy (non-cancerous) cells. Disease conditions suitable for use in the methods of this invention include, but are not limited to, diseases or disorders associated with inflammatory or autoimmune reactions in individuals. The methods described above can be used to modulate inflammatory or autoimmune responses in individuals. Diseases and conditions resulting from inflammatory and autoimmune disorders that may be diagnosed and/or treated using the compositions and methods of the present invention include, by way of illustration and not limitation, multiple sclerosis, meningitis, encephalitis, stroke, other injuries brain, inflammatory bowel disease, including ulcerative colitis and Crohn's disease, myasthenia gravis, lupus vulgaris, rheumatoid arthritis, asthma, acute juvenile diabetes, AIDS dementia, atherosclerosis, nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemia and acute, lymphocyte-mediated lung injury.

However, other indications for diagnostic and/or therapeutic use of the antibodies and other therapeutic agents of the present invention include administration to individuals at risk of organ or transplant rejection. In recent years, there has been a significant increase in the effectiveness of surgical methods for transplanting tissues and organs such as skin, kidney, liver, heart, lung, pancreas and bone marrow. Perhaps the main unresolved problem is the lack of satisfactory agents for inducing immunotolerance in the recipient to a transplanted allograft or organ. When allogeneic cells or organs are transplanted into a host (i.e., the donor and recipient are different individuals of the same species), the host's immune system will likely mount an immune response against foreign antigens in the graft (host-versus-graft response), resulting in destruction transplanted tissue.

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Anti-CD3 monoclonal antibodies generated using the methods described herein can be used to determine the presence or absence of human cancer stem cells in a number of tissues. Cancer stem cells (CSCs) have been hypothesized to play a role in tumor growth and metastasis (Ghotra, V.P. et al. (2009) The Cancer Stem Cell Microenvironment And Anti-Cancer Therapy, Int. J. Radiat.

Biol. 85(11): 955-962; Gupta, P.B. et al. (2009) Cancer Stem Cells: Mirage Or Reality? Nat. Med. 15(9): 1010-1012; Lawson, J.C. et al. (2009) Cancer Stem Cells in Breast Cancer And Metastasis, Breast Cancer Res. Treat. 118(2): 241-254;

Hermann, P.C. et al. (2009) Pancreatic Cancer Stem Cells Insights And Perspectives, Expert Opin. Biol. Ther. 9(10) : 1271-1278; Schatton, T. et al. (2009) Identification And Targeting Of Cancer Stem Cells, Bioessays 31(10): 1038-1049; Mittal, S. et al. (2009) Cancer Stem Cells: The Other Face of Janus, Amer. J. Med. Sci. 338(2): 107-112; Alison, M.R. et al. (2009) Stem Cells And Lung Cancer: Future Therapeutic Targets? Expert Opin. Biol. Ther. 9(9): 1127-1141; Charafe Jauffret, E. et al. (2009) Breast Cancer Stem Cells: Tools And Models To Rely On, BMC Cancer 9:202; Scopelliti, A. et al.

(2009) Therapeutic Implications Of Cancer Initiating Cells, Expert Opin. Biol. Ther. 9(8): 1005-1016; publication of PCT application No. 2008/091908).

According to this hypothesis, CSCs provide a small, distinct subpopulation of cells within each tumor that are capable of unlimited self-renewal and development into more mature tumor cell(s) that are relatively limited in their ability to replicate. It has been hypothesized that these cancer stem cells may be more resistant to chemotherapeutic agents, radiation, or other toxic conditions, and therefore remain after clinical therapies and later develop into secondary tumors, metastasize, or are responsible for relapse. It has been proposed that CSCs may arise from either stem cells of normal tissues or progenitor cells of differentiated tissues.

The applications described in this application, which cover the use of anti-CD3 antibodies, also cover the use of other CD3 agonists, antagonists and modulators described herein for use in identifying and treating cancer stem cells. In such embodiments, anti-CD3 antibodies and other CD3 agonists, antagonists, and modulators are used to identify, diagnose, or therapeutically treat cancer stem cells using methods similar to the methods described, and modifications are made within the average skill of the practitioner to tailor the method to the identification /diagnosis or treatment of cancer stem cells.

Having now generally described the present invention, it will be easier to understand the same by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless otherwise noted.

Example 1: mAb 1 binds to both human and cynomolgus CD3

Capture ELISA was performed to evaluate the ability of mAb1 to bind to human CD3. The plates were coated with 1 μg/ml soluble cynomolgus CD3 (sCD3) and incubated in the presence of various concentrations of the chimeric mAb1 variant (ch-mAb1) (containing mAb1 variable region sequences and human antibody constant regions), the humanized variant (h-mAb1), and the antibody , consisting of the light chain of the chimeric antibody mAb1 and the heavy chain of the humanized version of mAb1. The results of this experiment are presented in Fig. 1A. This experiment demonstrates the ability of mAb1 to bind to non-human mammalian CD3. In addition, the binding of the chimeric mAb1 antibody was compared with that of an antibody comprising the humanized mAb1 LC-2 and the mAb1 heavy chain. The results of this experiment are presented in Fig. 1B. This experiment demonstrates the ability of mAb1 to bind to human CD3. Thus, FIG. 1A and 1B show that the humanized mAb1 was capable of binding to both human and non-human mammalian CD3. The humanized mAb demonstrated binding to sCD3 and hCD3 similar to that of the chimeric mAb1.

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Example 2. Humanization of mAbl

Humanized mAbl derivatives were prepared. The amino acid sequences of these humanized derivatives and the polynucleotide sequences encoding them are presented below. CDRs are bold and underlined.

Amino acid sequence of humanized mAb1 light chain variable region variant 1 (SEQ ID NO: 10):

DIQMTQSPSS LSASVGDRVT ITCSASSSVS YMNWYQQKPG KAPKRLIYDS SKLASGVPSR FSGSGSGTEF TLTISSLQPE DFATYYCQQW SRNPPTFGGG TKVEIK

Polynucleotide sequence encoding humanized mAb1 light chain variable region variant 1 (SEQ ID NO: 11):

gacatccaga tgacccagtc cccctccagc ctgtccgcct ctgtgggcga cagagtgaca atcacctgtt ccgccagctc ctccgtgtcc tacatgaact ggtatcagca gaagcccggc aaggccccca agcggctgat ctacgactcc tccaagctgg cctccggcgt gccctccaga ttctccgg ct ctggctccgg caccgagttc accctgacca tctccagcct gcagcccgag gacttcgcca cctactactg ccagcagtgg tcccggaacc cccctacctt cggcggaggc accaaggtgg aaatcaag

Amino acid sequence of humanized mAb1 light chain variable region variant 2 (LC-2 mAb1) (SEQ ID NO: 12):

DWMTQSPAI MSAFPGEKVT ITCSASSSVS YMNWYQQKPG KAPKRWIYDS SKLASGVPSR FSGSGSGTEF TLTISSLQPE DFATYYCQQW SRNPPTFGGG TKVEIK

Polynucleotide sequence encoding humanized mAb1 light chain variable region variant 2 (SEQ ID NO: 13): gacgtggtga tgacccagtc tcctgccatc atgagtgctt tcccaggcga gaaagtgacc attacatgct ctgcttccag ctctgtgtcc tacatgaact ggtatcagca gaagccaggg aaagcacc ca agaggtggat ctacgactcc tccaagctgg cctccggcgt gccaagccgg ttctctggta gtggctcagg aaccgagttt actctgacca tttccagcct gcagcctgaa gatttcgcaa catactattg tcagcagtgg tccagaaatc cccctacatt tggcggaggg actaaagtgg aaatcaag

Humanized mAb1 heavy chain variable region amino acid sequence (SEQ ID NO: 14):

QVQLVQSGAE VKKPGASVKV SCKASGYTFT RSTMHWVRQA PGQGLEWIGY INPSSAYTNY NQKFKDRVTI TADKSTSTAY MELSSLRSED TAVYYCASPQ VHYDYNGFPY WGQGTLVTVS S

Polynucleotide sequence encoding the heavy chain variable region of the humanized mAb1 (SEQ ID NO: 15):

caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgcctc cgtgaaggtg tcctgcaagg cctccggcta caccttcacc cggtccacca tgcactgggt gcgacaggcc ccaggccagg gactggaatg gatcggctac atcaacccct ccagcgccta caccaactac aaccagaaat tcaa ggaccg cgtgaccatc accgccgaca agtccaccag caccgcctac atggaactgt ctagcctgcg gagcgaggac accgccgtgt actactgcgc ctccccccag gtgcactacg actacaacgg cttccctac tggggccagg gcaccctggt gacagtgtcc tcc

Example 3: Humanization of mAb2

Humanized mAb2 derivatives were prepared. The amino acid sequences of these humanized derivatives and the polynucleotide sequences encoding them are presented below. CDRs are bold and underlined.

Amino acid sequence of humanized mAb2 light chain variable region variant 1 (VL-1 h-mAb2) (SEQ ID NO: 16):

IQAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQQ KPGQAPRTLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 1 (VL-1 h-mAb2) (SEQ ID NO: 17):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg gttccagcag aagccaggac aggcaccaag gaccctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt tttctgg aag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

Amino acid sequence of humanized mAb2 light chain variable region variant 2 (VL-2 h-mAb2) (SEQ ID NO: 18):

- 40 045070

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRTLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 2 (VL-2 h-mAb2) (SEQ ID NO: 19):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag aagccaggac aggcaccaag gaccctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt tttct ggaag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

Amino acid sequence of humanized mAb2 light chain variable region variant 3 (VL-3 h-mAb2) (SEQ ID NO: 20):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQE KPGQAPRTLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 3 (VL-3 h-mAb2) (SEQ ID NO: 21):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg gttccaggag aagccaggac aggcaccaag gaccctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt tttctgga ag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

Amino acid sequence of humanized mAb2 light chain variable region variant 4 (VL-4 h-mAb2) (SEQ ID NO: 22):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 4 (VL-4 h-mAb2) (SEQ ID NO: 23):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg gttccagcag aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt tttct ggaag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

Amino acid sequence of humanized mAb2 light chain variable region variant 5 (VL-5 h-mAb2) (SEQ ID NO: 24):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQE KPGQAPRTLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 5 (VL-5 h-mAb2) (SEQ ID NO: 25):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcaggag aagccaggac aggcaccaag gaccctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt tttctgg aag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

Amino acid sequence of humanized mAb2 light chain variable region variant 6 (VL-6 h-mAb2) (SEQ ID NO: 26):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 6 (VL-6 h-mAb2) (SEQ ID NO: 27):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcagcag aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt ttt ctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

- 41 045070

Amino acid sequence of humanized mAb2 light chain variable region variant 7 (VL-7 h-mAb2) (SEQ ID NO: 28):

I QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQE KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 7 (VL-7 h-mAb2) (SEQ ID NO: 29):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg gttccaggag aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt tttctgg aag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

Amino acid sequence of humanized mAb2 light chain variable region variant 8 (VL-8 h-mAb2) (SEQ ID NO: 30):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQE KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 8 (VL-8 h-mAb2) (SEQ ID NO: 31):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcaggag aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt tttct ggaag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

Amino acid sequence of humanized mAb2 light chain variable region variant 9 (VL-9 h-mAb2) (SEQ ID NO: 32):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQE KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 9 (VL-9 h-mAb2) (SEQ ID NO: 33):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcacagg cgcagtgacc acatctaact acgccaattg ggtgcaggag aagccaggac aggcattcag gggcctgatc gggggtacaa acaaaagggc tccctggacc cctgcacggt ttt ctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

Amino acid sequence of humanized mAb2 light chain variable region variant 10 (VL-10 h-mAb2) (SEQ ID NO: 34):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQQ KPDHLFTGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG

Polynucleotide sequence encoding humanized mAb2 light chain variable region variant 10 (VL-10 h-mAb2) (SEQ ID NO: 35):

caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ccagcactgg agcagtgact acctctaact acgctaattg gttccagcag aagcccgacc acctgttcac tgggctgatc ggcggaacca acaaaagggc tccctggacc cctgca cggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc gggggtggca caaaactgac tgtgctggga

Amino acid sequence of humanized mAb2 heavy chain variable region variant 1 (VH-1 h-mAb2) (SEQ ID NO: 36):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCAR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding humanized mAb2 heavy chain variable region variant 1 (VH-1 h-mAb2) (SEQ ID NO: 37):

- 42 045070 gaggtgcagc tggtggaaag cggcggagga ctggtgcagc caggtggcag cctgcgactg tcttgcgccg ctagtggctt caccttttct acatacgcca tgaactgggt gaggcaggct cctggaaagg ggctggagtg ggtgggcagg atcaggtcca agtacaa caa ttatgcaacc tactatgccg actcagtgaa ggatagattc acaatttccc gcgacgattc taaaaacagt ctgtatctgc agatgaactc cctgaagact gaagacaccg ccgtgtacta ttgtgcaaga cacggaaact tcggcaactc ctacgtgtcc tggtttgcat attggggtca gggcac actg gtgaccgtgt ccagc

Amino acid sequence of humanized mAb2 heavy chain variable region variant 2 (VH-2 h-mAb2) (SEQ ID NO: 38):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCAR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding humanized mAb2 heavy chain variable region variant 2 (VH-2 h-mAb2) (SEQ ID NO: 39):

gaggtgcagc tggtggaaag cggcggagga ctggtgcagc caggtggcag cctgcgactg tcttgcgccg ctagtggctt cacctttaac acatacgcca tgaactgggt gaggcaggct cctggaaagg ggctggagtg ggtgggcagg atcaggtcca agtacaacaa ttatgcaacc tactatg ccg actcagtgaa ggatagattc acaatttccc gcgacgattc taaaaacagt ctgtatctgc agatgaactc cctgaagact gaagacaccg ccgtgtacta ttgtgcaaga cacggaaact tcggcaactc ctacgtgtcc tggtttgcat attggggtca gggcacactg gtgaccgtgt cca gc

Amino acid sequence of humanized mAb2 heavy chain variable region variant 3 (VH-3 h-mAb2) (SEQ ID NO: 40):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCAR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding humanized mAb2 heavy chain variable region variant 3 (VH-3 h-mAb2) (SEQ ID NO: 41):

gaggtgcagc tggtggaaag cggcggagga ctggtgcagc caggtggcag cctgcgactg tcttgcgccg ctagtggctt caccttttct acatacgcca tgaactgggt gaggcaggct cctggaaagg ggctggagtg ggtggccagg atcaggtcca agtacaacaa ttatgcaacc tactatg ccg actcagtgaa ggatagattc acaatttccc gcgacgattc taaaaacagt ctgtatctgc agatgaactc cctgaagact gaagacaccg ccgtgtacta ttgtgcaaga cacggaaact tcggcaactc ctacgtgtcc tggtttgcat attggggtca gggcacactg gtgaccgtgt cca gc

Amino acid sequence of humanized mAb2 heavy chain variable region variant 4 (VH-4 h-mAb2) (SEQ ID NO: 42):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding humanized mAb2 heavy chain variable region variant 4 (VH-4 h-mAb2) (SEQ ID NO: 43):

gaggtgcagc tggtggaaag cggcggagga ctggtgcagc caggtggcag cctgcgactg tcttgcgccg ctagtggctt caccttttct acatacgcca tgaactgggt gaggcaggct cctggaaagg ggctggagtg ggtgggcagg atcaggtcca agtacaacaa ttatgcaacc tactat gccg actcagtgaa ggatagattc acaatttccc gcgacgattc taaaaacagt ctgtatctgc agatgaactc cctgaagact gaagacaccg ccgtgtacta ttgtgtgaga cacggaaact tcggcaactc ctacgtgtcc tggtttgcat attggggtca gggcacactg gtgaccgtgt ccagc

Amino acid sequence of humanized mAb2 heavy chain variable region variant 5 (VH-5 h-mAb2) (SEQ ID NO: 44):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCAR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding humanized mAb2 heavy chain variable region variant 5 (VH-5 h-mAb2) (SEQ ID NO: 45):

gaggtgcagc tggtggaaag cggcggagga ctggtgcagc caggtggcag cctgcgactg tcttgcgccg ctagtggctt cacctttaac acatacgcca tgaactgggt gaggcaggct cctggaaagg ggctggagtg ggtggccagg atcaggtcca agtacaacaa ttatgcaacc tactatgcc g actcagtgaa ggatagattc acaatttccc gcgacgattc taaaaacagt ctgtatctgc agatgaactc cctgaagact gaagacaccg ccgtgtacta ttgtgcaaga cacggaaact tcggcaactc ctacgtgtcc tggtttgcat attggggtca gggcacactg gtgaccgtgt ccag c

Amino acid sequence of humanized mAb2 heavy chain variable region variant 6 (VH-6 h-mAb2) (SEQ ID NO: 46):

- 43 045070

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding humanized mAb2 heavy chain variable region variant 6 (VH-6 h-mAb2) (SEQ ID NO: 47):

gaggtgcagc tggtggaaag cggcggagga ctggtgcagc caggtggcag cctgcgactg tcttgcgccg ctagtggctt cacctttaac acatacgcca tgaactgggt gaggcaggct cctggaaagg ggctggagtg ggtgggcagg atcaggtcca agtacaacaa ttatgcaacc tactatg ccg actcagtgaa ggatagattc acaatttccc gcgacgattc taaaaacagt ctgtatctgc agatgaactc cctgaagact gaagacaccg ccgtgtacta ttgtgtgaga cacggaaact tcggcaactc ctacgtgtcc tggtttgcat attggggtca gggcacactg gtgaccgtgt ccagc

Amino acid sequence of humanized mAb2 heavy chain variable region variant 7 (VH-7 h-mAb2) (SEQ ID NO: 48):

EVQLVESGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding humanized mAb2 heavy chain variable region variant 7 (VH-7 h-mAb2) (SEQ ID NO: 49): gaggtgcagc tggtggaaag cggcggagga ctggtgcagc caggtggcag cctgcgactg tcttgcgccg ctagtggctt caccttttct acatacgcca tgaactgg gt gaggcaggct cctggaaagg ggctggagtg ggtggccagg atcaggtcca agtacaacaa ttatgcaacc tactatgccg actcagtgaa ggatagattc acaatttccc gcgacgattc taaaaacagt ctgtatctgc agatgaactc cctgaagact gaagacaccg ccgtgtacta ttgtgtgaga cacggaaact tcggcaactc ctacgtgtcc tggtttgcat attggggtca gggcacactg gtgaccgtgt ccagc

Amino acid sequence of humanized mAb2 heavy chain variable region variant 8 (VH-8 h-mAb2) (SEQ ID NO: 50):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding humanized mAb2 heavy chain variable region variant 8 (VH-8 h-mAb2) (SEQ ID NO: 51): gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggagggtc cctgagactc tcctgtgcag cctctggatt caccttcaac acatacgcta tgaattgggt ccgccaggct ccagggaagg ggctggagtg ggttgcaagg atcaggtcca agtacaacaa ttatgcaacc tactatgccg actctgtgaa ggatagattc accatctcaa gagatgattc aaagaactca ctgtatctgc aaatgaacag cctgaaaacc gaggacacgg ccgtgtatta ctgtgtgaga cacggtaact tcggcaattc ttacgtgtct tggtttgctt attggggaca ggggacactg gtgactgtgt cttcc

Amino acid sequence of the humanized mAb2 heavy chain variable region QV variant (VH-QV h-mAb2) (SEQ ID NO: 52):

EVQLVESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYADSVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Polynucleotide sequence encoding the heavy chain variable region QV variant of humanized mAb2 (VH-QV h-mAb2) (SEQ ID NO: 53): gaggtgcagc tggtggaaag cggcggagga ctggtgcagc caaagggatc actgaaactg tcctgcgccg cctccggctt cacctttaac acatacgcta tgaattggg t gcgacaggca cctggcaagg gcctggagtg ggtggcaagg atcaggtcca agtacaacaa ttatgcaacc tactatgccg actctgtgaa ggatagattc acaatcagtc gcgacgattc ccagagcatt ctgtatctgc agatgaacaa tctgaaaact gaagacaccg ccatgtacta ttgtgtgcgg cacggtaact tcggcaattc ttacgtgtct tggtttgctt attggggaca ggggacactg gtgactgtgt cttcc

Example 4.

mAb2 binds to both human and cynomolgus CD3 As discussed above, mAb2 was originally isolated based on its ability to bind to human CD3. Capture ELISA was performed to evaluate the ability of mAb2 to bind to non-human CD3. The plates were coated with 1 μg/ml CD3 (either human or cynomolgus) and incubated in the presence of various concentrations of a chimeric mAb2 antibody variant (ch-mAb2) (containing mAb2 variable region and human antibody constant region sequences). As a control, plates were also coated with an antibody consisting of a light chain of a humanized mAb1 and a heavy chain of a chimeric antibody. The results of this experiment are presented in Fig. 2A and 2B show that the chimeric mAb2 exhibited equal binding to human CD3 and cynomolgus CD3.

- 44 045070

Example 5. Analysis of the binding properties of h-mAb2 light and heavy chain variants

An analysis was performed to determine the effect of residue variations in the light chain framework regions of mAb2. In table 2 shows the studied substitutions.

table 2

Light chain Balance number according to Kabat: 36 38 41 42 43 44 45 46 SEQ ID NO: SEQ ID NO:5 Residue No.: 38 40 43 44 45 46 47 48 Option mAb2-VL V E D H L F T G 5 h-mAb2 VL-1 F Q G Q A P R T 16 h-mAb2 VL-2 V 18 h-mAb2 VL-3 E 20 h-mAb2 VL-4 G 22 h-mAb2 VL-5 V E 24 h-mAb2 VL-6 V G 26 h-mAb2 VL-7 E G 28 h-mAb2 VL-8 V E G thirty h-mAb2 VL-9 V E F G 32 h-mAb2 VL-10 D H L F T G 34 Heavy chain Balance number according to Kabat: thirty 49 52a 58 93 SEQ ID NO: SEQ ID NO:7 Residue No.: thirty 49 53 61 99 Option mVH N A Y V 7 hVH-1 S G A 36 hVH-2 N 38 hVH-3 A 40 hVH-4 V 42 hVH-5 N A 44 hVH-6 N V 46 hVH-6L N E V 54 hVH-6M N N E V 72 hVH-7 A V 48 hVH-8 N A V 50 hVH-8L N A E V 55 hVH-8M N A N E V 74

Antibodies were generated having mAb2 light chains of SEQ ID NO:11 but containing substitutions (using Kabat numbering) D41G, H42Q, L43A, F44P, T45R or G46T, and chimeric mAb2 heavy chains (CDR mAb2 with hFRl-mFR2- hFR3-4) and their binding was assessed using capture ELISA. The plates were coated with 1 μg/ml human CD3 extracellular domain (soluble hCD3 or shCD3) and incubated in the presence of various concentrations of antibody. The results (Fig. 3) indicate that the T substitution at position 46 of Kabat abolished the ability of the antibody to bind to shCD3.

Additional studies were conducted to determine the effect of variations in Kabat light chain positions 36, 38, 44 and 46. Antibodies containing the light chain variable region of VL-8 h-mAb2, VL-9 h-mAb2 or VL-10 h-mAb2 and the heavy chain of the chimeric mAb2 antibody were generated and assessed using the capture ELISA described above. The results of this experiment are presented in Fig. 4 and show that binding to shCD3 antibody containing the hVL-8 light chain variable region was similar to that of the chimeric mAb2 light chain antibody.

Antibodies containing the light chain variable region of VL-6 h-mAb2, VL-7 h-mAb2, or VL-8 h-mAb2 and the heavy chain of the chimeric mAb2 antibody were also generated and assessed using the capture ELISA described above (except plates were coated with 0.5 μg/ml shCD3 in phosphate buffered saline) to determine the effect of additional substitutions at positions 36, 38, and 46. The results of this experiment are presented in FIG. 5 and show that substitutions F36V and T46G using Kabat numbering were sufficient to produce an antibody whose binding to shCD3 was similar to that of the chimeric mAb2 light chain antibody.

The effect of substitutions in the mAb2 heavy chain sequence was assessed by generating antibodies containing the light chain of the chimeric mAb2 antibody and the VH-5 h-mAb2, VH-6 h-mAb2 or VH-7 h-mAb2 heavy chain variable region and assessing binding using the described above capture ELISA (using 1 μg/ml shCD3 coating). The results of these studies are presented in Fig. 6. In addition, antibodies were generated containing the light chain of the chimeric antibody mAb2 and

- 45 045070 humanized variant of the heavy chain variable region of VH-4 h-mAb2, VH-7 h-mAb2 or VH-9 h-mAb2. The binding of such antibodies was assessed using the capture ELISA described above. The results of these studies are presented in Fig. 7.

hVH-6L (and its variant hVH-6M) and hVH-8L (and its variant VH-8M) heavy chains are particularly preferred for the production of antibodies having less affinity for CD3 than antibodies comprising hVH-1, hVH -2, hVH-3, hVH-4, hVH-5, hVH-6, hVH-7 or hVH-8 table. 2. Such reduced affinity antibodies will preferably include either hVH-6L heavy chain or VH-8L heavy chain in combination with any of VL-1 light chain h-mAb2, VL-2 h-mAb2, VL-3 hmAb2 , VL-4 h-mAb2, VL-5 h-mAb2, VL-6 h-mAb2, VL-7 h-mAb2, VL-8 h-mAb2, VL-9 h-mAb2 or VL-10 h-mAb2. A particularly preferred deimmunized antibody will consist of hVH-6L (or its variant hVH-BM) heavy chain and VL-6 light chain h-mAb2, or hVH-8L (or its variant hVH-8M) heavy chain and VL-6 light chain h-mAb2. Below are the sequences of such polypeptides:

Amino acid sequence of hVH-6L (SEQ ID NO: 54):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVGR IRNKYNNYAT EYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS

Amino acid sequence of hVH-8L (SEQ ID NO: 55):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR IRNKYNNYAT EYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS hVH-6L and hVH-8L heavy chains were further modified to produce variants containing the substitution modification to asparagine at position 52a (S52aN). The amino acid sequences of these modified heavy chain variable regions and the corresponding polynucleotide sequences encoding them are presented below:

Amino acid sequence of hVH-6M (SEQ ID NO: 72):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT EYAASVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding the heavy chain variable region of hVH-6M (SEQ ID NO: 73):

gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggagggtc cctgagactc tcctgtgcag cctctggatt caccttcaac acatacgcta tgaattgggt ccgccaggct ccagggaagg ggctggagtg ggttggaagg atcaggtcca agtacaacaa ttatgcaacc gagtatg ccg actctgtgaa ggatagattc accatctcaa gagatgattc aaagaactca ctgtatctgc aaatgaacag cctgaaaacc gaggacacgg ccgtgtatta ctgtgtgaga cacggtaact tcggcaattc ttacgtgtct tggtttgctt attggggaca ggggacactg gtgactgtgt cttcc

Amino acid sequence of hVH-8M (SEQ ID NO: 74):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR IRNKYNNYAT EYAASVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS

Polynucleotide sequence encoding the heavy chain variable region of hVH-8M (SEQ ID NO: 75):

gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggagggtc cctgagactc tcctgtgcag cctctggatt caccttcaac acatacgcta tgaattgggt ccgccaggct ccagggaagg ggctggagtg ggttgcaagg atcaggaaca agtacaacaa ttatgcaacc gagtat gccg actctgtgaa ggatagattc accatctcaa gagatgattc aaagaactca ctgtatctgc aaatgaacag cctgaaaacc gaggacacgg ccgtgtatta ctgtgtgaga cacggtaact tcggcaattc ttacgtgtct tggtttgctt attggggaca ggggacactg gtgactgtgt cttcc hVH-8 di-1 and hVH-8 di- 2 heavy chains are particularly preferred for producing antibodies that are less immunogenic than antibodies comprising hVH-1, hVH-2, hVH-3, hVH-4, hVH-5, hVH-6, hVH7 or hVH- 8 tables 2. Such deimmunized antibodies will preferably consist of either hVH-8 di-1 heavy chain or hVH-8 di-2 heavy chain in combination with any of VL-1 light chain h-mAb2, VL-2 h-mAb2, VL -3 h-mAb2, VL-4 h-mAb2, VL-5 h-mAb2, VL-6 hmAb2, VL-7 h-mAb2, VL-8 h-mAb2, VL-9 h-mAb2 or VL-10 h -mAb2. A particularly preferred deimmunized antibody will consist of hVH-8 di-1 heavy chain and VL-6 h-mAb2 light chain, or hVH-8 di-2 heavy chain and VL-6 h-mAb2 light chain.

Amino acid sequence of hXR32VH-8 di-1 (SEQ ID NO: 56):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKANSYTT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCAR HGNFGNSYVS WFAYWGQGTL VTVSS

Amino acid sequence of hXR32VH-8 di-2 (SEQ ID NO: 57):

- 46 045070

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVGR TRSKANSYTT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCAR HGNFGNSYVS WFAYWGQGTL VTVSS

Such deimmunized antibodies will preferably consist of either hVH-8 di-1 heavy chain or hVH-8 di-2 heavy chain in combination with any of VL-1 light chain h-mAb2, VL-2 h-mAb2, VL-3 h-mAb2, VL-4 h-mAb2, VL-5 h-mAb2, VL-6 h-mAb2, VL-7 h-mAb2, VL-8 h-mAb2, VL-9 h-mAb2 or VL-10 h -mAb2. A particularly preferred deimmunized antibody will consist of hVH-8 di-1 heavy chain and VL-6 h-mAb2 light chain, or hVH-8 di-2 heavy chain and VL-6 hmAb2 light chain.

Additional humanized variants of the heavy chain variable region of the murine monoclonal antibody mAb2 (SEQ ID NO: 7) were also created. The amino acid sequences of such variants are presented below, with changes from SEQ ID NO: 7 shown in bold and underlined.

Amino acid sequence of variant a (I51T Y52cA) of the humanized heavy chain variable region of the murine monoclonal antibody mAb2 (SEQ ID NO: 76):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TASKANNYAT YYADSVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of variant b (I51T N54S) of the humanized heavy chain variable region of the murine monoclonal antibody mAb2 (SEQ ID NO: 77):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKYNSYAT YYADSVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of variant c (I51T A56T) of the variable region of the heavy chain of the mouse monoclonal antibody mAb2 (SEQ ID NO: 78):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKYNNYTT YYADSVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of the d(I51T Y52cA N54S) variant of the humanized heavy chain variable region of the murine monoclonal antibody mAb2 (SEQ ID NO: 79):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKANSYAT YYADSVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of variant e (I51T N54S A56T) of the humanized heavy chain variable region of the mouse monoclonal antibody mAb2 (SEQ ID NO: 80):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKYNSYTT YYADSVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of variant f'(I51T Y52cA N54S A56T) of the humanized heavy chain variable region of the mouse monoclonal antibody mAb2 (SEQ ID NO: 81):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKANSYTT YYADSVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of the humanized heavy chain variable region of the murine monoclonal antibody mAb2 (SEQ ID NO: 82) variant g(I51T D61A):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKYNNYAT YYAASVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of the humanized heavy chain variable region of the murine monoclonal antibody mAb2 (SEQ ID NO: 83) variant h(I51T D65G):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKYNNYAT YYADSVKGRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of variant i (I51T Y52cA N54S D61A) of the humanized heavy chain variable region of the mouse monoclonal antibody mAb2 (SEQ ID NO: 84):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKANSYAT YYAASVKDRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of variant j (I51T Y52cA N54S D65G) of the humanized heavy chain variable region of the mouse monoclonal antibody mAb2 (SEQ ID NO: 85):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKANSYAT YYADSVKGRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

- 47 045070

Amino acid sequence of variant k(I51T Y52cA N54S D61A D65G) of the humanized heavy chain variable region of the murine monoclonal antibody mAb2 (SEQ ID NO: 86):

EVKLLESGGG LVQPKGSLKL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKANSYAT YYAASVKGRF TISRDDSQSI LYLQMNNLKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGTL VTVSA

Amino acid sequence of the humanized heavy chain variable region of the mouse monoclonal antibody mAb2 (SEQ ID NO: 87) variant 2k(I51T Y52cA N54S D61A D65G (VH8-A49G V93A)):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVGR TRSKANSYTT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCAR HGNFGNSYVS WFAYWGQGTL VTVSS

Amino acid sequence of the 5k variant (I51T Y52cA N54S D61A D65G (VH8-V93A)) of the humanized heavy chain variable region of the mouse monoclonal antibody mAb2 (SEQ ID NO: 88):

EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR TRSKANSYTT YYAASVKGRF TISRDDSKNS LYLQMNSLKT EDTAVYYCAR HGNFGNSYVS WFAYWGQGTL VTVSS

All such additional humanized variants of the murine monoclonal antibody mAb2 heavy chain variable region can be used to form deimmunized antibodies of the present invention. Such additional deimmunized and humanized antibodies will preferably include a heavy chain variable region: a, b, c, d, e, f, g, h, i, j, k, 2k or 5k, in combination with any of the lung variable regions circuits: VL-1 h-mAb2, VL-2 hmAb2, VL-3 h-mAb2, VL-4 h-mAb2, VL-5 h-mAb2, VL-6 h-mAb2, VL-7 h-mAb2, VL -8 h-mAb2, VL-9 hmAb2 or VL-10 h-mAb2. A particularly preferred deimmunized antibody will comprise a 2k or 5k heavy chain variable region and a VL-6 hmAb2 light chain variable region, or an hVH-8M8L di-2 heavy chain variable region and a VL-6 h-mAb2 light chain variable region. The 2k and 5k variants bind to protein A in the variable region, thereby facilitating the purification of molecules (such as diabodies) that may lack Fc regions or other domains that can be used to separate such molecules from other molecules. Variants hVH-8M, hVH8L, hVH-6M and hVH-6L exhibit reduced immunogenicity compared to the corresponding parental polypeptides.

The present invention particularly relates to deimmunized and humanized antibodies comprising hVH-8 heavy chain and VL-6 light chain. The present invention further relates to deimmunized and humanized antibodies comprising hVH-4 heavy chain and VL-6 light chain. In addition, the present invention particularly relates to deimmunized and humanized antibodies comprising hVH-2k heavy chain and VL-6 light chain.

Example 6. Analysis of the binding properties of light and heavy chain variants of chimeric and humanized mAb2

Capture ELISA was performed to evaluate the ability of the chimeric and humanized mAb2 to bind to non-human CD3. The plates were coated with 1 μl/ml extracellular domain CD3 (soluble CD3) (either human or cynomolgus) and incubated in the presence of various concentrations of antibody. The results of this experiment are presented in Fig. 8A and 8B show that mAb2 and its humanized variant exhibited equal binding to soluble human CD3 and soluble cynomolgus CD3.

Example 7 Quantitative analysis of mAb2 binding to human CD3 and cynomolgus CD3

BIACORE™ assays were performed to determine the extent of binding between mAb2 and human or cynomolgus CD3. BIACORE™ assays measure the dissociation rate, kd. The affinity (KD) of an antibody for its target depends on the kinetic constants of association (rate of association, k _a ) and dissociation (rate of dissociation, kd) according to the equation KD=k _d /k _a . BIACORE™ assays use surface plasmon resonance to directly determine these kinetic parameters. Anti-CD3 mAb2 (6.3-100 nM) was immobilized onto a support using anti-EK antibodies and incubated in the presence of soluble human CD3 (shCD3) or soluble cynomolgus CD3 (scCD3). The dynamics of dissociation were determined and the data were approximated to the divalent nature of the antibodies. The results of the BIACORE™ assays are presented in FIG. 9A-9D. The kinetics-related data are summarized in Table. 3.

- 48 045070

Table 3

shCD3 Antibody k _a k _d K _D ch-mAb2 1.7x10 ⁵ M- ¹ sect ¹ 2.5xl0- ³ sect ¹ 14.7 nM h-mAb2 1.9xl0 ⁵ M- ¹ sect ¹ 3, 8x10-3 sect ¹ 2 0.0 nM scCD3 Antibody k _a k _d K _D ch-mAb2 1, 6xl0 ⁵ M- ¹ sect ¹ 2.3x10-3 sec ^{- 1} 14.4 nM h-mAb2 1.7xl0 ⁵ M- ¹ sect ¹ 4.1x10-z sec - ¹ 24.1 nM

Example 8: Bispecific Binding Data for DART™ Diabodies Containing the h-mAb2 CDR

The humanized mAb2 (h-mAb2) CDR was used to generate a series of DART™ diabodies containing a first epitope-binding site capable of binding to CD3 and a second epitope-binding site capable of binding to Her2/neu (DART™ diabody Her2-h-mAb2), or with CD 19 (diabody DART™ CD19-h-mAb2), or with epidermal growth factor receptor (EGFR) (diabody DART™ ERBITUX™ -h-mAb2).

Diabody DART™ Her2/neu-h-mAb2

Amino acid sequence of hXR32VL-Her-2VH-E-helix diabody DART™ Her2-h-mAb2 (linkers between the hXR32VL sequence and the Her2VH sequence and between the Her2VH sequence and the E-helix sequence are underlined) (SEQ ID NO: 58):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QLQQSGPELV KPGASLKLSC TASGFNIKDT YIHWVKQRPE QGLEWIGRIY PTNGYTRY DP KFQDKATITA DTSSNTAYLQ VSRLTSEDTA VYYCSRWGGD GFYAMDYWGQ GASVTVSSGG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE

Amino acid sequence of Her2VLhXR32VH-K-helix diabody DART™ Her2-h-mAb2 (linkers between the Her2VL sequence and the hXR32VH sequence and between the hXR32VH sequence and the K-helix sequence are underlined) (SEQ ID NO: 59):

DIVMTQSHKF MSTSVGDRVS ITCKASQDVN TAVAWYQQKP GHSPKLLIYS

ASFRYTGVPD RFTGNRSGTD FTFTISSVQA ADLAVYYCQQ HYTTPPTFGG

GTKLEIKRAG GGSGGGGEVQ LVESGGGLVQ PGGSLRLSCA ASGTFNTYA

MNWVRQAPGK GLEWVARIRS KYNNYATYYA DSVKDRFTIS RDDSKNSLYL

QMNSLKTEDT AVYYCVRHGN FGNSYVSWFA YWGQGTLVTV SSGGCGGGEV

AALEKEVAAL EKEVAALEKE VAALEK

Diabody DART™ CD19-h-mAb2

Amino acid sequence of CD19VL-hXR32VH-E-helix diabody DART™ CD19-hmAb2 (linkers between the CD19VL sequence and the hXR32VH sequence and between the hXR32VH sequence and the E-helix sequence are underlined) (SEQ ID NO: 60):

DIQLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSYLNWY QQIPGQPPKL LIYDASNLVS GIPPRFSGSG SGTDFTLNIH PVEKVDAATY HCQQSTEDPW TFGGGTKLEI KGGGSGGGGGE VQLVESGGGL VQPGGSLRLS CAASGFTFNT YAMNWVRQAP GKGLEWVARI RSKYNNYAT Y YADSVKDRFT ISRDDSKNSL YLQMNSLKTE DTAVYYCVRH GNFGNSYVSW FAYWGQGTLV TVSSGGCGGG EVAALEKEVA ALEKEVAALE KEVAALEK

Amino acid sequence of hXR32VL-CD19VH-K-helix diabody DART™ CD19-hmAb2 (linkers between the hXR32VL sequence and the CD19VH sequence and between the CD19VH sequence and the K-helix sequence are underlined) (SEQ ID NO: 61):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QLQQSGAELV RPGSSVKISC KASGYAFSSY WMNWVKQRPG QGLEWIGQIW PGDG DTNYNG KFKGKATLTA DESSSTAYMQ LSSLASEDSA VYFCARRETT TVGRYYYAMD YWGQGTTVTV SSGGCGGGKV AALKEKVAAL KEKVAALKEK VAALKE

Diabody DART™ ERBITUX™-h-mAb2

Amino acid sequence of hXR32VL-EGFRVH-E-helix diabody DART™ ERBITUX™h-mAb2 (linkers between the hXR32VL sequence and the EGFRVH sequence and between the EGFRVH sequence and the E-helix sequence are underlined) (SEQ ID NO: 62):

- 49 045070

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QLKQSGPGLV QPSQSLSITC TVSGFSLTNY GVHWVRQSPG KGLEWLGVIW SGGNTDYNTP F TSRLSINKD NSKSQVFFKM NSLQSNDTAI YYCARALTYY DYEFAYWGQG TLVTVSSGGC GGGEVAALEK EVAALEKEVA ALEKEVAALE K

Amino acid sequence of EGFRVL-hXR32VH-K-helix diabody DART™ ERBITUX ^TM h-mAb2 (linkers between the EGFRVL sequence and the hXR32VH sequence and between the hXR32VH sequence and the K-helix sequence are underlined) (SEQ ID NO: 63):

DILLTQSPVI LSVSPGERVS FSCRASQSIG TNIHWYQQRT NGSPRLLIKY ASESISGIPS RFSGSGSGTD FTLSINSVES EDIADYYCQQ NNNWPTTFGA GTKLELKGGG SGGGGEVQLV ESGGGLVQPG GSLRLSCAAS GFTFNTYAMN WVRQAPGKGL EWVARIRSKY NNYATYYADS VKDR FTISRD DSKNSLYLQM NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS GGCGGGKVAA LKEKVAALKE KVAALKEKVA ALKE

These DART™ diabodies were found to be capable of binding to cynomolgus CD3 (FIGS. 10A-10C).

The humanized mAb2 (h-mAb2) CDR was used to generate a series of DART™ diabodies containing a first epitope-binding site capable of binding to CD3 and a second epitope-binding site capable of binding to B7-H3 (DART™ diabody B7-H3-1-h -mAb2 and B7-H3-2-h-mAb2).

Diabody DART™ B7-H3-1-h-mAb2

Amino acid sequence of hBRCA69DVL-hXR32VH-E-helix diabody DART™ B7-H31-h-mAb2 (linkers between the hBRCA69DVL sequence and the hXR32VH sequence and between the hXR32VH sequence and the E-helix sequence are underlined) (SEQ ID NO: 64):

DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLHSGVPS RFSGSGSGTD FTLTISSLQP EDIATYYCQQ GNTLPPTFGG GTKLEIKGGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFNTYAM NWVRQAPGKG LEWVARIRSK YNNYATY YAD SVKDRFTISR DDSKNSLYLQ MNSLKTEDTA VYYCVRHGNF GNSYVSWFAY WGQGTLVTVS SGGCGGGEVA ALEKEVAALE KEVAALEKEV AALEK

Amino acid sequence of hXR32VL-hBRCA69DVH-K-helix diabody DART™ B7-H31-h-mAb2 (linkers between the hXR32VL sequence and the hBRCA69DVH sequence and between the hBRCA69DVH sequence and the K-helix sequence are underlined) (SEQ ID NO: 65):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QLVQSGAEVK KPGASVKVSC KASGYTFTSY WMQWVRQAPG QGLEWMGTIY PGDGDTRY TQ KFKGRVTITA DKSTSTAYME LSSLRSEDTA VYYCARRGIP RLWYFDVWGQ GTTVTVSSGG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE

Diathelo DART™ B7-H3-2-h-mAb2

Amino acid sequence of hBRCA84DVL-hXR32VH-E-helix diabody DART™ B7-H32-h-mAb2 (linkers between the hBRCA84DVL sequence and the hXR32VH sequence and between the hXR32VH sequence and the E-helix sequence are underlined) (SEQ ID NO: 66):

DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNNYPFTFGQ GTKLEIKGGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFNTYAM NWVRQAPGKG LEWVARIRSK YNNYATYYAD SVKDRFTISR DDSKNSLYLQ MNSLKTEDTA VYYCVRHGNF GNSYVSWFAY WGQGTLVTVS SGGCGGGEVA ALEKEVAALE KEVAALEKEV AALEK

Amino acid sequence of hXR32VL-hBRCA84DVH-K-helix diabody DART™ B7-H32-h-mAb2 (linkers between the hXR32VL sequence and the hBRCA84DVH sequence and between the hBRCA84DVH sequence and the K-helix sequence are underlined) (SEQ ID NO: 67):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGEV QLVESGGGLV QPGGSLRLSC AASGTFSSF GMHWVRQAPG KGLEWVAYIS SDSSAIYYAD TVKGRFTI SR DNAKNSLYLQ MNSLRDEDTA VYYCGRGREN IYYGSRLDYW GQGTTVTVSS GGCGGGKVAA LKEKVAALKE KVAALKEKVA ALKE

These DART™ antibodies were found to be capable of binding to soluble cynomolgus CD3 (FIG. 10D).

Example 9: Dual Affinity Retargeting Reagents (DART™) Specific for HER2/neu and CD3 Mediate Potent Redirected T Cell-Mediated Killing

Dual Affinity Retargeting Reagent Reagents (DART™) diabodies specific for HER2/neu and CD3 were prepared. These DART™ diabodies have the ability to localize

a T cell (as a result of binding of such a T cell to the CD3 binding portion of the CD3 binding diabody DART™) in the position of a tumor cell (as a result of binding of such a cancer cell to

HER2/neu-binding portion of the DART™ diabody). The localized T cell can then kill the tumor cell in a process referred to here as redirected killing.

A dual-affinity retargeting reagent (DART™) specific for HER2/neu and CD3 was constructed, containing the HER2/neu-binding variable domains of trastuzumab and the CD3-binding variable domains VH-8 h-mab2 and VL -6 h-mab2 (SEQ ID NO:58-59).

To demonstrate the ability of DART™ diabodies to mediate redirected killing of cancer cells, the above-described HER2/neu x CD3 bispecific DART™ diabody was incubated at various concentrations with target tumor cells (SKOV-3 tumor cells, SKBR-3 tumor cells, A549 tumor cells and MCF-7 tumor cells) and resting PBMC effectors (E:T ratio=30:1), and cytotoxicity was determined (LDH assay). The results of these studies demonstrate the ability of the HER2/neu x CD3 bispecific diabody DART™ to mediate redirected tumor cell killing.

Example 10 Anti-TCR Monoclonal Antibody Therapy for Patients with Autoimmune Diabetes

Patients:

Forty patients with type 1 diabetes were recruited according to the following criteria: age between 7 and 20 years, diagnosis within 6 weeks according to American Diabetes Association criteria, and evidence of autoantibodies against GAD65, ICA512, and/or insulin. Patients remain under the care of their personal physicians throughout the study.

Eligible patients are randomly assigned to a control group and a humanized anti-CD3 antibody (N297Q) treatment group (comprising VH-8 hmab2 and VL-6 h-mab2). After randomization, blood samples are collected to establish baseline HA1c levels, pre-treatment C-peptide response is established in the MMTT, and pre-treatment FPIR is performed in the IGTT. Patients in both groups are hospitalized to receive either a 6-day course of treatment with a humanized monoclonal anti-CD3 antibody (N297Q) or placebo. The antibody is administered intravenously at the following dose: 17 µg/ ^m2 on day 1, 34.3 µg/ ^m2 on day 2, 69 µg/ ^m2 on day 3, 137.6 µg/ ^m2 on day 4 and 275.3 µg/m ² on days 5 and 6. Alternatively, the antibody can be administered intravenously at the following dose: 1.6 µg/kg/day on day 1; 3.2 mcg/kg/day on day 2; 6.5 mcg/kg/day on day 3; 13 mcg/kg/day on day 4 and 26 mcg/kg/day on days 5-14. In dose escalation studies, treatment may be, for example, 1.42 mcg/kg/day on day 1; 5.7 mcg/kg/day on day 2; 11 mcg/kg/day on day 3; 26 mcg/kg/day on day 4 and 45.4 mcg/kg/day on days 5-14. In subsequent studies, therapy is changed by increasing the dose and/or decreasing the treatment period. For example, in subsequent studies, patients may be given a 4-day treatment: 6.4 mcg/kg/day on day 1; 13 mcg/kg/day on day 2 and 26 mcg/kg/day on days 3 and 4; during additional studies using dose escalation, treatment may be 8 mcg/kg/day on day 1; 16 mcg/kg/day on day 2 and 32 mcg/kg/day on days 3 and 4.

During initial studies, the antibody dose is administered in the first three days of treatment by slow intravenous infusion over 20 hours to monitor for adverse reactions. In subsequent studies, the administration time will be reduced and/or the dose will be divided into 2-4 equal parts, which will be administered as bolus injections evenly distributed over a period of 12 hours. Patients in the control group undergo metabolic and immunological studies, but not obtain monoclonal antibodies. The immunosuppressive effects of the anti-CD3 monoclonal antibody (N297Q) in patients are monitored throughout the study.

Patients are followed for 18 months after treatment. β-cell function is assessed every 6 months in the case of reduced glucose tolerance and every 12 months in the case of normal glucose tolerance. Patients are allowed to maintain a normal diet and remain under the supervision of their personal physicians throughout the study. Immunological tests are repeated at intervals of 6 months. Patients will receive insulin therapy as prescribed by their personal physicians.

β-cell functioning will be analyzed according to changes in Speptide levels determined by radioimmunoassay. After sampling to establish baseline C-peptide and glucose levels, patients are given a mixed meal. C-peptide levels were determined in samples taken at 15, 30, 60, 90, 120, 150, 180, 210 and 240 min. The response in the form of an increase in Speptide in the mixed food tolerance test (MMTT) is presented as the total area under the response curve (AUC). A response change is considered to have occurred if the response differs by more than 7.5% from the response at entry into the study. Responses in the form of an increase in C-peptide in patients in

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MMTT is continuously reviewed at 6 months, 9 months, 12 months, 15 months and 18 months after treatment. Alternatively, β-cell function is assessed using FPIR (first phase insulin release) in the IGTT. Serum insulin levels were determined using a modification of the dual antibody radioimmunoassay method using monoiodinated tyrosine A14-labeled insulin (Amersham Pharmacia). FPIR is calculated as the sum of insulin levels 1 and 3 minutes after glucose administration (0.5 g/kg). Glycated hemoglobin levels are determined using a latex agglutination inhibition assay.

Immunological testing: Levels of autoantibodies against GAD65, IA2/ICA512 and insulin are determined using radiobinding assays known in the art (eg, Woo et al., 2000, J. Immunol Methods 244: 91-103). Genotyping of HLA-DQA and HLA-DQB is performed by direct sequencing of exon 2 polymorphisms after PCR amplification. Serum cytokine levels after monoclonal antibody administration are determined using an enzyme-linked immunosorbent assay (ELISA). Anti-idiotypic antibody production is monitored by ELISA using plate-bound anti-CD3 antibody (N297Q) or by flow cytometry to determine whether anti-CD3-FITC blocks binding to the TCR CD3 chain.

Statistical analysis:

Analyzes of data regarding residual beta cell function, autoantibody levels, cytokine levels, and glycosylated hemoglobin levels will be performed. χ ² analysis will be performed to test the effect of drug treatment before and after drug administration. Comparisons between the control group and the treatment group will be made using the Mann-Whitney U test.

Example 11: Dual Affinity Retargeting Reagents (DART™) Specific for B7H3 and CD3 Mediate Potent Retargeting T Cell-Mediated Killing

Dual affinity redirecting reagent (DART™) diabodies specific for B7H3 and CD3 were prepared. B7H3 has been immunohistologically detected in tumor cell lines (Chapoval, A. et al. (2001) B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production, Nature Immunol. 2: 269-274; Saatian, B. et al. (2004) Expression Of Genes For B7-H3 And Other T Cell Ligands By Nasal Epithelial Cells During Differentiation And Activation, Amer. J. Physiol. Lung Cell. Mol. Physiol. 287: L217-L225; Castriconi et al. ( 2004) Identification Of 4Ig-B7-H3 As A NeuroblastomaAssociated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis, Proc. Natl. Acad. Sci. (U.S.A.) 101(34): 12640-12645); Sun, M. et al. (2002) Characterization of Mouse and Human B7-H3 Genes, J. Immunol. 168: 6294-6297). Several independent studies have shown that human malignant tumor cells show a clear increase in B7-H3 protein expression, and that this increased expression is associated with increased disease severity (Zang, X. et al. (2007) The B7 Family And Cancer Therapy: Costimulation And Coinhibition , Clin. Cancer Res. 13: 5271-5279), which suggests that B7-H3 is used by tumors as an immune escape route (Hofmeyer, K. et al. (2008) The Contrasting Role Of B7- H3, Proc Natl Acad Sci (U.S.A.) 105(30): 10277-10278).

The CD3 binding portion of these DART™ antibodies consisted of the light and heavy chain variable regions of the humanized anti-CD3 mAb2 (VH-8 h-mAb2 and VL-6 h-mAb2) described above. The B7H3 binding portion of these DART™ antibodies consisted of hBRCA84D-2 light chain and hBRCA84D-2 heavy chain (SEQ ID NO:64-65).

Such DART™ diabodies have the ability to localize a T cell (as a result of binding of such T cell to the CD3 binding portion of the CD3 binding portion of the DART™ diabody) to the location of a tumor cell (as a result of binding of such cancer cell to the B7H3 binding portion of the DART™ diabody) . The localized T cell can then mediate the killing of the tumor cell through a process of redirected killing.

To demonstrate the ability of these DART™ diabodies to mediate such redirected killing of cancer cells, DART™ diabodies were incubated at various concentrations with target tumor cells (A498 tumor cells, RECA905021E tumor cells) and resting PBMC effectors (E:T ratio = 30:1) , and cytotoxicity was determined (LDH assay). The DART™ diabody (4420-h-mAb2), which has dual specificities for CD3 (h-mAb2) and fluorescein (4420 antibody), was used as a control.

Diathelo DART™ 4420-h-mAb2

Amino acid sequence 4420VL-hXR32VH-E-helix of DART™ 4420-h-mAb2 diabody (linkers between the 4420VL sequence and the hXR32VH sequence and between the hXR32VH sequence and the E-helix sequence are underlined) (SEQ ID NO: 68):

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DWMTQTPFS LPVSLGDQAS ISCRSSQSLV HSNGNTYLRW YLQKPGQSPK IVLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP WTFGGGTKLE IKGGGSGGGG EVQLVESGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR IRSKYNNY AT YYADSVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSSGGCGG GEVAALEKEV AALEKEVAAL EKEVAALEK

Amino acid sequence of hXR32VL-4420VH-K-helix diabody DART™ 4420-h-mAb2 (linkers between the hXR32VL sequence and the 4420VH sequence and between the 4420VH sequence and the K-helix sequence are underlined) (SEQ ID NO: 69):

QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRGLI

GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF

GGGTKLTVLG GGGSGGGGEV KLDETGGGLV QPGRPMKLSC VASGFTFSDY

WMNWVRQSPE KGLEWVAQIR NKPYNYETYY SDSVKGRFTI SRDDSKSSVY

LQMNNLRVED MGIYYCTGSY YGMDYWGQGT SVTVSSGGCG GGKVAALKEK

VAALKEKVAA LKEKVAALKE

The results of these studies (FIGS. 11A-11B) indicate the ability of the B7H3 x CD3 bispecific diabody DART™ to mediate redirected killing of B7H3-expressing tumor cells.

Example 12: Dual Affinity Retargeting Reagents (DART™) Specific for A33 and CD3 Mediate Potent Retargeting T Cell-Mediated Killing

Dual affinity redirecting reagent (DART™) diabodies specific for A33 and CD3 were prepared (DART™ diabody A33-h-mAb2). A33 is a membrane antigen that is expressed in normal human colon and small intestinal epithelium and in >95% of human colon cancers (Heath, J.K. et al. (1997) The Human A33 Antigen Is A Transmembrane Glycoprotein And A Novel Member Of The Immunoglobulin Superfamily, Proc. Natl. Acad. Sci. (USA) 94: 469-474).

Such DART™ diabodies have the ability to localize a T cell (as a result of binding of such T cell to the CD3 binding portion of the CD3 binding portion of the DART™ diabody) at the location of a tumor cell (as a result of binding of such cancer cell to the A33 binding portion of the DART™ diabody) . The localized T cell can then mediate the killing of the tumor cell through a process of redirected killing.

The CD3 binding portion of these DART™ diabodies consisted of the humanized mAb2 light and heavy chain variable regions (VH-8 h-mAb2 and VL-6 h-mAb2) described above. The A33-binding portion of these DART™ diabodies consisted of the RECA47 antibody.

Diathelo DART™ A33-h-mAb2

Amino acid sequence of RECA47VL-hXR32VH-K-helix diabody DART™ A33-hmAb2 (linkers between RECA47VL sequence and hXR32VH sequence and between hXR32VH sequence and K-helix sequence are underlined) (SEQ ID NO: 70): QIVLTQSPAI MSASPGERVT MTCSARSSIS FMYWYQQK PG SSPRLLIYDT SNLASGVPVR FSGSGSGTSY SLTISRMEAE DAATYYCQQW SSYPLTFGSG TKLELKRGGG SGGGGEVQLV ESGGGLVQPG GSLRLSCAAS GFTFNTYAMN WVRQAPGKGL EWVARIRSKY NNYATYYADS VKDRFTISRD DSKNSLYLQM NSLKTEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS GGCGGGKVAA LKEKVAAL KE KVAALKEKVA ALKE

Amino acid sequence of hXR32VL-RECA47VH-E-helix diabody DART™ A33-hmAb2 (linkers between the hXR32VL sequence and the RECA47VH sequence and between the RECA47VH sequence and the E-helix sequence are underlined) (SEQ ID NO: 71): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQ Q KPGQAPRGLI GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QLQQSGPELV KPGASVKISC KASGYTFSGS WMNWVKQRPG QGLEWIGRIY PGDGETNYNG KFKDKATLTA DKSSTTAYME LSSLTSVDSA VYFCARIYGN NVYFDVWGAG TTVTVSSGGC GGGEVAALEK EVAA LEKEVA ALEKEVAALE K

To demonstrate the ability of such DART™ diabodies to mediate such redirected killing of cancer cells, DART™ diabody was incubated at various concentrations with target tumor cells (Colo205 tumor cells, RECA905021E tumor cells) and resting PBMC effector cells (E:T ratio = 30:1 ), and cytotoxicity was determined (LDH assay). The results of these studies (FIGS. 12A-12E) demonstrate the ability of A33 x CD3 bispecific DART™ diabodies to mediate redirected killing of A33-expressing tumor cells.

Example 13 CD3-specific dual affinity redirecting reagents (DART™) diabodies result in redirected T cell-mediated killing equivalent to other human CD3-specific diabodies

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To further evaluate the CD3-specific dual-affinity redirecting reagents (DART™) diabodies of the present invention, the ability of the above-described DART™ diabody CD19-h-mAb2 to induce redirected T cell-mediated killing was compared with that of CD19 x CD3 -bispecific diabody DART Moore, R.A. et al. (2011) (Application Of Dual Affinity Retargeting Molecules To Achieve Optimally Redirected T-Cell Killing Of B-Cell Lymphoma, Blood 117(17): 4542-4551). The DART™ CD19-h-mAb2 diabody exhibits specificity for human CD3 as well as non-human CD3; CD19 x CD3 bispecific diabody DART Moore, P.A. et al. (2011) shows specificity for only human CD3.

Accordingly, Raji human B-cell lymphoma cells (see Drexler, H.G. et al. (1998) History And Classification Of Human Leukemia-Lymphoma Cell Lines, Leuk. Lymphoma 31(3-4): 305-316; Arndt, R. ( 1984) Demonstration Of C3-Binding Circulating Immune Complexes Using Raji, Conglutinin And Anti-C3 Assays - A Critical Review, Immun. Infekt. 12(1): 3-11) or lymphoma cells from human tissue cells JeKo-1 (Salaverria, I. et al (2006) Mantle Cell Lymphoma: From Pathology And Molecular Pathogenesis To New Therapeutic Perspectives, Haematologica 91: 11-16; Jeon, H. J. et al (1998) Establishment And Characterization Of A Mantle Cell Lymphoma Cell Line, Br J Haematol 102(5): 1323-1326) were incubated in the presence of DART™ diabody and resting peripheral blood mononuclear cells (PBMC) (E:T=30:1). The results of this experiment (FIGS. 13A and 13B) show that the DART™ CD19-hmAb2 diabody of the present invention results in redirected T cell-mediated killing that is equivalent to that observed with the human CD3-specific CD19 x CD3 bispecific DART diabody . Thus, the expansion of specificity for non-human CD3 homologs did not reduce the ability of the DART™ diabody to mediate redirected killing.

Example 14 Redirected Cytolysis Using CD3-Specific Dual Affinity Retargeting Reagent Diabodies (DART™) Cross-Reactive with Cynomolgus CD3

The ability of the DART™ CD19-h-mAb2 DART™ diabody described above to induce redirected T cell-mediated killing in the presence of either human or cynomolgus T cells was tested.

Human Colon Cancer Cells HT-29 (Marchis-Mouren, G. et al. (1988) HT 29, A Model Cell Line: Stimulation By The Vasoactive Intestinal Peptide (VIP); VIP Receptor Structure And Metabolism, Biochimie 70(5) : 663-671; Fogh, J. et al. (1975) In: J. Fogh (ed.), HUMAN TUMOR CELLS IN VITRO, New York: Plenum Press. 1975) were incubated in the presence of human or cynomolgus T cells ( E:T ratio=30:1) and either the DART™ CD19-h-mAb2 diabody described above, or a CD19 x CD3 bispecific DART diabody whose CD3 binding sequences are derived from the FN-18 antibody. The FN-18 antibody shows specificity only for cynomolgus CD3 (Nooij, F.J. et al. (1986) Differentiation Antigens On Rhesus Monkey Lymphocytes. I. Identification Of T Cells Bearing CD3 And CD8, And Of A Subset Of CD8-Bearing Cells, Eur J Immunol 16(8): 975-979 Meng, G et al (1998) The Effect Of Anti-CD3-Immunotoxin On T Lymphocyte Function in vitro, Transpl Immunol 6(1): 53 -59). The resulting percentage of cytotoxicity was determined depending on the concentration of the diabody. The results (FIGS. 14A and 14B) show that the DART™ CD19-h-mAb2 diabody is capable of mediating cytolysis in the presence of either human or cynomolgus effector T cells. In contrast, the FN-18 diabody was capable of mediating cytolysis only in the presence of cynomolgus T cells.

Example 15: Dual Affinity Retargeting Reagents (DART™) Require to Contact Target Cells

To demonstrate that the observed redirected killing mediated by the CD3-specific DART™ diabody of the present invention was specific, the extent of killing in the presence and absence of target cells was determined.

Human PBMCs were incubated in the presence of the above-described DART™ ERBITUX™-hmAb2, (ERBITUX™-T-cell receptor)-bispecific DART™ diabody (capable of binding to EGFR (epidermal growth factor receptor) and T-cell receptor) or DART diabody ™ ERBITUX™-CD3 FN18 (capable of binding to EGFR and cynomolgus CD3). Incubations were performed in the presence or absence of A498 target renal cancer cells (Giard, D. J. et al. (1973) In vitro Cultivation Of Human Tumors: Establishment Of Cell Lines Derived From A Series Of Solid Tumors, J. Natl. Cancer Inst. 51 : 1417-1423; Fogh, J. (1978) Cultivation, Characterization, And Identification Of Human Tumor Cells With Emphasis On Kidney, Testis And Bladder Tumors, Natl. Cancer Inst. Monogr. 49: 5-9).

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes, present on cells of most hematopoietic lineages, including neutrophils, after stimulation (Atzenia, F. et al. (2002) Induction Of CD69 Activation Molecule On Human Neutrophils by GM-CSF, IFN -γ, and IFN-α, Cellular Immunol. 220(1): 20-29). For this reason, as a way to assess immune system activation

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Claims (24)

determined the mean fluorescence intensity (MFI) of CD69 (depending on the concentration of diabodies) (see, for example, Ampel, N.M. et al. (2002) In Vitro Whole-Blood Analysis of Cellular Immunity in Patients with Active Coccidioidomycosis by Using the Antigen Preparation T27K, Clinical Diagnostic Laboratory Immunology 9 (5): 1039-1043). The results (FIGS. 15A and 15B) show that immune system activation (as measured by CD69 MFI) was increased only when CD4+ or CD8+ T cells were incubated with the DART™ ERBITUX™-h-mAb2 diabody of the present invention or (ERBITUX™-T -cell receptor) bispecific diabody DART™ (capable of binding to EGFR and T-cell receptor). The DART™ ERBITUX™-CD3 FN18 diabody (capable of binding to EGFR and cynomolgus CD3) did not cause an increase in CD69 MFI. Example 16 Redirected Killing Using Humanized Cynomolgus/Human CD3 Reactive DART™ Diabodies To further demonstrate the ability of the DART™ diabodies of the present invention to mediate redirected killing, targeted A498 kidney cancer cells or A431 squamous cell carcinoma cells were used (Lee, CM et al (2010) The Distribution Of The Therapeutic Monoclonal Antibodies Cetuximab And Trastuzumab Within Solid Tumors BMC Cancer 10: 255; pages 1-11; Bryant, J. A. et al (2004) EGF Activates Intracellular And Intercellular Calcium Signaling By Distinct Pathways In Tumor Cells, Cancer Biol Ther. 3(12): 1243-1249) and determined the extent of redirected killing mediated by various DART™ diabodies in the presence of PMBC effector cells (E:T=30:1). Cells were incubated in the presence of either DART™ ERBITUX™-h-mAb2 diabody, DART™ ERBITUX™-m-mAb2 diabody or DART™ 4420-h-mAb2 diabody (as a negative control) or a control secondary antibody. Binding to target cells was determined using MFI measurement. Redirected killing was determined by measuring % cytotoxicity. The results of this study are presented in Fig. 16A-16D. Diabodies with specificity for CD3 and EGFR were found to be capable of binding to A498 or A431 cells (FIGS. 16A and 16C, respectively) and mediating redirected killing of these cells (FIGS. 16B and 16D, respectively). All publications and patents mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and separately stated to be incorporated by reference in its entirety. Although the present invention has been described in connection with specific embodiments, it will be understood that further modifications are possible and that this application is intended to cover any variations, applications or adaptations of the present invention following generally the principles of the present invention and including such deviations from the description of the present invention, which fall within known or common practice in the field of technology to which the present invention relates, and which may be applicable to the essential features set forth above. CLAIM (1) said domains (A) and (E) are linked to form said antigen binding domain that is capable of immunospecifically binding to both human CD3 and non-human mammalian CD3; 1. A CD3-binding molecule containing an antigen-binding fragment of an antibody that contains a CD3-specific VL domain of an antibody and a CD3-specific VH domain of an antibody, wherein the CD3-specific VL domain and the CD3-specific VH domain form an antigen-binding domain capable of immunospecifically binding to both an epitope human CD3 and with a non-human mammalian CD3 epitope, where: (I) said CD3-specific VL domain contains three complementarity determining regions (CDRs) of SEQ ID NO: 5 and (II) said CD3-specific VH domain contains three complementarity determining regions (CDRs) of SEQ ID NO: 7, modified such that contain one or more substitutions selected from the group consisting of: (i) I51T; (ii) S52aN; (iii) Y52cA; (iv) N54S; (v) A56T; (vi) Y58E; (vii) D61A and (viii) D65G; where the indicated numbering corresponds to the numbering according to Kabat. (2) said domains (B) and (D) are associated to form a binding site that immunospecifically binds to a second epitope, wherein said second epitope differs from the CD3 epitope in that it binds to an antigen binding domain formed by said domains (A) and (E) ); and (3) said domains (C) and (F) are covalently linked to each other. 2. The CD3 binding molecule according to claim 1, wherein said CD3-specific VL domain is selected from the group consisting of h-mab2 VL-4 (SEQ ID NO: 22), h-mab2 VL-6 (SEQ ID NO: 26 ), - 55 045070 h-mab2 VL-7 (SEQ ID NO: 28), h-mab2 VL-8 (SEQ ID NO: 30), h-mab2 VL-9 (SEQ ID NO: 32) and h-mab2 VL- 10 (SEQ ID NO: 34). 3. The CD3-binding molecule according to any one of claims 1 to 2, wherein said CD3-specific VH domain is selected from the group consisting of h-mab2 VH-6L (SEQ ID NO: 54), h-mab2 VH-8L (SEQ ID NO: 55), h-mab2 VH-8 di-1 (SEQ ID NO: 56), h-mab2 VH-8 di-2 (SEQ ID NO: 57), h-mab2 VH-6M (SEQ ID NO : 72), h-mab2 VH-8M (SEQ ID NO: 74), h-mab2 VH-2k (SEQ ID NO: 87) and h-mab2 VH-5k (SEQ ID NO: 88). 4. The CD3-binding molecule according to any one of claims 1 to 2, wherein said CD3-specific VH domain contains an amino acid sequence that differs from the amino acid sequences of h-mab2 VH-1 (SEQ ID NO: 36), h-mab2 VH- 2 (SEQ ID NO: 38), h-mab2 VH-3 (SEQ ID NO: 40), hmab2 VH-4 (SEQ ID NO: 42), h-mab2 VH-5 (SEQ ID NO: 44), h -mab2 VH-6 (SEQ ID NO:46), h-mab2 VH-7 (SEQ ID NO: 48) and h-mab2 VH-8 (SEQ ID NO: 50) in that it contains the indicated amino acid substitution. 5. A CD3-binding molecule according to any one of claims 1 to 4, wherein said CD3-specific molecule is an antibody. 6. The CD3 binding molecule according to claim 5, wherein in said antibody: (A) lacks an Fc region, or (B) contains an Fc region that: (i) does not exhibit effector functions; or (ii) has reduced effector function; or (iii) impairs the ability of the Fc region of said antibody to bind to the Fc receptor; wherein the indicated lack of effector function, reduced effector function and impaired binding capacity are compared to the wild type Fc receptor. 7. A CD3 binding molecule according to any one of claims 1 to 4, which is a CD3 binding diabody that contains a first polypeptide chain and a second polypeptide chain, wherein said chains are covalently linked to each other, wherein: (I) said first polypeptide chain contains an amino (N-) terminus and a carboxy (C-) terminus and from the N terminus to the C terminus: (i) domain (A) containing said CD3-specific VL domain; (ii) domain (B) containing the binding region of the second immunoglobulin heavy chain variable domain (VH2); and (iii) domain (C); wherein said domains (A) and (B) are not linked to each other to form an epitope binding site; and (II) said second polypeptide chain contains an amino (N-) terminus and a carboxy (C-) terminus and from the N terminus to the C terminus: (i) a domain (D) containing the light chain variable domain binding region of said second immunoglobulin (VL2); (ii) domain (E) containing said CD3-specific VH domain; and (iii) domain (F); wherein said domains (D) and (E) are not linked to each other to form an epitope binding site; and where: 8. The CD3 binding molecule of claim 7, wherein said second epitope is not a CD3 epitope. 9. The CD3 binding molecule of claim 7, wherein said second epitope is a CD3 epitope that differs from the CD3 epitope in that it binds to the antigen binding domain formed by said domains (A) and (E). 10. CD3 binding molecule according to any one of claims 1 to 9, which is humanized. 11. CD3-binding molecule according to any one of claims 1-4 or 7-10, which is capable of immunospecific - 56 045070 simultaneously contact: (i) CD3 and (ii)(a) a tumor antigen or (ii)(b) a cell surface antigen, receptor or receptor ligand. 12. The CD3-binding molecule according to claim 11, which is capable of immunospecifically binding to CD3 and to a tumor antigen expressed on a tumor cell, wherein said tumor cell is a tumor cell of a malignant tumor selected from the group consisting of breast cancer, prostate cancer , stomach cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cancer, pharynx cancer, esophageal cancer, larynx cancer, bone cancer, skin cancer, melanoma, cancer uterine, testicular cancer, bladder cancer, kidney cancer, brain cancer, glioblastoma, thyroid cancer, lymphoma, myeloma and leukemia. 13. The CD3 binding molecule of claim 11, capable of immunospecifically binding to CD3 and to a cell surface antigen, receptor or receptor ligand, wherein the cell surface antigen, receptor or receptor ligand is HER2/neu, B7-H3, CD20, PSMA, IGF1R, EGFR, A33, CD19 or Ep-CAM. 14. The CD3 binding molecule of claim 11, capable of immunospecifically binding to CD3 and to a cell surface antigen, receptor or receptor ligand, wherein the cell surface antigen, receptor or receptor ligand is a molecule involved in T cell B cell binding, wherein said molecule involved in T cell-B cell binding is selected from the group consisting of CD19, CD20, CD22, CD23, CD27, CD32B, CD38, CD40, CD79a, CD79b, CD80, CD86, LFA-I, LFA-3 and CFA-I. 15. CD3-binding molecule according to any one of claims 7 to 9, where: (A) said domain (B) contains amino acid residues 119-238 of SEQ ID NO: 65 and (B) said domain (D) contains amino acid residues 1-107 of SEQ ID NO: 64. 16. CD3 binding molecule according to any one of claims 7 to 9, where: (A) said domain (B) contains amino acid residues 119-240 of SEQ ID NO: 67 and (B) said domain (D) contains amino acid residues 1-107 of SEQ ID NO: 66. 17. The CD3 binding molecule according to any one of claims 7 to 16, wherein said CD3-specific VL domain is h-mab2 VL-6 (SEQ ID NO: 26). 18. CD3-binding molecule according to any one of claims 7-9 or 15-17, which contains an Fc domain or part thereof. 19. CD3 binding molecule according to any one of claims 7-9 or 15-18, where: (A) said first polypeptide chain further comprises an E-helix sequence and said second polypeptide chain further comprises a K-helix sequence; or (B) said first polypeptide chain further comprises a K-helix sequence and said second polypeptide chain further comprises an E-helix sequence; wherein said E-helix sequence corresponds to amino acid residues 244-271 of SEQ ID NO: 62, and said K-helix sequence corresponds to amino acid sequences 247-274 of SEQ ID NO: 63. 20. The CD3 binding molecule according to claim 19, which contains an Fc domain or part thereof. 21. A pharmaceutical composition comprising a CD3 binding molecule according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier, excipient or diluent. 22. Use of a CD3-binding molecule according to any one of claims 1 to 20, or a pharmaceutical composition according to claim 21, for the preparation of a medicament for the treatment of a malignant neoplasm or an autoimmune or inflammatory disease. 23. Use according to claim 22, wherein said autoimmune or inflammatory disease is selected from the group consisting of insulin-dependent type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, celiac disease, Sjögren's syndrome, Grave's disease, Crohn's disease, autoimmune hepatitis, psoriasis, psoriatic arthritis, asthma, allergic rhinitis, effects after organ transplantation and graft-versus-host disease (GVHD). 24. Use according to claim 23, wherein said autoimmune or inflammatory disease is insulin-dependent type I diabetes. -