NZ618021B2 - Cd3-binding molecules capable of binding to human and non-human cd3 - Google Patents

Abstract

Disclosed is a CD3-binding molecule comprising an antigen-binding fragment of an antibody, wherein the antigen-binding fragment comprises an antibody CD3-specific VL domain and an antibody CD3-specific VH domain, 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 of human CD3 and to an epitope of the CD3 of a non-human mammal, wherein the VH and VL chains are of the sequences as defined in the specification. ng domain capable of immunospecifically binding to both an epitope of human CD3 and to an epitope of the CD3 of a non-human mammal, wherein the VH and VL chains are of the sequences as defined in the specification.

Description

recogn1zed by the system as De1ng Iore1gn to the body. ln st, the cellular imm11111111fom imtwnltwon flan makiliwnh‘n“ n‘P noflnin nolln fawn/<1 'T‘ nolln flanf nowvo 0 Title of the Invention: CD3-Binding Molecules Capable of g to Human and man CD3 Cross-Reference to Related Applications: This application claims priority to United States Patent Applications Nos. 61/488,716 (filed on May 21, 2011; pending) and 61/530,353 (filed on September 1, 2011; pending), each of which applications is herein incorporated by reference in its entirety.

Reference to Sequence Listing: This application includes one or more Sequence Listings pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in both paper and computer-readable media, and which paper and computer-readable disclosures are herein incorporated by reference in their entirety. ound of the Invention: Field of the ion: The present invention relates to CD3-binding molecules capable of binding to human and non-human CD3, and in particular to such molecules that are cross- reactive with CD3 of a non-human mammal (e.g., a cynomolgus monkey). The invention also pertains to uses of such antibodies and antigen-binding fragments in the treatment of cancer, autoimmune and/or inflammatory diseases and other conditions.

Description of Related Art: The body's immune system serves as a defense against a variety of conditions, including, e.g., injury, ion and sia, and is mediated by two separate but interrelated systems: the cellular and l immune systems.

Generally speaking, the humoral system is mediated by soluble products odies or immunoglobulins) that have the ability to combine with and neutralize products ized by the system as being foreign to the body. In contrast, the cellular immune system involves the mobilization of certain cells, termed T cells, that serve a Lnnnnnnyununubnyw; Lvuyunnuv uv yvavavLLu MAL“ Lu; ‘4;be uwvuuvuxvvu Lvuvu VAL v" u characteristics: the ite specificity of the immune response for antigen variety of therapeutic roles. T cells are lymphocytes that are derived from the thymus and ate between the tissues, lymphatic system and the circulatory system. They act t, or in response to, a variety of foreign structures (antigens). In many instances these foreign antigens are expressed on host cells as a result of neoplasia or infection. Although T cells do not themselves secrete antibodies, they are usually required for antibody secretion by the second class of lymphocytes, B cells (which derive from bone marrow). Critically, T cells t extraordinary immunological specificity so as to be capable of discerning one n from another).

A naive T cell, e.g., a T cell which 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 hage or a dendritic cell. When a naive T cell encounters a specific peptide:MHC complex on an n presenting cell, a signal is delivered through the T-cell receptor which induces a change in the conformation of the T cell’s lymphocyte function associated n (LFA) molecules, and increases their affinity for intercellular adhesion molecules (ICAMs) present on the surface of the antigen presenting cell. The signal generated by the ction of the T cell with an antigen presenting cell is necessary, but not sufficient, to activate a naive T cell. A second co-stimulatory signal is required. The naive T cell can be activated only by an antigen-presenting cell carrying both a specific peptide MHC complex and a co-stimulatory molecule on its surface. Antigen recognition by a naive T cell in the absence of co-stimulation results in the T cell becoming anergic. The need for two signals to activate T cells and B cells such that they achieve an adaptive immune response may e a mechanism for avoiding responses to self-antigens that may be present on an antigen presenting cell at locations in the system where it can be recognized by a T cell. Where contact of a T cell with an n presenting cell s in the generation of only one of two required s, the T cell does not become activated and an ve immune response does not occur.

The efficiency with which humans and other mammals develop an immunological se to pathogens and foreign substances rests on two characteristics: the exquisite specificity of the immune response for antigen complex has such a large number of ITAMS (10 in all), and these ITAMS are arrayed in tandem (due to the dimerization of the tuent chains), phosphorylation of the recognition, and the immunological memory that allows for faster and more Vigorous responses upon re-activation with the same n (Portoles, P. et al. (2009) “The 3 Complex: Opening the Gate to sful Vaccination,” Current Pharmaceutical Design 15:3290-3300; Guy, C.S. et al. (2009) ization of Proximal Signal Initiation at the TCR:CD3 Complex,” Immunol ReV. 232(l):7-2l).

The city of the response of T-cells is mediated by the recognition of antigen (displayed on Antigen-Presenting Cells (APCs) by a molecular complex involving the T Cell Receptor ) and the cell surface receptor ligand, CD3. The TCR is a covalently linked heterodimer of or and B chains (“TCRaB”). These chains are class I membrane polypeptides of 259 (or) and 296 ([3) amino acids in length. The CD3 molecule is a complex containing a CD3 y chain, a CD3 5 chain, and two CD3 8 chains associated as three dimers (83/, 88, CC) (Guy, C.S. et al. (2009) “Organization of al Signal Initiation at the TCR:CD3 x,” Immunol ReV. :7-2l; 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 73:209-2l2). The TCR and CD3 complex, along with the CD3 5; chain zeta chain (also known as T-cell receptor T3 zeta chain or CD247) comprise the TCR complex (van der Merwe, P.A. etc. (epub Dec. 3, 2010) “Mechanisms For T Cell Receptor ring,” Nat. ReV. Immunol. 11:47-55; Wucherpfennig, KW. 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). The complex is particularly significant since it contains a large number (ten) of immunoreceptor tyrosine-based activation motifs (ITAMs).

In mature T cells, TClVCD3 activation by foreign antigenic peptides associated to self-MHC molecules is the first step needed for the ion of antigen-specific T cells, and their differentiation into effector or memory T lymphocytes. These processes involve the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR complex. Because the TCR complex has such a large number of ITAMS (10 in all), and these ITAMS are d in tandem (due to the dimerization of the constituent chains), phosphorylation of the vuvv Allvllvuvlvuuuvt/t/I vuquV Avarvv, ALwLLuyLwLLuwuLuLL IA.:./v 1.2.2, L'ALuvvvuv, L‘. V» w!»- (2003) “Individualized T Cell Monitored Administration Of ATG Versus OKT3 In relevant tyrosine residues upon TCR ligation creates paired docking sites for ns that contain Src homology 2 (SH2) domains such as the f; chain-associated protein of 70 kDa (ZAP-70), and thereby initiate an amplifying signaling cascade which 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):7-2l).

The outcome of these processes is ted by the intensity and quality of the antigen stimulus, as well as by the nature of accompanying signals delivered by co-receptor and co-stimulatory surface molecules, or by 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 l(3):23l- 240). Although TCR stimulation is a prerequisite for T-cell activation, it is well recognized that engagement of co-stimulatory molecules, such as CD28, is necessary for filll T-cell activation and differentiation (Guy, C.S. et al. (2009) ization of Proximal Signal Initiation at the D3 Complex,” Immunol Rev. 232(1):7-2l).

Due to the fundamental nature of CD3 in initiating an anti-antigen response, monoclonal antibodies against this receptor have been proposed as being 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 dies were the first antibody approved for the human therapy (St. Clair E.W. (2009) “Novel Targeted Therapies for Autoimmunity,” Curr. Opin. Immunol. 648-657). Anti- CD3 antibody (marketed as LONE TM OKT3TM by Janssen-Cilag) has been administered to reduce acute rejection in ts with organ transplants and as a treatment for lymphoblastic ia (Cosimi, AB. et al. (1981) “Use Of Monoclonal dies To T-Cell Subsets For Immunologic Monitoring And Treatment In Reczpients OfRenal Allografts,” N. Engl. J. Med. 8-314; Kung, P. et al. (1979) Monoclonal antibodies defining distinctive human T cell surface antigens,” Science 7-349; Vigeral, P. et al. (1986) “Prophylactic Use OfOKT3 Monoclonal Antibody In Cadaver Kidney Reczpients. Utilization Of OKT3 As The Sole Immunosuppressive Agent,” lantation 41:730-733; Midtvedt, K. et al. (2003) idualized T Cell Monitored Administration Of ATG Versus OKT3 In Interleukin-2, And Gamma-Interferon In Serum After Injection 0f OKT3 Monoclonal Antibody In Kidney Transplant Recipients.” Transplantation 47:606-608; Ferran. C. et Steroid-Resistant Kidney Graft Rejection,” Clin. Transplant. 17(1):69-74; Gramatzki, M. et al. (1995) “Therapy With 0KT3 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 0f ic Anti-CD3 Are Nonmitogenic to T cells,” J. Immunol. 159(7):3613-3621; Cole, M.S. et al. (1999) “I-Ium291, A Humanized Anti-CD3 Antibody, Is Immunosuppressive To T Cells While ting Reduced Mitogenicity in vitro,” lantation 68:563-571; US. Patent Nos. 6,491,916; ,585,097 and 6,706,265). r, such anti-CD3 treatment has not proven to be specific enough to avoid side effects gsson, J. (2009) “The Role nomodulation Therapy in Autoimmune Diabetes,” J. Diabetes Sci. Technol. 3(2):320-330). Repeated daily administration of OKT3 results in profound suppression and provides effective treatment of rejection following renal transplantation. The in vivo administration of OKT3 results in both T cell activation and suppression of immune responses. However, the use of OKT3 has been hampered by a first toxic dose reaction syndrome that is related to initial T-cell activation events and to the ensuing release of cytokines that occurs before immunosuppression of T cell responses. The ed side effects that follow the first and sometimes the second injection of this mouse monoclonal antibody include a “flu-like” syndrome consisting of high fever, , headache, and gastrointestinal symptoms (vomiting and diarrhea) and in severe cases pulmonary edema within hours of treatment has been noted (Thistlethwaite, J.R.

Jr. et al. (1988) “Complications and Monitoring of 0KT3 y,” Am. J. Kidney Dis. 11:112-119). This syndrome is believed to reflect OKT3-mediated cross-linking of the TCIVCD3 complex on the T cell surface and the resultant release of cytokines (e.g, tumor necrosis factor alpha (TNFu), interferon-y, interleukins IL-2, IL-3, IL-4, IL-6, IL-10 and granulocyte-macrophage colony-stimulating factor (Masharani, U.B. et al. (2010) “Teplizumab y For Type I Diabetes,” Expert Opin. Biol. Ther. (3):459-465; Abramowicz, D. et al. (1989) “Release 0f Tumor Necrosis Factor, Interleukin-2, And Gamma-Interferon In Serum After ion 0f OKT3 Monoclonal Antibody In Kidney lant Recipients,” Transplantation -608; Ferran, C. et above. Therefore, preclinical data generated in rodents are of d predictive “A-“A“ "724.1, «AMAAAL LA LLA 4...”. nn«,;l.‘,;ln4.,\ 'T‘LA ..,.,\n,.L AL‘ ALAIAA 13,“. "ADAA.-. LANL£«,. In al. (1990) “Cytokine-Related Syndrome Following Injection 0fAnti—CD3 Monoclonal Antibody: Further ce For Transient In Vivo T Cell Activation,” Eur. J.

Immunol. 20:509-515; Hirsch, R. et al. (12989) “Eflects OfIn Vivo Administration 0f Anti-CD3 Monoclonal Antibody 0n T Cell Function In Mice. II. In Vivo Activation Of T Cells,” J. l. 142:737-743). The use of anti-CD3 antibodies is disclosed in United States Patents Nos. 7,883,703; 7,728,114; 472; 7,575,923; and 7,381,903, and in United States Patent Publications Nos. 2010/0150918; 2010/0209437; 2010/0183554; 2010/0015142, 2008/0095766, 077246 and in PCT Publication /119567.

A particular limitation of prior antibodies is their specificity for only human CD3. This limitation is a significant impediment to the pment of such antibodies as therapeutic agents for the ent of human diseases. In order to obtain market approval any new candidate medication must pass through rigorous testing. This testing can be subdivided into preclinical and al phases. Whereas the latter — filrther subdivided into the generally known clinical phases 1, II and III — is performed in human patients, the former is performed in animals. The aim of preclinical testing is to prove that the drug candidate has the desired activity and most importantly is safe. Only when the safety in animals and possible effectiveness of the drug candidate has been ished in nical testing this drug candidate will be approved for clinical testing in humans by the respective regulatory authority. Drug candidates can be tested for safety in animals in the following three ways, (i) in a relevant species, i.e., in a species where the drug candidates can recognize the ortholog antigens, (ii) in a transgenic animal containing the human antigens and (iii) by use of a surrogate for the drug candidate that can bind the ortholog antigens present in the animal. Limitations of enic animals are that this technology is typically limited to rodents. However, rodents and humans have significant differences in logy that may complicate the extrapolation of safety data obtained in s to predict safety in . The limitations of a surrogate for the drug candidate are the different composition of matter compared to the actual drug candidate and often the animals used are rodents with the limitation as discussed above. Therefore, preclinical data generated in rodents are of limited predictive power with respect to the drug candidate. The approach of choice for safety testing is group consisting of h-mab2 VH-l (SEQ ID NO:36), h-mab2 VH-2 (SEQ ID the use of a relevant species, preferably a lower primate. The limitation now of the CD3 binding molecules suitable for therapeutic intervention in man described in the art is that the relevant species are higher primates, in particular cynomolgus monkeys. ingly, an anti-CD3 antibody capable of binding to both human and primate CD3 is highly desirable. Such antibodies have been described in United States Patent Publication No. 20100150918 and in PCT Publication /119567.

Despite such es, a need remains for anti-human CD3 antibodies and their antigen-binding fragments that are capable of cross-reacting with CD3 of a non- human mammal (e.g., a cynomolgous monkey). The present invention addresses this need and the need for ed therapeutics for cancer, autoimmunity and inflammatory diseases. y of the Invention: The present invention relates to CD3-binding molecules capable of g to human and man CD3, and in particular to such molecules that are cross- reactive with CD3 of a non-human mammal (e.g., a lgus monkey). The invention also pertains to uses of such antibodies and antigen-binding fragments in the treatment of cancer, autoimmune and/or inflammatory es and other conditions.

In detail, the invention provides a CD3-binding molecule comprising an antigen-binding nt of an antibody, wherein the antigen-binding fragment comprises an antibody CD3-specific VL domain and an antibody ecific VH domain, wherein the CD3-specific VL domain and the CD3-specific VH domain form an antigen-binding domain capable of immunospecif1cally binding to both an epitope of human CD3 and to an epitope of the CD3 of a non-human mammal, wherein: (I) the CD3-specific VL domain is selected from the group consisting of h-mab2 VL-1 (SEQ ID NO:16), h-mab2 VL-2 (SEQ ID NO:18), h-mab2 VL-3 (SEQ ID NO:20), h-mab2 VL-4 (SEQ ID , h-mab2 VL-S (SEQ ID NO:24), h-mab2 VL-6 (SEQ ID NO:26), 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), and said CD3-specific VH domain is selected from the group consisting of h-mab2 VH-l (SEQ ID NO:36), h-mab2 VH-2 (SEQ ID [CITIIIIIUS 'dIILl II'Ol'II 1V -[CI'1'IIIIIUS [O L-[CITIIIIIUSI (i) a domain (A) comprising the CD3-specif1c VL domain; NO:38), h-mab2 VH-3 (SEQ ID NO:40), h-mab2 VH-4 (SEQ ID , h- mab2 VH-S (SEQ ID NO:44), h-mab2 VH-6 (SEQ ID NO:46), h-mab2 VH- 6L (SEQ ID NO:54), h-mab2 VH-7 (SEQ ID NO:48), h-mab2 VH-8 (SEQ ID NO:50), h-mab2 VH-8L (SEQ ID NO:55), h-mab2 VH-8 di-l (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-Zk (SEQ ID NO:87), and h-mab2 VH-Sk (SEQ ID NO:88); or (II) the CD3-specif1c VL domain is selected from the group consisting of h-mabl VL-l (SEQ ID NO:10) and h-mabl VL-2 (SEQ ID NO:12), and the CD3- specific VH domain is h-mabl VH (SEQ ID NO:14).

The invention particularly concerns the embodiment of the above-described CD3-binding molecule wherein the ecif1c VL domain is h-mab2 VL-6 (SEQ ID NO:26).

The ion further concerns the embodiment of the above-described CD3- binding molecules wherein the CD3-specif1c VH domain is h-mab2 VH-8 (SEQ ID NO:50), h-mab2 VH-6 (SEQ ID NO:46), or h-mab2 VH-Zk (SEQ ID NO:87).

The invention ularly concerns the embodiment of the above-described CD3-binding molecule wherein the molecule is an antibody, and particularly, wherein the antibody lacks an Fc region or ses an Fc region that: (A) lacks or fianction or has reduced effector fianction; or (B) s the ability of the Fc region of the antibody to bind to an Fc receptor; wherein the reduction in or function and the impairment of binding ability is ve to that of a wild-type Fc receptor.

The invention r concerns the embodiment of the above-described CD3- binding molecules wherein the molecule is a CD3-binding diabody that comprises a first polypeptide chain and a second polypeptide chain, the chains being covalently bonded to one another, wherein: I. the first polypeptide chain comprises an amino us and a carboxy terminus and from N-terminus to C-terminus: (i) a domain (A) comprising the CD3-specif1c VL domain; (ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2); and (iii) a domain (C); wherein the domains (A) and (B) do not associate with one another to form an epitope binding site; (H) the second ptide chain comprises an amino terminus and a carboxy terminus and from N-terminus to C-terminus: (i) a domain (D) comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2); (ii) a domain (E) comprising the CD3-specific VH domain; and (iii) a domain (F); wherein the domains (D) and (E) do not associate with one another to form an epitope binding site; and wherein: (1) the domains (A) and (E) associate to form the antigen-binding domain that is capable of immunospecif1cally binding to both human CD3 and to the CD3 of a non-human ; (2) the domains (B) and (D) associate to form a binding site that immunospecifically binds to a second epitope, the second epitope being different from the CD3 epitope bound by the antigen-binding domain formed from the association of the domains (A) and (E); and (3) the domains (C) and (F) are covalently associated together.

The invention further concerns the embodiment of the above-described CD3- binding les n the second epitope is not an epitope of CD3.

The invention further concerns the embodiment of the above-described CD3- binding molecules wherein the second e is an epitope of CD3 that is different from the CD3 epitope bound by the antigen-binding domain formed from the association of the domains (A) and (E). association and the molecule involved in the T cell — B cell association is selected The invention further concerns the embodiment of the above-described CD3- binding molecules or antibodies or diabodies in which such le humanized.

The ion further concerns the embodiment of the above-described CD3- binding molecules or antibodies or diabodies in which such molecule is capable of immunospecifically binding to CD3 and to fluorescein.

The invention further concerns the embodiment of the above-described CD3- binding molecules or diabodies in which such molecule is capable of immunospecif1cally binding to both: (i) CD3 and (ii)(a) a tumor antigen, or (ii)(b) a cell surface antigen, receptor or receptor ligand.

The invention further concerns the embodiment of the above-described CD3- binding molecules or ies in which the molecule or diabody is capable of immunospecif1cally binding to CD3 and to a tumor antigen expressed on a tumor cell, wherein the tumor cell is a tumor cell from a cancer selected from the group consisting of: breast cancer, prostate , c cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cavity , pharyngeal 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.

The invention further concerns the embodiment of the above-described CD3- g molecules or diabodies in which the molecule or diabody is capable of specif1cally g 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, IGF-lR., Ep-CAM, or is a molecule involved in a T cell — B cell association that leads to T cell or B cell activation in an adaptive immune se.

The invention further concerns the embodiment of the described CD3- binding molecules or ies in which the le or diabody is e of immunospecif1cally binding to CD3 and to a molecule involved in the T cell — B cell association and the molecule involved in the T cell — B cell association 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 invention further concerns a pharmaceutical ition comprising any of the above-descibed CD3-binding molecules, antibodies or diabodies, and a pharmaceutically acceptable carrier, excipient or diluent.

The invention further concerns the above-described pharmaceutical ition for use in the treatment of cancer or an autoimmune or inflammatory disease.

The invention further concerns the above-described pharmaceutical composition for use in the treatment of an autoimmune or inflammatory disease selected from the group ting of: type I insulin-dependent diabetes, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, celiac’s disease, Sjogren's syndrome, Grave’s e, Crohn’s disease, autoimmune tis, psoriasis, psoriatic arthritis, asthma, allergic rhinitis, effects from organ transplantation, or graft vs. host disease (GVHD). The invention particularly concerns the above-described pharmaceutical composition for use in the treatment of type I insulin-dependent diabetes.

Brief Description of the Drawings: Figures lA-lB show the results of a capture ELISA in which the ability of anti-CD3 antibody mABl (Figure 1A) or a chimeric derivative of antibody mABl (ch-mAbl) (Figure 1B) was assessed using human soluble CD3 3”).

Figures 2A-2B show the results of a capture ELISA in which the ability of anti-CD3 antibody mAB2 (Figure 2A) or a chimeric derivative of dy mAB2 (ch-mAb2) (Figure 2B) was ed using human soluble CD3 (“shCD3”) or soluble cynomolgus monkey CD3 (“scCD3”).

Figure 3 show the s of es to determine the effect of variations in Kabat numbered framework residues 41-46 of the light chain ofmAb2. 1)’ lllpllulllu UVLLD’ 1' [Sui b 1.)”. 1 Mull VULVU 1\1111115 UL J V1\U_ 1 llulllull illullLLV UVLL lymphoma cells Figure 4 show the s of analyses to determine the effect of variations in Kabat numbered framework residues 36, 38, 44 and 46 of the light chain ofmAb2.

Figure 5 show the results of es to determine the effect of variations in Kabat numbered framework es 36, 38 and 46 of the light chain .

Figure 6 show the results of analyses to determine the effect of variations in Kabat numbered framework residues 30, 49 and 93 of the heavy chain ofmAb2.

Figure 7 show the results of additional es conducted to determine the effect of variations in Kabat numbered framework residues 30, 49 and 93 of the heavy chain of mAb2.

Figures 8A-8B show the results of analyses conducted to assess the ability of chimeric and humanized mAb2 to bind to non-human CD3.

Figures 9A-9D show gram tracings of BIACORETM analyses done to determine the kinetics of the binding of ch-mAB2 or h-mAb2 and scCD3 or scCD3.

Figures 10A-10D show the results of capture ELISAs performed on DARTTM ies having an anti-CD3 first epitope binding site and second epitope binding site that bind to either Her2/neu, CD19, EGFR, or B7-H3.

Figures 11A-11B show the ability of B7H3 x CD3 DARTTM diabodies to mediate redirected killing of tumor cells expressing B7H3.

Figures 12A-12E show the ability of A33 x CD3 DARTTM diabodies to mediate redirected killing of tumor cells expressing A33.

Figures 13A and 133 show the results of a comparison of the capacity of a CD19-h-mAb2 DARTTM and a CD19 x CD3 DART diabody to cause redirected T- ediated killing. The CD19-h-mAb2 DARTTM diabody exhibits specificity to human as well as non-human CD3; the CD19 x CD3 DART diabody o exhibits specificity only to human CD3. Figure 13A: redirected g of Raji human B-cell lymphoma cells; Figure 13B: redirected killing of JeKo-l human mantle cell lymphoma cells antigen-binding domain exhibits reactivity such that it will immunospecif1cally Figures 14A and 143 show that the CD19-h-mAb2 DARTTM diabody of the present invention was able to e sis in the presence of either human or non-human T-cell effector cells.

Figures 15A and ISB show the ability of the ERBITUXTM-h-mAbZ DARTTM diabody of the present invention or an ERBITUXTM-T-Cell Receptor DARTTM y to mediate an increase in CD69 MFI upon incubation with CD4+ or CD8+ T cells; A control ERBITUXTM-FN18 CD3 DARTTM diabody (capable of binding to EGFR and to cynolmolgus monkey CD3) failed to induce an increase in the CD69 MFI.

Figures 16A-16D show the results of investigations into the binding of either ERBITUXTM-h-mAbZ DARTTM diabody, ERBITUXTM-m-mAbZ DARTTM diabody or 4420-h-mAb2 DARTTM diabody ive control) or a l secondary to A498 or A431 cells (Figure 16A and 16C, tively), and to mediate redirected killing of such cells (Figure 16B and 16D, respectively).

Detailed Description of the Invention: The present invention relates to anti-human CD3 antibodies and their antigen-binding fragments, and in particular to such antibodies that are cross-reactive with CD3 of a non-human mammal (e.g., a cynomolgous monkey). The invention also pertains to uses of such antibodies and antigen-binding nts in the treatment of cancer, autoimmune and/or inflammatory diseases and other conditions. 1. ions As used herein, the term “CD3-binding molecule” denotes a molecule capable of immunospecific binding to both human CD3 and to the CD3 of a non- human mammal through at least one antigen recognition site (e.g., an antigen-binding domain of an antibody) located in the variable region of the molecule. As used herein such lity to immunospecifically bind to both human CD3 and to the CD3 of a man mammal is not intended to denote a capacity of a single antigen binding domain to simultaneously bind to both such CD3 molecules, but rather that such an antigen-binding domain exhibits cross-reactivity such that it will immunospecif1cally v “LLWVLV vanunn u; w unnnvnvnnu \v.b., ULLV UVUVLLu/ “A AL‘A “LWVVuJ FunjtjvtjunuLLLLLLLLLu form an epitope binding site. DARTTM ies may be monospecific, bispecif1c, bind to human CD3 when incubated in the presence of human CD3 and will immunospecif1cally bind to the CD3 of a non-human mammal when incubated in the presence of such non-human mammalian CD3.

As used , the term inding molecule” asses 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 an antibody n with an antigen ition site of the ed specificity, humanized antibodies, chimeric antibodies, “BiTEs®,” “DARTTM” diabody molecules and any other modified configuration of the immunoglobulin molecule that comprises an antigen ition site of the required specificity. The term “BiTEs” (bi-specific T-cell engagers) refers to a single polypeptide chain molecule that haVing 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 a target ( WO 547; Baeuerle, P et al. (2008) “BiTE®.' A New Class OfAntibodies That Recruit T Cells,” Drugs of the Future 33: 137-147; , et al. 2008) “Tumor Regression in Cancer ts by Very Low Doses of a T Cell- Engaging Antibody,” Science 321: 974-977).

The term “DARTTM” (Dual Affinity ReTargeting reagent) y refers to an immunoglobulin le that comprises at least two polypeptide chains that associate (especially through a covalent interaction) to form at least two epitope binding sites, which may recognize the same or different epitopes. Each of the polypeptide chains of a DARTTM diabody se 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. Rather, the immunoglobulin heavy chain variable region of one (e.g, the first) of the DARTTM diabody polypeptide chains interacts with the immunoglobulin light chain variable region of a different (e.g., the second) DARTTM polypeptide chain to form an epitope binding site.

Similarly, the immunoglobulin light chain variable region of one (e.g, the first) of the DARTTM diabody polypeptide chains interacts with the immunoglobulin heavy chain variable region of a different (e.g., the second) DARTTM diabody polypeptide chain to form an epitope binding site. DARTTM diabodies may be monospecific, bispecif1c, rcinoma antigen, LEA, lung adenocarcinoma F3 antigen, malignant human lvmphocvte antigen-APO-l. melanoma antigen gn75. melanoma-associated antigen trispecific, etc., thus being able to simultaneously bind one, two, three or more different epitopes (which may be of the same or of different antigens). DARTTM diabodies may additionally be lent, bivalent, trivalent, tetravalent, pentavalent, hexavelent, etc., thus being able to simultaneously bind one, two, three, four, five, six or more molecules. These two attributes of DARTTM diabodies , degree of specificity and valency may be combined, for example to produce if1c antibodies (i.e., capable of binding two epitopes) that are tetravalent (i.e., capable of binding four sets of epitopes), etc. DARTTM diabody molecules are disclosed in PCT Publications WO 13665, , and .

The bispecific (or trispecific or multispecif1c) molecules of the present invention will be capable of binding to both human CD3 and the CD3 of a non-human mammal (e.g., lgous monkey), and also to a second (or additional) and different n(s) or epitope(s). The second antigen or epitope is preferably a tumor antigen expressed on a tumor cell. Such tumor cells may be from cancers, for example, breast cancer, prostate cancer, gastric cancer, lung cancer, h cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, n cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer, laryngeal cancer, bone cancer, skin cancer, melanoma, uterine , testicular cancer, bladder cancer, kidney cancer, brain cancer, astoma, thyroid cancer, lymphoma, myeloma, or leukemia. The additional antigens or es are preferably cell surface tumor antigens or epitopes (such as: 17-1A, A33, adult erythrocyte primary endoderm I antigen, alpha fetoprotein, an envelope antigen of an RNA tumor virus, r tumor oncofetal antigen, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, Burkitt’s lymphoma antigen- 38.13, CA125, CD18, CD19, human B-lymphoma antigen-CD20, CD22, CD33, CD44, CD52, CEA, COl7-1A, CTA-1, CTLA-4, epidermal growth factor receptor, Ep-CAM, EphA2, fetal erythrocyte I antigen, f1brosarcoma antigen, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, GICA 19-9, gp IIIb/IIIa, gp72, HERl, neu, HER3, HER4, high molecular weight melanoma antigen, HLA- DR antigen, human leukemia T cell antigen-Gp37, human lung carcinoma antigen L20, human lung carcinoma n L6, human milk fat globule antigen, IgE, KS 1/4 pan-carcinoma antigen, LEA, lung adenocarcinoma F3 antigen, malignant human lymphocyte n-APO-l, melanoma antigen gp75, melanoma-associated antigen immunoglobulins as well as the fragments etc. described above under the definition of “onh‘knr‘xr ” p97, neoglycoprotein, nuC242, polymorphic epithelial mucin antigen, prostate specific antigen, prostate specific membrane antigen, prostatic acid phosphate, SK-l n, TAG-72, T-antigen, tumor n CAlZS, tumor antigen MUCl, tumor- specific transplantation type of cell-surface antigen, vascular endothelial growth , vascular endothelial growth factor-receptor, and (va3). Alternatively, such additional antigens or epitopes may be associated with a pathogen (such as: hepatitis type A, hepatitis type B, hepatitis type C, influenza, lla, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, galovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, small pox, Epstein Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type 11 (HIVII ), viral miningitis, viral encephalitis, dengue, small pox; mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia burgclorferi, us anthracis, Streptococcus, Staphylococcus, Mycobacterium, tetanus, pertissus, cholera, plague, diptheria, chlamydia, and legionella; leishmania, kokzidioa, trypanosoma or malaria; chlamydia and tsia.

The term “monoclonal antibody” refers to a homogeneous antibody population wherein the onal antibody is comprised of amino acids (naturally ing and non- naturally occurring) that are involved in the selective binding of an antigen. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The term “monoclonal antibody” encompasses not only intact monoclonal antibodies and filll- length monoclonal antibodies, but also nts thereof (such as Fab, Fab', F(ab')2 Fv), single chain (ScFv), mutants thereof, fusion proteins sing an antibody portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified ration of the immunoglobulin molecule that ses an antigen ition site of the required specificity and the ability to bind to an antigen. It is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e. g., by oma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of “antibody.” 88:4181-4185; Tempest, P.R. et al. (1991) “Reshaping A Human Monoclonal The term “humanized antibody” refer to a chimeric molecule, generally prepared using recombinant techniques, having an antigen binding site d from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the le based upon the structure and /or sequence of a human immunoglobulin. The antigen-binding site may comprise either te variable domains fused onto nt domains or only the complementarity ining regions (CDRs) grafted onto appropriate framework regions in the variable domains.

Antigen binding sites may be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A.F. et al. (1989) “Mouse/Human Chimeric Monoclonal Antibody In Man.‘ Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (USA) 86:4220- 4224). r approach focuses not only on providing human-derived constant regions, but modifying the variable regions as well so as to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity- determining regions (CDRs) which vary in response to the ns in on and ine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide scaffolding for the CDRs. When nonhuman antibodies are prepared with t to a particular antigen, the variable regions can be “reshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53:851-856.

Riechmann, L. et al. (1988) ping Human Antibodies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: ng An Antilysozyme Activity,” Science 239: 536; Kettleborough, C. A. et al. (1991) “Humanization OfA Mouse Monoclonal Antibody By CDR-Grafting.‘ The Importance OfFramework Residues 0n 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 dy,” Proc. Natl. Acad. Sci. (USA) 88:4181-4185; Tempest, P.R. et al. (1991) ping A Human Monoclonal (e.g, an anti-CD3 antibody) to bind to the epitope under different conditions, for Antibody To t Human Respiratory ial Virus Infection in vivo,” Bio/Technology 9:266-271; Co, M. S. et al. (1991) “Hamanized Antibodies For Antiviral Therapy,” Proc. Natl. Acad. Sci. (USA) 88:2869-2873; Carter, P. et al. (1992) “Hamanization OfAn Anti-p185her2 dy For Human Cancer Therapy,” Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co, MS. et al. (1992) “Chimeric And Hamanized Antibodies With Specificity For The CD33 Antigen,” J. Immunol. 148:1149-1154.

In some embodiments, humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanized dies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the al antibody. As disclosed below, the preferred antibodies of the present invention have ic identified CDRs. The present invention, however, contemplates equivalent antibodies having altered CDRs.

As used herein, an antibody or a polypeptide is said to “immunospecif1cally” or equivalently, “specifically” bind a region of another molecule (i.e., an e) if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with that e ve to ative epitopes. For example, an antibody that specifically binds to a CD3 epitope is an antibody that binds this CD3 epitope with greater affinity, avidity, more readily, and /or with greater duration than it binds to other CD3 epitopes or non-CD3 epitopes. It is also understood by reading this tion that, for e, an antibody (or moiety or epitope) that specif1cally binds to a first target may or may not specifically or preferentially bind to a second target. As such, “immunospecific binding” does not necessarily require (although it can include) “exclusive” binding. Generally, but not necessarily, reference to binding means “immunospecif1c” binding.

As used herein, the term “immunologically active” in reference to an epitope being or “remaining immunologically active” refers to the capability of an antibody (e.g, an anti-CD3 antibody) to bind to the epitope under different conditions, for uvxnkunvv, “LLULVVuJ LwaLALVLLw, w VLI/WLALLLL uvxnkunvv, w VMLVVLLJuwaV, w uvzxLLL, u; w chemotherapeutic compound. Various compounds can be synthesized, for example, example, after the epitope has been subjected to reducing and ring conditions.

For example, if the antibody is no longer able to bind a denatured epitope, that epitope is said to have been rendered immunologically inactive.

Different biological fianctions are associated with the anti-CD3 antibodies of the present ion, and such antibodies may t any or all of the following attributes, or may lack, one, two, three or more such attributes: an ability to specifically bind human CD3 as endogenously expressed on the surface of a normal human T cell; an ability to specifically bind human CD3 as endogenously expressed on the surface of a human leukemic T cell; an ability to specifically bind non-human mammal (e.g., cynomolgus monkey) CD3 as endogenously expressed on the surface of a normal non-human mammal T cell; an y to specifically bind non-human CD3 as endogenously expressed on the e of a normal non-human T cell; an ability to specifically bind a man CD3 as endogenously expressed on the surface of a non-human leukemic T cell; an y to neutralize (2'.e., block or interfere with binding) the formation of the CD3 complex; an ability to neutralize the formation of the TCR complex; an ability to te (either antagonistically or agonistically) signaling by the TCR complex; an ability to bind the Fc receptor; an ability to competitively inhibit preferential binding of a known D3 antibody to CD3, including the ability to preferentially bind to the same CD3 epitope to which the original antibody preferentially binds; an ability to bind to a portion of CD3 that is exposed on the surface of a liVing cell in vitro or in viva; an ability to bind to a portion of CD3 that is exposed on the surface of a living cancer cell; an y to deliver a chemotherapeutic agent into a cancerous T cell; and/or an ability to deliver a therapeutic agent, toxin or detectable marker into a T cell. As discussed herein, polypeptides (including antibodies) of the invention may have any one or more of these characteristics.

As used herein, the term “agent” refers to a biological, pharmaceutical, or chemical compound. Non-limiting examples include simple or complex organic or nic le, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a vitamin tive, a ydrate, a toxin, or a chemotherapeutic compound. s compounds can be synthesized, for example, ULLVLALVULLVLWIJVMI/LU “5V“"l VJ “LLVVU VLALuLLLb u; LLLuLLVVU VLALuLLLb vuw “wt/MULLLALVLL» uv w common platform, such that the antibody directs the localization of the agent to the small molecules and oligomers (e. g., oligopeptides and oligonucleotides), and synthetic organic compounds based on various core structures. In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. Agents that are employed in the methods of this invention can be randomly selected or rationally selected or designed. As used , an agent is said to be randomly selected when the agent is chosen without prior eration or knowledge of the specific amino acid or other chemical moieties involved in the association of the molecule with its native binding r(s) or known antibodies.

An example of a randomly selected agent is an agent that is identified h the use and screening of a chemical library or a peptide combinatorial library. As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a non-random basis that takes into account the sequence of the target site and /or its conformation in connection with the agent's action. Agents can be rationally selected or ally designed by utilizing the peptide sequences that make up the contact sites of the receptor /ligand and/or CD3/anti-CD3 antibody complex. For example, a rationally selected peptide agent can be a peptide whose amino acid ce is identical to an epitope appearing on CD3 as it is exposed on the surface of a living cell in its native environment. Such an agent will reduce or block the ation of the anti-CD3 dy with CD3, or the association of CD3 with its native ligand, as desired, by binding to the anti-CD3 antibody or to the native ligand.

As used herein, the term “labeled,” with regard to an antibody, is intended to encompass direct labeling of the antibody by coupling (z'.e., physically linking) a detectable substance, such as a radioactive agent or a hore (e. g. phycoerythrin (PE) or cein isothiocyanate (also known as fluoroisothiocyanate or FITC)) to the dy, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance.

As used herein, the term “association,” with regard to an antibody, includes covalent and non-covalent attachment or binding of an agent (e. g., chemotherapeutic agent) to the dy. The antibody can be associated with an agent (e.g., chemotherapeutic agent) by direct g or indirect binding via attachment to a common rm, such that the antibody directs the localization of the agent to the such as via targeting and /or internalization, delaying the progression of the disease, cancerous cell to which the antibody binds and wherein the antibody and agent do not substantially dissociate under physiological conditions such that the agent is not targeted to the same cancerous cell to which the antibody binds or such that the s potency is not decreased.

The term gical sample” encompasses a variety of sample types obtained from an individual and can be used in a stic or monitoring assay. The definition encompasses saliva, blood and other liquid samples of biological origin, solid tissue samples such as a biopsy en or tissue cultures or cells d rom, and the progeny thereof, for example, cells ed from a tissue sample ted from an individual suspected of having cancer, in preferred embodiments from ovary, lung, prostate, pancreas, colon, and breast tissue. The definition also includes samples that have been manipulated in any way after their ement, such as by ent with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides, or embedding in a semi-solid or solid matrix for sectioning purposes. The term “biological sample” encompasses a clinical sample, and also includes cells in e, cell supematants, cell lysates, serum, plasma, biological fluid, and tissue samples.

The term “host cell” includes an individual cell or cell culture that can be or has been a ent for vector(s) for incorporation of polynucleotide s. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a cleotide(s) of this invention.

As used herein, an “effective amount” of a pharmaceutical composition, in one embodiment, is an amount sufficient to effect beneficial or desired results including, without limitation, clinical results such as shrinking the size or rate of growth of a tumor, delaying or attenuating an inflammatory reaction, increasing the quality of life of those suffering from a disease, decreasing the dose of other medications required to treat such disease, enhancing the effect of another medication such as via targeting and /or internalization, delaying the progression of the disease, denotes a human. and/ or prolonging survival of individuals. Such effective amount can be administered in one or more administrations. For purposes of this invention, an ive amount of drug, compound, or pharmaceutical composition is an amount sufficient to rate a clinical observable 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 ered in the context of administering one or more additional agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved. While individual needs vary, determination of optimal ranges of ive amounts of each component is within the skill of the art. Typical dosage administered to a patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a 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 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight. The dosage and frequency of administration of molecules of the invention may be reduced or altered by ing uptake and tissue penetration of the molecules of the invention by modifications such as, for example, tion.

As used herein, a nucleic acid molecule or agent, antibody, composition or cell, etc, is said to be “isolated” when that nucleic acid molecule, agent, antibody, composition, or cell, etc. is ntially separated from inant nucleic acid molecules, antibodies, agents, itions, or cells, etc. naturally t in its original source.

The term “individual” refers to a vertebrate animal, preferably a mammal.

Mammals include, but are not limited to, humans, farm animals, sport animals, pets, es, mice and rats. In the most preferred embodiment, the term individual denotes a human. obtaining a beneficial or desired result including and preferably a beneficial or desired The terms “polypeptide,” “oligopeptide, 3, ide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling ent. Also included within the definition are, for example, polypeptides ning one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the ptides of this invention are based upon an antibody, the polypeptides can occur as single chains or as ated chains.

As used herein, the term antially pure” refers to material that is at least 50% pure (i.e., free from contaminants), 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 ably greater than 99% pure.

As used herein, the term “toxin” refers to any substance which effects an adverse response within a cell. For example, a toxin ed to a ous cell would have an adverse, sometimes deleterious effect, on the cancerous cell.

Examples of toxins include, but are not limited to, a taxane, a maytansinoid, an auristatin (e.g., monomethyl auristatin (MMAE), monomethyl auristatin F (MMAF), auristatin E (AE), etc.) (such as those disclosed in United States s Nos. ,208,020; 5,416,064; 6,333,410; 701; 6,372,738; 6,436,931; 6,441,163; 6,596,757; 7,276,497; 7,585,857; or 7,851,432), a calicheamicin, an anthracycline (e.g., doxorubicin), a CC-1065 , docetaxel,; cathepsin B or E; ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, and RNase; radiolabeled dies (e.g., tiuxetan-conjugated or labeled with a toxic radioisotope (for example, 90Y; 1311, 177Lu, 186 188 211 212225 Re, Re, At, B1, B1, Ac, etc.).

As used herein, the terms “treatment” or “treating” denote an approach for obtaining a beneficial or desired result including and preferably a beneficial or desired suspension. ing B-cells, or all dissociated spleen cells, can then be fused with mveloma cells (e.g.. X63- A98.653 and those from the Salk Institute. Cell clinical result. Such beneficial or d clinical results include, but are not limited to, one or more of the following: reducing inflammation or an autoimmune se, reducing the proliferation of (or destroying) cancerous cells or other diseased cells, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and /or prolonging al of duals. 11. Methods of Making the Antibodies And Polypeptides 0f the Present Invention Methods of making onal antibodies are known in the art. One method which may be employed is the method of Kohler, G. et al. (1975) “Continuous Cultures d Cells Secreting Antibody 0fPredefined Specificity,” Nature 256:495-497 or a modification thereof. lly, onal antibodies are developed in non-human species, such as mice. In general, a mouse or rat is used for immunization but other animals may also be used. The antibodies are produced by immunizing mice with an immunogenic amount of cells, cell extracts, or n preparations that contain human CD3. The immunogen can be, but is not limited to, primary cells, cultured cell lines, cancerous cells, nucleic acids, or tissue.

In one embodiment, monoclonal antibodies that bind to CD3 are obtained by using host cells that over-express CD3 as an immunogen. Such cells e, by way of example and not by limitation, human T cells.

To monitor the antibody response, a small biological sample (e. g., blood) may be obtained from the animal and tested for antibody titer against the gen.

The spleen and /or several large lymph nodes can be removed and dissociated into single cells. If d, the spleen cells may be screened (after removal of non- specifically adherent cells) by applying a cell suspension to a plate or to a well coated with the antigen. B-cells, expressing membrane-bound immunoglobulin specific for the antigen, will bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, can then be fused with myeloma cells (e.g., X63- Ag8.653 and those from the Salk Institute, Cell four general steps to humanize a monoclonal antibody. These are: (l) determining the Distribution Center, San Diego, CA). Polyethylene glycol (PEG) may be used to fuse spleen or lymphocytes with myeloma cells to form a hybridoma. The hybridoma is then cultured in a selective medium (e. g., hypoxanthine, aminopterin, thymidine medium, otherwise known as “HAT medium”). The resulting hybridomas are then plated by limiting dilution, and are d for the production of antibodies that bind specifically to the immunogen, using, for e, FACS (fluorescence activated cell g) or immunohistochemistry (IHC) screening. The selected onal antibody-secreting hybridomas are then cultured either in vitro (e. g., in tissue culture s or hollow fiber reactors), or in vivo (e. g., as ascites in mice).

As another alternative to the cell ‘l technique, Epstein-Barr Virus (EBV)—immortalized B cells may be used to produce monoclonal antibodies of the subject ion. The hybridomas are expanded and subcloned, if desired, and supematants are assayed for anti-immunogen activity by conventional assay procedures (e. g., FACS, IHC, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, etc.).

In another alternative, anti-CD3 monoclonal antibody and any other equivalent antibodies can be sequenced and produced recombinantly by any means known in the art (e. g., humanization, use of transgenic mice to produce fully human dies, phage display technology, etc.). In one embodiment, anti-CD3 monoclonal antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation. The sequence encoding the antibody of st may be maintained in a vector in a host cell and the host cell can then be expanded and frozen for fiJture use.

The cleotide ce of anti-CD3 onal antibody and any other equivalent antibodies may be used for genetic manipulation to generate a “humanized” antibody, to improve the affinity, or other characteristics of the antibody. The general principle in humanizing an antibody involves retaining the basic sequence of the antigen-binding portion of the antibody, while swapping the man remainder of the antibody with human antibody sequences. There are four general steps to humanize a monoclonal antibody. These are: (l) determining the Daugherty et al. (1991) “Polymerase Chain Reaction Facilitates The Cloning, CDR- Grafting. And Ranid Expression OfA Murine Monoclonal Antibody Directed Against nucleotide and predicted amino acid ce of the starting antibody light and heavy variable domains (2) designing the humanized antibody, i. e., deciding which antibody ork region to use during the humanizing process (3) the actual humanizing methodologies /techniques and (4) the transfection and expression of the zed antibody. See, for example, US. s Nos. 4,816,567; 5,807,715; 692; and 6,33 1,415.

A number of “humanized” antibody molecules comprising an antigenbinding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent or modified rodent V regions and their associated complementarity determining regions (CDRs) fiased to human constant domains (see, for example, Winter et al. (1991) “Man-made Antibodies,” Nature 349:293-299; Lobuglio et al. (1989) “Mouse/Human Chimeric Monoclonal dy 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 (I 7-IA) To A Colon Cancer Tumor-Associated Antigen,” J.

Immunol. 138:4534-4538, and Brown et al. (1987) “Tumor-Specific Genetically Engineered /Human Chimeric Monoclonal Antibody,” Cancer Res. 47:3577- 3583). Other references be rodent CDRs grafted into a human supporting framework region (FR) prior to fusion with an appropriate human antibody nt domain (see, for example, Riechmann, L. et al. (1988) “Reshaping Human dies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,” e 239:1534-1536; and Jones et al. (1986) “Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse,” Nature 321:522-525). Another reference describes rodent CDRs supported by recombinantly veneered rodent framework regions. See, for example, European Patent Publication No. 519,596. These “humanized” molecules are designed to minimize unwanted immunological response toward rodent anti-human antibody molecules, which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. Other s of zing antibodies that may also be utilized are disclosed by rty et al. (1991) “Polymerase Chain Reaction Facilitates The Cloning, CDR- Grafting, And Rapid Expression OfA Murine onal Antibody Directed Against vv VAL Mu FULJtJthvLuvu "qu; UULLVL tjvuu wLwLLUwaLVLLVI/L LALVuLLvat/LVLLU’ ”mun; WU, LVL v‘annntjnv, The CD18 Component 0fLeukocyte Integrins,” Nucl. Acids Res. l9:247l-2476 and in US. Patents Nos. 6,180,377; 6,054,297; 5,997,867; and 5,866,692.

The invention also asses single chain le region fragments (“scFV”) of antibodies of this invention, such as mu-anti-CD3. Single chain variable region fragments are made by linking light and/ or heavy chain le regions by using a short linking peptide. Bird et al. (1988) (“Single-Chain Antigen-Binding Proteins,” Science 3-426) describes example of linking peptides which bridge approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used (Bird et al. (1988) “Single-Chain Antigen-Binding Proteins,” Science 242:423-426). Linkers can in turn be modified for additional filnctions, such as attachment of drugs or attachment to solid supports. The single chain variants can be ed either recombinantly or synthetically. For tic production of scFV, an automated synthesizer can be used. For recombinant production of scFV, a suitable plasmid containing 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 made by routine manipulations such as ligation of polynucleotides. The resultant scFV can be isolated using rd protein purification techniques known in the art.

The invention includes ations to anti-CD3 antibodies and their binding nts. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of d polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the onal activity, or use of chemical analogs. Amino acid residues which can 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/tryosine.

These polypeptides also include glycosylated and cosylated polypeptides, as well as ptides with other ranslational modifications, such as, for example, glycosylation with ent sugars, acetylation, and phosphorylation. Preferably, the SF. (1991) “Amino Acid tution Matrices From An ation Theoretic Dnnnnnnnfinn ” T “linl D1n1 910 <4474A< pnwnnflxr Han mnaf orqxronnnr‘ DT OQTTR/f amino acid substitutions would be conservative, i.e., the substituted amino acid would possess similar al properties as that of the original amino acid. Such conservative substitutions are known in the art, and examples have been provided above. Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable region. Changes in the variable region can alter g y and/or specificity. Other methods of modification include using coupling ques known in the art, including, but not limited to, tic means, oxidative substitution and chelation. Modifications can be used, for example, for attachment of labels for immunoassay, such as the attachment of radioactive moieties for radioimmunoassay. Modified polypeptides are made using established procedures in the art and can be screened using standard assays known in the art.

The fact that a single amino acid alteration of a CDR residue can result in loss of functional binding (Rudikoff, S. etc. (1982) e Amino Acid Substitution Altering Antigen-Binding Specificity,” Proc. Natl. Acad. Sci. (USA) 79(6): 1979-1983) provides a means for systematically identifying alternative functional CDR sequences.

In one preferred method for obtaining such variant CDRs, a polynucleotide encoding the CDR is mutagenized (for example via random mutagenesis or by a site-directed method (e.g., polymerase mediated amplification with primers that encode the mutated locus)) to produce a CDR having a substituted amino acid residue. By comparing the identity of the relevant residue in the al (filnctional) CDR sequence to the identity of the substituted unctional) variant CDR sequence, the BLOSUM62.iij substitution score for that substitution can be identified. The BLOSUM system provides a matrix of amino acid substitutions created by analyzing a database of sequences for trusted alignments (Eddy, SR. (2004) “Where Did The BLOSUM62 ent 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) 15-10919; Karlin, S. et al. (1990) “Methods For Assessing The Statistical Significance 0f Molecular ce Features By Using General Scoring Schemes,” Proc. Natl. Acad. Sci. (USA) 87:2264-2268; Altschul, SF. (1991) “Amino Acid Substitution Matrices From An Information Theoretic Perspective,” J. Mol. Biol. 219, 555-565. Currently, the most advanced BLOSUM rather than single nucleotides results in a semi-randomized repertoire of amino acid database is the BLOSUM62 database (BLOSUM62.iij). Table 1 presents the BLOSUM62.iij substitution scores (the higher the score the more conservative the substitution and thus the more likely the substitution will not affect function). If an antigen-binding fragment comprising the ant CDR fails to bind to CD3, then the BLOSUM62.iij substitution score is deemed to be insufficiently vative, and a new candidate substitution is selected and produced having a higher substitution score. Thus, for e, if the original residue was glutamate (E), and the non- functional substitute residue was histidine (H), then the BLOSUM62.iij tution score will be 0, and more conservative changes (such as to aspartate, asparagine, glutamine, or lysine) are preferred.

Table 1 —----EIIIIP -l 0 -2 -l -l -l -l -2 -l 0 -2 0 -3 -2 +2 -l -3 -2 +1 -3 -3 0 -2 -3 -2 —1 —1 —3 —4 —1 —3 — 3 -l —3 —3 —1 —1 —3 —1 —2 -3 —2 0 —3 —2 --1 0 — 3 -l —2 0 —3 —3 --1 —2 -3 -l 0 6 —2 —4 —4 —2 -3 -3 -2 0 —2 +8 -3 —3 —1 -2 - 1 _2 -l 4 2 -3 --l 0 -3 2 4 -2 --2 -3 -2 -l -3 -l 0 2 "5 0 -2 0 -3 0 +6 -4 - -l -2 —4 7 —2 —2 0 —1 —2 — l 4 -2 -2 —1 —1 —1 —1 —2 —1 1 -2 -2 —3 —2 —3 —1 --1 —4 —3 — -2 -2 -3 -3 +3 +1 -2 +1 - -2 The invention thus contemplates the use of random mutagenesis to identify improved CDRs. Phage display technology can alternatively be used to se (or decrease) CDR affinity. This technology, ed to as affinity maturation, employs mutagenesis or “CDR walking” and re-selection uses the target antigen or an antigenic fragment thereof to identify antibodies having CDRs that bind with higher (or lower) affinity to the n when compared with the initial or parental antibody (See, e.g. Glaser et al. (1992) J. Immunology 149:3903). Mutagenizing entire codons rather than single nucleotides results in a semi-randomized repertoire of amino acid mutations. Libraries can be ucted consisting of a pool of variant clones each of “Affinity maturation of antibodies assisted by in silico modeling,” Proc. Natl. Acad.

Qni {TTQA\ 1n<l9£\-On90,0n’1/‘ 114 o nrnpnwnr‘ nmlanr‘limnnf «“111fome 14101-00 moxr Ian which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue.

Mutants with increased (or decreased) g affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant dies with increased or decreased affinity to the antigen (e.g., ELISA) (See Wu et al. 1998, Proc Natl. Acad Sci. USA 95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDR walking which randomizes the light chain may be used possible (See Schier et al., 1996, J. Mol. Bio. 263 :55 1).

Methods for accomplishing such affinity maturation are described for example in: Krause, J.C. et al. (2011) “An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function OfA Human Antibody,” MBio. 2(1) pii: -10. doi: 10.1128/mBio.00345-10; Kuan, C.T. et al. (2010) “Afiinity-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 apes,” J.

Mol. Biol. 401(1):84-96; Montgomery, D.L. et al. (2009) “Afiinity Maturation And Characterization Of A Human Monoclonal Antibody Against HIV-I gp4I,” MAbs 1(5):462-474; Gustchina, E. et al. (2009) “Afiinity Maturation By Targeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab d From A Synthetic Naive Human Antibody Library And Directed Against The Internal Trimeric Coiled-Coil 0f Gp4I Yields A Set Of Fabs With Improved HIV-I Neutralization Potency And Breadth,” Virology 393(1):112-119; Finlay, W.J. et al. (2009) “Afiinity Maturation OfA Humanized Rat Antibody For Anti-RAGE Therapy.‘ hensive Mutagenesis Reveals A High Level OfMutational Plasticity Both Inside And e The Complementarity-Determining Regions,” J. Mol. Biol. 388(3):541-558; m, J. et al. (2009) ving Antibody g y And Specificity For Therapeutic Development,” Methods Mol. Biol. 525:353-376; , S. et al. (2008) “In Vitro Afiinity Maturation Of Human GM—CSF Antibodies By Targeted CDR- Diversification,” Mol. Immunol. 46(1):135-144; and as, 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, multi-well plates may be Lnnvvnnununn9 “LL “LLwLVVu AMULULL L UULLUWLLLU ULLV u; LALULV u; v wLuv “ULALWLLLU IzLLVI/I/ specifically bind to CD3 and another amino acid sequence to which it is not attached coated with a selected CD3 antibody (e. g., 100 ng/well in carbonate buffer at room ature for 2 hrs) and subsequently incubated with soluble CD3 added at a dilution of 1/10 and incubated at room temperature for 16 hrs or diluted to a concentration of 50 ng/ml in PBS-T-BSA (0.05 ml added to each well and incubated for at least 2 h at room temperature). The plate is then washed and dilutions of recombinant antibodies starting at 0.5 ug/ml in PBS-T-BSA are then added and incubated for 1 hr at room temp. Binding of recombinant antibodies to the captured antigen is then measured using, for example, an anti-human IgG-HRP conjugate and TMB substrate. After stopping color development using dilute sulfuric acid, the plate is read at 450 nM and higher affinity antibodies identified (see, e.g., United States Patent No. 7,351,803).

The invention includes polypeptides comprising an amino acid sequence of the dies of this invention. The ptides of this invention can be made by procedures known in the art. The polypeptides can be produced by proteolytic or other degradation of the antibodies, by recombinant methods (i.e., single or fusion polypeptides) as described above or by chemical synthesis. Polypeptides of the dies, especially shorter polypeptides up to about 50 amino acids, are conveniently made by chemical sis. Methods of chemical synthesis are known in the art and are cially available. For example, an anti-CD3 polypeptide could be produced by an automated polypeptide synthesizer employing the solid phase method.

The invention also encompasses 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 that comprises at least 10 uous amino acids of variable light chain region and at least 10 amino acids of variable heavy chain region. In another embodiment, the fusion polypeptide contains a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide ns a light chain variable region and a heavy chain variable region of an antibody ed from a publicly-deposited oma. For es of this invention, an antibody fusion n contains one or more polypeptide s that specifically bind to CD3 and another amino acid sequence to which it is not attached 1501'dtlng Ule 'dIlIlDOLlleS rnaue II'Ol'Il HOST 'dIlll'Il'dlS, ODI'dlIllIlg Ule gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g., in the native le, for example, a heterologous sequence or a homologous sequence from r region.

An anti-CD3 polypeptide, and other CD3 agonists, antagonists and modulators can be created by s known in the art, for example, tically or recombinantly. One method of producing such molecules involves chemical synthesis of the polypeptide, followed by treatment under oxidizing conditions appropriate to obtain the native mation, that is, the correct disulfide bond linkages. This can be accomplished using methodologies well known to those skilled in the art (see, e. g., 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 United States s Nos. 603; 3,972,859; 3,842,067; and 3,862,925).

Polypeptides of the invention may be conveniently prepared using solid phase peptide synthesis (Merrifield, B. (1986) “Solid Phase Synthesis,” e 232(4748):341-347; en, RA. (1985) “General Method For The Rapid Solid- Phase Synthesis 0f Large Numbers 0f Peptides: Specificity 0f Antigen-Antibody Interaction At The Level OfIndividual Amino Acids,” Proc. Natl. Acad. Sci. (USA) 82(15):5131-5135; Ganesan, A. (2006) “Solid-Phase sis In The -First y,” Mini Rev. Med. Chem. 6(1):3-10).

In yet another alternative, fully human antibodies may be obtained through the use of commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XENOMOUSETM (Abgenix, Inc., Fremont, CA) and HUMAB-MOUSE® and TC M (both from Medarex, Inc., Princeton, NJ).

In an alternative, antibodies may be made recombinantly and expressed using any method known in the art. Antibodies may be made recombinantly by first isolating the antibodies made from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g., CHO cells). Another method that may be employed is to express the antibody ce in plants {e.g., tobacco) or transgenic milk. Suitable methods for expressing antibodies recombinantly in plants or milk have been sed (see, for example, Peeters et al. (2001) “Production 0fAntibodies 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.(l999) “Transgenic Milk As A Method For The Production 0f Recombinant Antibodies,” J. Immunol Methods 231:147-157). Suitable methods for making derivatives of antibodies, e. g., humanized, single chain, etc. are known in the art. In another alternative, antibodies may be made recombinantly by phage display technology (see, for example, US.

Patent Nos. 332; 5,580,717; 5,733,743; 6,265,150; and Winter, G. et al. (1994) “Making dies By Phage Display Technology,” Annu. Rev. Immunol. 12.433- 455).

The antibodies or protein of interest may be subjected to sequencing by Edman degradation, which is well known to those of skill in the art. The peptide information generated from mass ometry or Edman degradation can be used to design probes or primers that are used to clone the protein of interest.

An alternative method of cloning the n of interest is by “panning” using purified CD3 or portions thereof for cells expressing the antibody or protein of interest. The “panning” ure may be conducted by obtaining a cDNA library from tissues or cells that express CD3, over-expressing the cDNAs in a second cell type, and ing the transfected cells of the second cell type for a specific binding to CD3. Detailed ptions of the s used in cloning mammalian genes coding for cell surface proteins by ng” can be found in the art (see, for example, Aruffo, A. et al. (1987) “Molecular Cloning OfA CD28 cDNA By A High- Efiiciency COS Cell Expression System,” Proc. Natl. Acad. Sci. (USA) 84:8573- 8577 and Stephan, J. et al. (1999) tive Cloning 0f Cell Surface Proteins Involved In Organ Development: Epithelial Glycoprotein Is Involved In Normal Epithelial Difi’erentiation,” Endocrinol. 140:5841-5854). tJLthLLL u; LLLUVLVL’U vaL vv LuVLLuLLLvu. cDNAs encoding anti-CD3 antibodies, and other CD3 peptide agonists, nists and modulators can be obtained by reverse transcribing the mRNAs from a particular cell type according to standard s in the art. Specifically, mRNA can be isolated using various lytic enzymes or chemical solutions according to procedures set forth in, for example, MOLECULAR CLONING: A LABORATORY MANUAL, Third Edition (Sambrook et al. Eds., 2001) Cold Spring Harbor Press, Cold Spring Harbor, NY) or ted using commercially available nucleic-acid-binding resins following the accompanying instructions provided by manufacturers (e.g., Qiagen, Invitrogen, Promega). The synthesized cDNAs may then be introduced into an expression vector to produce the dy or protein of interest in cells of a second type. It is implied that an expression vector must be replicable in the host cells either as an episome or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, associated viruses, retroviruses, and cosmids.

The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, m phosphate, DEAE- n, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.

Any host cells capable of xpressing logous DNAs can be used for the purpose of isolating the genes ng the antibody, polypeptide or n 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 cDNAs at a level of about 5-fold , more preferably 10-fold higher, even more preferably -fold higher than that of the corresponding endogenous dy or protein of interest, if present, in the host cells. Screening the host cells for a specific binding to CD3 is effected by an immunoassay or FACS. A cell over-expressing the antibody or n of interest can be identified.

III. Methods for Screening Polypeptides and onal Antibodies Several methods may be used to screen polypeptides and monoclonal antibodies that bind to CD3. It is tood that “binding” refers to biologically or immunologically relevant specific binding, and does not refer to non-specific binding that may occur, for e, when an immunoglobulin is used at a very high concentration against a ecific target. In one embodiment, monoclonal antibodies are screened for binding to CD3 using standard screening techniques. In this manner, anti-CD3 monoclonal antibody was obtained. The preferred hybridomas of the present invention are those that produce antibodies mAbl and mAb2, or ic or humanized tives thereof. However, additional monoclonal antibodies that bind to CD3 may be identified. For this purpose, monoclonal antibodies are screened for their differential ability to bind to human CD3 as well as a primate CD3.

Any of several different detection s may be utilized to detect binding of antibodies to tissue section. Typically, immunohistochemistry involves the binding of a primary antibody to the tissue and then a secondary antibody ve against the s from the primary antibody was generated and conjugated to a detectable marker (e.g., horseradish peroxidase (HRP), or obenzedine (DAB)). One alternative method that may be used is polyclonal mirror image complementary antibodies or polyMICATM (polyclonal Mirror Image Complementary Antibodies; The Binding Site Limited, Birmingham, UK; Mangham, D.C. et al. (1999) “A Novel Immunohistochemical Detection System Using Mirror Image Complementary Antibodies (MICA),” athology 35(2):129-33). The PonMICATM technique can be used to test binding of primary antibodies (e. g., anti-CD3 antibodies) to normal and cancerous tissue. Several kinds of polyMICATM Detection kits are commercially available: Product No. HK004.D is a polyMICATM ion kit which uses DAB gen; Product No. HK004.A is a polyMICATM Detection kit which uses AEC chromagen. Alternatively, the primary antibody may be directly labeled with the detectable marker.

DUPPULL LIIL/ unvuuvu LullL/LLUII U1 LUIIULLUIID UL [JIM PGLLLUUIGI \JUJ PVPLIUV “SULLLDL, antagonist or modulator. These conjugates include CD3 peptide agonist, antagonist or IV. s of Characterizing Anti-CD3 Antibodies Any of several s can be used to terize anti-CD3 antibodies.

One method is to identify the epitope to which it binds. Epitope mapping is commercially available from various sources, for example, Pepscan Systems (Lelystad, The Netherlands). Epitope mapping can be used to determine the sequence to which an anti-CD3 antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a dimensional ction of amino acids that may not necessarily be contained in a single h.

Peptides of varying s (e.g., preferably at least 4-6 amino acids long) can be isolated or synthesized {e.g., recombinantly) and used for binding assays with anti-CD3 antibody. The epitope to which anti-CD3 antibody binds can be ined in a atic screening by using overlapping peptides derived from the extracellular sequence and determining binding by anti-CD3 antibody.

Yet r method that can be used to characterize an anti-CD3 antibody is to use ition assays with other antibodies known to bind to the same antigen, i.e., CD3 to determine if anti-CD3 antibodies binds to the same epitope as other antibodies. Examples of cially available antibodies to CD3 may be available and may be identified using the binding assays taught herein. Competition assays are well known to those of skill in the art, and such procedures and illustrative data are detailed fiarther in the Examples. Anti-CD3 antibodies can be fiarther characterized by the tissues, type of cancer or type of tumor to which they bind.

V. red Compositions of the Present Invention The present invention encompasses compositions, including pharmaceutical compositions, comprising anti-CD3 antibodies, polypeptides derived from anti-CD3 antibodies, polynucleotides comprising sequence encoding anti-CD3 antibodies, and other agents as described herein. The invention fiarther es for ates of any CD3 peptide agonist, antagonist or modulator, and additional al structures that support the intended fianction or functions of the particular CD3 peptide agonist, antagonist or modulator. These conjugates include CD3 peptide agonist, antagonist or ing by the TCR complex; (7) an ability to bind the Fc or; modulator covalently bound to a macromolecule such as any insoluble, solid support matrix used in the diagnostic, screening or purification procedures discussed herein.

Suitable matrix materials include any nce that is chemically inert, has high porosity and has large numbers of functional groups capable of forming covalent linkages with peptide ligands. es of matrix materials and procedures for preparation of matrix-ligand conjugates are described in Dean et al. (Eds) AFFINITY CHROMATOGRAPHY: A PRACTICAL APPROACH, IRL Press (1985); Lowe, “An Introduction to Afi‘mz’ty Chromatography”, in Work et al. (eds) LABORATORY TECHNIQUES IN MISTRY AND MOLECULAR Y, Vol. 7, Part 11, North- Holland (1979); Porath et al., “Biospecz'fic Afi‘mz’ty 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. NY (1984).

Also ed herein are conjugates of CD3 peptide agonist, antagonist or modulator and any reporter moiety used in the diagnostic procedures discussed herein.

The CD3 peptide agonist, antagonist or modulator agents, polypeptides and ns of this invention, including anti-CD3 antibodies, are further identified and characterized by any (one or more) of the following criteria: (1) an ability to specifically bind human CD3 as endogenously expressed on the surface of a normal human T cell; (2) an ability to specifically bind human CD3 as endogenously expressed on the surface of a human leukemic T cell; (3) an ability to specifically bind non-human CD3 (e.g., CD3 of cynomolgus monkey) as endogenously sed on the surface of a normal non-human T cell; (4) an ability to specifically bind a non-human CD3 as endogenously expressed on the surface of a non-human leukemic T cell; (5) an ability to neutralize (z'.e. , block or interfere with binding) the formation of the CD3 complex; an ability to neutralize the formation of the TCR complex; (6) an ability to modulate (either antagonistically or tically) ing by the TCR complex; (7) an y to bind the Fc receptor; FVLJtJvtjt/Luvu UVLLLIJLLULLLb “LLJ u; wLLvuv LwaLALVLLt/u “AV LuvLLwLLLVu “LL“ VLLwvavaLuvu VJ anv (one or more) of the criteria described above. (8) an y to competitively inhibit preferential binding of a known anti- CD3 antibody to CD3, ing the ability to preferentially bind to the same CD3 epitope to which the original antibody preferentially binds; (9) an ability to bind to a portion of CD3 that is exposed on the surface of a living cell in vitro or in viva; an ability to bind to a n of CD3 that is exposed on the surface of a living cancer cell; (10) an ability to deliver a chemotherapeutic agent into a cancerous T cell; and/or (1 1) an ability to deliver a therapeutic agent, toxin or detectable marker into a T cell.

A red antibody of the invention will exhibit differential IHC ng of tumor tissue relative to normal, non-cancerous tissue, and will moreover be capable of testing in primate (and particularly cynomolgus monkey) models of antibody efficacy.

Preferred antibodies of the present invention will additionally exhibit ble levels of affinity and antigen specificity. Preferred antibodies of the present invention will additionally exhibit desirable levels of modulatory activity and cellular internalization.

In some embodiments, the antibody of the invention is an dy that is produced by hybridoma mAbl or oma mAb2, which respectively express murine antibody mAbl and murine antibody mAb2, or progeny thereof The present invention also encompasses various formulations of antibodies produced by these hybridomas and lent antibodies or polypeptide fragments (e.g., Fab, Fab', F(ab')2 Fv, Fc, etc.), chimeric antibodies, single chain (scFv), mutants thereof, fusion proteins comprising an antibody n, humanized antibodies, and any other d configuration of any of these or equivalent antibodies that comprises an n (CD3), recognition site of the required specificity. The invention also provides human antibodies displaying one or more of the biological characteristics of an anti-CD3 family member. The equivalent antibodies of the anti-CD3 family (including humanized antibodies and human antibodies), polypeptide nts, and polypeptides comprising any of these fragments are identified and characterized by any (one or more) of the criteria described above. of the antibody that has any of the following: at least 5 contiguous amino acids of a AAAAAAAA AL‘LLA A«I,.I«n1 n«4..'1.,\,;l-. n4. 1AA"; 0 AA«L.‘,.“A“" ,\ “"24" n4. 1AA"; ALA“; 1n Accordingly, the invention provides any of the following (or compositions, including pharmaceutical compositions, comprising any of the following): (a) an antibody produced by the host cell or its progeny; (b) a humanized form of such an antibody; (c) an dy sing one or more of the light chain and /or heavy chain variable regions of such an antibody; (d) a ic antibody sing variable regions homologous or derived from variable regions of a heavy chain and a light chain of such an antibody, and constant regions homologous or derived from constant regions of a heavy chain and a light chain of a human antibody; (e) an dy comprising one or more of the light chain and /or heavy chain CDRs (at least one, two, three, four, five, or six) of such an antibody; (f) an antibody comprising a heavy and /or a light chain of such an antibody; (g) a human antibody that is equivalent to such an antibody. A humanized form of the dy may or may not have CDRs identical to that original antibody, or antibody produced by the host cell identified above. Determination of CDR regions is well within the skill of the art.

Other embodiments include antibodies that have at least two, three, four, five, or six CDR(s) that are substantially homologous to at least two, three, four, five or six CDRs of an antibody produced from a hybridoma deposited as identified herein, or derived from such an antibody. It is tood that, for purposes of this invention, binding specificity and/or overall actiVity is generally retained, although the extent of actiVity may vary compared to an dy produced by a ted hybridoma (may be greater or lesser). The invention also provides methods of making any of these antibodies. Methods of making dies are known in the art and are described herein.

The invention also provides polypeptides comprising an amino acid sequence of the antibodies of the invention. In some embodiments, the ptide comprises one or more of the light chain and /or heavy chain variable regions of the antibody. In some embodiments, the polypeptide comprises one or more of the light chain and /or heavy chain CDRs of the antibody. In some embodiments, the polypeptide comprises three CDRs of the light chain and /or heavy chain of the antibody. In some embodiments, the polypeptide comprises an amino acid sequence of the antibody that has any of the following: at least 5 contiguous amino acids of a sequence of the al antibody, at least 8 contiguous amino acids, at least about 10 Cauuaguuau Luuuugauua LCLCCLCCQL ggaaauugag guua cctactactg ccagcagtgg tcccggaacc cccctacctt cggcggaggc accaagctgc agatcaccag a contiguous amino acids, at least about 15 uous amino acids, at least about 20 uous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids, wherein at least 3 of the amino acids are from a variable region of the antibody. In one embodiment, the variable region is from a light chain of the al antibody. In another embodiment, the variable region is from a heavy chain of the antibody. In another embodiment, the 5 (or more) contiguous amino acids are from a 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 or other CD3-binding polypeptides of this invention are administered directly to an individual to modulate in viva CD3 biological activity.

] The preferred anti-CD3 antibodies of the present invention are mAbl and mAb2, and humanized or chimeric derivatives and antigen-binding fragments f that are reactive toward the human and cynomolgus CD3 molecule. The amino acid and encoding polynucleotide sequences of the le light chain and variable heavy chain of murine antibodies mAbl and mAb2 are shown below. The sequences of the CDRs of the exemplary antibodies (mAbl and mAb2) are shown in boldface and underlined.

A. Sequences of Variable Regions of Murine Monoclonal Antibody mAbl Amino Acid Sequence of Murine Monoclonal Antibody mAbl Variable Light Chain (SEQ ID NO:1): QVVLTQSPA" MSAEPGLKVT MTCSASSSVS QKSG IYE§ SKLASGVPAR FSGSGSGTSY SET SSMflTfl DAATYYCQQW SRNPPTFGGG TKLQITR Polynucleotide ce Encoding VIurine Monoclonal Antibody mAbl Variable Light Chain (SEQ ID NO:2): caggeggegc Lgacccagtc ccccgccaec gcce eccccggcga gaaagtgaca atgacctgc: ccgcceccec chc eacaLgaacL ggeaecagca gaagtccggc acctccccca agcggeggae cLacgachc tccaagc:gg cctccggcg: gcccgccaga Lececeggce ccggctccgg ceac Lcccegacca echceccaL ggaaaccgag gacgccgcca cc:actactg ccagcagtgg :cccggaacc cccctacct: cggcggaggc accaagc:gc agatcaccag a gaggtgaagc egc eggaaag cggcggagga ctgg:gcagc caaaggga":C achaaach ecc egcgccg cctccggctt cacc:ttaac gc ":6.

Amino Acid Sequence of Murine Monoclonal Antibody mAbl Variable Heavy Chain (SEQ ID NO:3): SGA'_‘'.L4 LARPGASVKM SCKASGYTFT RSTMHWVKQa PGQGT.«.W G: INPSSAYTNY NQKFKDKATL TADKSSSTAY MQLSSLTS'.L'.4 3 SAVYYCASP_Q VHYDYNGFPY VTVS S ] Polynucleotide Sequence Encoding mAbl Murine Monoclonal Antibody Variable Heavy Chain (SEQ ID NO:4): caggtgcagc :gcagcag:c tggcgccgag ctggccagac ctggcgcctc chgaagaeg echgcaagg cc:ccggcta caccttcacc acca ggg: gaaacagcgg cc:ggacagg gcctggaatg gatcggc:ac a:caacccc: ccagcgcc:a caccaac:ac aaccagaagt tcaaggacaa ggccaccc:g accgccgaca agtcctccag caccgchac aegcagcege cctccc:gac ctccgaggac tccgccgtgt actactgcgc thcccccag gegcaceacg actacaacgg cLecccceac ngggccagg gcaccctgg: gacagtgtcc tcc B. Sequences of Variable Regions of Murine Monoclonal Antibody mAbZ Amino Acid Sequence Of Murine Monoclonal Antibody mAb2 Variable Light Chain (SEQ ID NO:5): QAVVTQESAL TTSPGETVTL TCRSSTGAVT WVQE KPDHLFTGL: GGTNKRAPGV PARFSGSL:G DKAALTITGA QTflDflA YhC ALWYSNLWVF GGGTKLTVLG Polynucleotide ce Encoding Murine Monoclonal Antibody n1Ab2 le Light Chain (SEQ ID NO:6): caggccgtgg agga chagceceg accacatccc caggcgaaac agegacheg achgcagaL ctgg agcagegace achceaacL acchaaeeg gngcaggag aagcccgacc accegeecac egggcegaec ggcggaacca acaaaagggc acccggegeg chgcccgge eeLchgcag Lcegaecgga gacaaggccg chLgacaae Laceggcgcc gagg aLgaagceaL LLacLLchL gcachegg aLagcaaLce gagggegLeL gggggtggca ccaaactgac agtgctggga Amino Acid Sequence of mAb2 Murine Monoclonal Antibody Variable Heavy Chain (SEQ ID NO:7): SGGG LVQPKGSLK; SCAASGFTFN TYAMNWVRQA WVAR IRSKYNNYAT YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCVR HGNFGNSYVS WFAYWGQGT; VTVSA Polynucleotide Sequence Encoding Murine Monoclonal Antibody n1Ab2 Variable Heavy Chain (SEQ ID NO:8): aagc egc eggaaag cggcggagga ctgg:gcagc caaaggga":C achaaach ecc g cctccggctt cacc:ttaac acatacgc ":6. tgaattgggt gcgacaggca cctggcaagg gccsggagsg aagg tcca agtacaacaa saLgcaacc sacsasgccg ac:ctgtgaa ggasagaLsc agtc attc ccagagcass csgsaschc agatgaacaa scsgaaaac: accg ccasgsacsa Lugsgsgcgg cacggtaact :cggcaassc sachgscL gcss assggggaca ggggacacsg ngachsgs c chc Position 40 of the heavy chain is a high affinity MHC class II binding peptide anchor residue. Positions 44, 48, 54, 94, 99 and108 of the heavy chain are moderate y MHC class II binding peptide anchor residues. Position 69 of the light chain is a high affinity MHC class II g peptide anchor residue. Position 59 of the light chain is a moderate affinity MHC class II binding peptide anchor residue.

These es may be substituted, using standard molecular biology techniques, to a residue in order to reduce or remove the MHC class II recognition site.

C. Fc-Engineered CD3 Antibodies In traditional immune function, the interaction of antibody-antigen complexes with cells of the immune system results in a wide array of responses, ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to modulatory signals such as regulating lymphocyte proliferation and antibody secretion. All of these ctions are initiated through the binding of the Fc domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses triggered by antibodies and immune complexes results from the structural heterogeneity of the three Fc receptors: FcyRI (CD64), FcyRII (CD32), and FcyRIII . FcyRI (CD64), FcyRIIA (CD32A) and FcyRIII (CD16) are activating (z'.e., immune system enhancing) receptors; FcyRIIB (CD32B) is an inhibiting (z'.e., immune system dampening) receptor. The amino acid sequence of the IgGl Fc region is shown 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), expressly incorporated herein by reference, and hereafter referred to as “Kabat EU”). Residues 230-341 are the Fc CH2 region. Residues 342-447 are the Fc CH3 region: lVVAA/J LLVLVLWVLJ’ I/LLV VLLLuLLLb FLUtJVLIILVU u; uLLv LALuvavau u; uLLv Lnnvvnnununn “AV SEQIDNO:9 PAPELLGGPS VFLFPPKPKD THM SRTPflV TCVVVDVSifl DPflVKhNWYV 230 240 250 260 270 DGVEVHNA<T KPRflflQYNST YRVVSVLTVL HQDWLNGKEY KC<VSNKALP 280 290 300 310 320 AP flKT S<A KGQPREPQVY THPPSRflflMT KNQVSLTCLV KGFYPSDIAV 330 340 350 360 370 flWflSNGQPflN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH 380 390 400 410 420 ?AHHNHYTQK SLSLSPGK 430 440 Since Fc receptor (FcR)-non-binding CD3-specific dies are minimally ing, it has been proposed that they may alter TCR s in a way that might induce immune tolerance (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 mune disease and host vs. graft tissue rejection. FcR non-binding CD3-specific antibodies have also been postulated to induce remission in type 1 diabetes mellitus tolerance (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).

] The present invention thus es antibodies that specifically bind to CD3 that comprise a variant Fc region haVing Fc regions that are modified (e.g., substitutions, deletions, insertions in one or more portions) so as to be unable or less able to bind to the Fc or (relative to an antibody haVing the same CDRs but a wild-type Fc region).

In one embodiment, such antibodies will be incapable of binding to any PC receptor. Alternatively, the Fc region of the antibody will be modified so as to permit it to bind to Fc receptors such as B that are inhibitory, but not to Fc receptors such as A, FcyRIIIA or IB that promote activation of the immune system.

Preferably, the binding properties of the molecules of the invention are characterized by in vitro filnctional assays for determining one or more FcyR mediator 1\11UVV11 LU U11V D1\111\/u 111 L11\/ “1L cuLu VAV111P1111VU 11V1V111. 111V nupp “DD“.YD UDVU 111 effector cell functions. The affinities and binding properties of the les, e.g., antibodies, of the ion for an FcyR can be determined using in vitro assays (biochemical or immunological based assays) known in the art for determining antibody-antigen or Fc-FcyR interactions, z'.e., specific binding of an antigen to an antibody or specific binding of an Fc region to an FcyR, respectively, including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays. In most preferred embodiments, the molecules of the invention have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays. However, the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in viva.

In some embodiments, the les of the invention comprising a variant Fc region comprise at least one amino acid modification (for example, possessing l, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH3 domain of the Fc region, which is defined as ing from amino acids 342-447. In other embodiments, the molecules of the invention comprising a variant Fc region comprise at least one amino acid ation (for example, possessing l, 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 extending from amino acids 231-341. In some embodiments, the molecules of the invention comprise at least two amino acid modifications (for example, possessing 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), wherein at least one such modification is in the CH3 region and at least one such ation is in the CH2 region. The invention filrther encompasses amino acid modification in the hinge region. In a particular ment, the invention encompasses amino acid modification in the CH1 domain of the Fc region, which is defined as extending from amino acids 2 1 6-230.

In particularly preferred embodiments, the ion asses molecules comprising a variant Fc region wherein said t confers or has a decreased ADCC ty and/or a decreased binding to A (CD32A), as measured using s known to one skilled in the art and exemplified herein. The ADCC assays used in Antibodies With Increased Activity To Recruit Complement,” J. Immunol. 1662571- accordance with the methods of the invention may be NK dependent or macrophage dependent.

In particularly preferred embodiments, the invention encompasses les comprising a variant Fc region n said variant confers or has a decreased ADCC activity and/or a decreased binding to IA (CDl6A), as measured using methods known to one skilled in the art and exemplified herein. The ADCC assays used in accordance with the methods of the invention may be NK dependent or macrophage dependent.

The PC variants of the present invention may be ed with other PC modifications, such as those disclosed in United States Patents Nos. 7,632,497; 7,521,542; 619; 7,416,727; 7,371,826; 7,355,008; 742; 7,332,581; 7,183,387; 7,122,637; and 6,737,056; in PCT Publications Nos. ; ; ; ; WO 06/088494; WO 05/115452; WO 05/110474; WO 04/1032269; and in 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 offucose on human IgG1 N-linked oligosaccharide improves binding to human chamma RIII 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 IgG1 variants with improved binding to the Fc gamma R,” J. Biol. Chem. 276(9):659l- 6604). The invention encompasses combining an Fc variant of the ion with other PC ations to e ve, synergistic, or novel properties to the modified antibody.

In other embodiments, the ion encompasses the use of any PC variant known in the art, such as those disclosed in Jefferis, B.J. et al. (2002) action Sites 0n Human IgG-Fc For chammaR.‘ 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. 1662571- 75; Shields, R.L. et al. (2001) “High Resolution Mapping Of The Binding Site On Human IgGI For Fc Gamma RI, Fc Gamma RII, Fc Gamma RIII, And FcRn And Design Of IgGI Variants With Improved Binding To The Fc gamma R,” J. Biol.

Chem. 276:6591-6604; Idusogic, E.E. et al. (2000) “Mapping Of The CIq Binding Site On Rituxan, A Chimeric Antibody With A Human IgG Fc,” J. Immunol. 164:4178-84; Roddy, M.P. et al. (2000) nation 0f Fc Receptor-Dependent r Functions Of A d IgG4 Monoclonal Antibody To Human CD4,” J.

Immunol. 164:1925-1933; Xu, D. et al. (2000) “In Vitro Characterization of Five Humanized OKT3 Eflector Function Variant dies,” Cell. Immunol. 200:16-26; Armour, K.L. et al. (1999) “Recombinant human IgG Molecules Lacking chamma Receptor I Binding And Monocyte Triggering Activities,” Eur. J. Immunol. - 24; Jeffcris, R. et al. (1996) “Modulation 0ch(Gamma)R And Human ment Activation By IgG3-Core Oligosaccharide Interactions,” Immunol. Lett. 54:101-04; Lund, J. et al. (1996) “Multiple Interactions OfIgG With Its Core Oligosaccharide Can Modulate Recognition By Complement And Human Fc Gamma Receptor I And Influence The Synthesis OfIts Oligosaccharide Chains,” J. Immunol. 157:4963-4969; Hutchins et al. (1995) “Improved tribution, Tumor Targeting, And Reduced Immunogenicity In Mice With A Gamma 4 Variant 0f Campath-IH,” Proc. Natl.

Acad. Sci. (USA) 92:11980-84; chfcris, R. et al. (1995) “Recognition Sites 0n Human IgG For Fc Gamma ors.‘ The Role Of Glycosylation,” Immunol. Lett. 44:111-17; Lund, J. et al. (1995) saccharide-Protein Interactions In IgG Can Modulate Recognition By Fc Gamma ors,” FASEB J. 9:115-19; Alcgrc, ML. et al. (1994) “A Non-Activating "Humanized"Anti-CD3 Monoclonal Antibody Retains Immunosuppressive ties In Vivo,” Transplantation 57:1537-1543; Lund et al. (1992) ple Binding Sites On The CH2 Domain OfIgG 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 0n Human IgG,” J.

Immunol. 147:2657-2662; Duncan, A.R. et al. (1988) “Localization Of The Binding Site For The Human High-Afiinity Fc Receptor 0n IgG,” Nature 332:563-564; US Patent Nos. 5,624,821; 5,885,573; 6,194,551; 7,276,586; and 7,317,091; and PCT Publications WO 00/42072 and PCT WO 99/5 8572.

In certain ments, the antibody of the invention comprises a variant Fc region (including an Fc derived from any human immunoglobulin type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass), wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, which variant Fc region exhibits d or hed binding to one or more effector ligands as determined by standard assays known in the art and disclosed herein, relative to a able molecule comprising the wild type Fc region. In certain embodiments, the variant Fc domain of the antibody of the invention comprises an amino acid modification (i.e., insertion, substitution, deletion) at one or more of the residues 233, 234, 235, 236, 237, 238, 265, 270, 297, 298, 299. In a specific embodiment, the one or more amino acid modifications which reduce or abolish binding to one or more effector ligands is a substitution with phenylalanine or proline at position 233; a substitution with alanine at position 234; a substitution with alanine or ic acid at position 235; a substitution with alanine at on 236, a substitution with alanine at position 237, a substitution with arginine at on 238; a substitution with alanine or glutamic acid at position 265; a substitution with alanine or asparagine at position 270; a tution with alanine or glutamine at position 297; a substitution with phenylalanine, asparagine or e at position 298; a substitution with any amino acid at position 299 other than serine or threonine; or a combination of two or more of the above-listed substitutions. In certain embodiments, the antibody of the invention comprises an Fc domain having a substitution with alanine at position 265 and at position 297; a tution with alanine at position 265 and with ine at position 297; a substitution with ic acid at position 265 and with alanine at position 297; or a substitution with glutamic acid at position 265 and with glutamine at position 297. In preferred embodiments, the antibody of the invention comprises an Fc domain having a modification (e.g., substitution, insertion, deletion) at on 234 and position 235 of the Fc region. In a specific example in accordance with this embodiment, the antibody of the invention comprises an Fc domain having a substitution at position 234 with e and a substitution at position 235 with glutamic acid. In a yet more preferred embodiment, the antibody of the invention comprises an Fc having a substitution with alanine at position 234 and a substitution with alanine at position 235.

In other embodiments, the antibody of the invention comprises a Fc region, which t Fc region exhibits reduced or abolished binding to one or more effector ligands as determined by standard assays known in the art and disclosed herein, relative to a comparable control molecule. In certain embodiments, the antibody of the invention has a Fc region that exhibits reduced or abolished binding to one or more effector s, which Fc region comprises a phenylalanine or proline at position 233; an alanine at position 234; an alanine or glutamic acid at position 235; an alanine at position 236, an alanine at position 237, an arginine at position 238; an alanine or glutamic acid at position 265; an alanine or asparagine at position 270; an alanine or glutamine at on 297; a alanine, asparagine or proline at position 298; any amino acid at position 299 other than serine or threonine; or a ation of two or more of the above-listed substitutions. In certain embodiments, the antibody of the ion comprises an Fc domain having an alanine at position 265 and at position 297; an alanine at position 265 and a glutamine at position 297; a glutamic acid at position 265 and an alanine at position 297; or a glutamic acid at position 265 and a glutamine at position 297. In certain embodiments, the antibody of the ion comprises an Fc domain having an alanine at 234 and a glutamic acid at position 235. In preferred embodiments, the antibody of the invention comprises an Fc having an alanine at position 234 and an alanine at position 235. dies of the invention that comprise and Fe domain having an alanine at positions ponding to 234 and 235 according to the ing scheme of Kabat are known as “ala-ala” dies. In certain embodiments, use of “ala-ala” Fc domains and/or other combinations of amino acid combinations herein bed ding combinations comprising “ala-ala” Fc domains) may abolish binding of the Fc domain to all FcyRs. The binding of a PC domain to one or more FcyRs may be determined by any method described herein and/or known in the art.

LAvauvu vu VJ LAvauvuLLLb UVVLvuLuLL u; VJ vaxLLLvu LLLULMuLLLb up.» LLVI/ LLLLLLUV“ uv ALLUVLLVML‘LLL -2 (IL-2). Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-l2 (IL-12), In certain embodiments, the one or more amino acid modifications which abolish binding to all FcyRs or reduce or abrogate binding to one or more or ligands comprise combinations of the modifications listed herein or combinations of the modifications listed herein with any that may confer null binding to any FcR (e.g.

FcyRIIIA, FcyRIIIB, FcyRIIA) as determined by the methods disclosed herein or known to one skilled in the art. As readily understood by one of skill in the art, such antibodies of the invention may find particular use in the treatment of an autoimmune disease in that the anti-CD3 antibodies and antigen-binding fragments serve to te immune filnction without the associated first-dose response common to anti- immune cell antibodies.

In certain embodiments, the anti-CD3 dies and antigen-binding fragments of the invention, or antigen binding fragments f, have diminished (such as, but not limited to, less than 50%, less than 40%, less than 30%, less than %, less than 10%, less than 5% or less than 1% as compared to binding by a protein sing a l Fc domain) or, more preferably, no detectable binding to one or more of any FcyR (e.g., FcyRI, FcyRII or I) via its Fc domain as determined by assays routine in the art. In addition or alternatively, the anti-CD3 antibodies and antigen-binding fragments of the ion, or antigen binding fragments thereof, may have diminished (such as, but not limited to, less than 50%, less than 40%, less than %, less than 20%, less than 10%, less than 5% or less than 1% as ed to binding by a control protein sing a l Fc domain) or, more preferably, no detectable binding to any complement receptors, such as, Clq, as determined in routinely used assays. In particular embodiments, the antibody is aglycosylated. In other embodiments, the antibody lacks an Fc domain (e.g., is a Fab fragment, F(ab’)2 or single chain antibody).

The antibodies of the invention are thus particularly useful because they have reduced or no in viva ty caused by lymphokine production or ne release.

Methods of measuring lymphokine production and cytokine release are known and routine in the art and encompassed . For example, cytokine release may be measured by measuring secretion of cytokines including but not limited to Interleukin -2 (IL-2). Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-l2 (IL-12), domains (A) and (E) associate to form a binding site that binds epitope (1); said Interleukin-16 (IL-16), PDGF, TGF-OL, TGF-B, TNF- 0t, TNF- B, GCSF, GM-CSF, MCSF, IFN— 0t, IFN— [3, TEN-y, IGF-I, IGF-II. For example, see, Isaacs et al., 2001, Rheumatology, 40: 8; Soubrane et a1., 1993, Blood, 81(1): 15-19; each of which is incorporated herein by reference in its entirety.

D. CD3 DARTTM Diabodies As discussed above, the present invention additionally encompasses ific, trispecific and mutispecific antibodies. A ularly preferred example of such antibodies comprise M” diabody molecules that comprise at least two polypeptide chains which form at least two epitope binding sites, at least one of which specifically binds to CD3. Exemplary M” diabody molecules are disclosed in US20100174053, US20090060910, US20070004909, EP2158221, EP1868650, WO2010080538, WO2008157379, and WO2006113665.

In preferred embodiments, the first polypeptide chain of the DARTTM diabody ses: (i) a domain (A) comprising a binding region of a light chain variable domain of a first immunoglobulin (VLl) specific for an epitope (1); (ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2); (iii) a domain (C).

The second polypeptide chain of such a DARTTM diabody comprises: (i) a domain (D) comprising a binding region of a light chain le domain of the second immunoglobulin (VL2) c for epitope (2); (ii) a domain (E) comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VH1) specific for epitope (1); and (iii) a domain (F).

The DARTTM diabody domains (A) and (B) do not ate with one another to form an epitope g site. Similarly, the DARTTM diabody domains (D) and (E) do not associate with one another to form an epitope binding site. Rather, DARTTM diabody domains (A) and (E) associate to form a binding site that binds epitope (1); said any affinity based method known in the art or exemplified herein, e.g., affinity tography.

DARTTM diabody domains (B) and (D) associate to form a binding site that binds said epitope (2). Domains (C) and (F) are covalently associated together.

Each polypeptide chain of the DARTTM diabody molecule comprises a VL domain and a VH domain, which are covalently linked such that the domains are constrained from self-assembly. Interaction of two of the polypeptide chains will produce two VL-VH pairings, forming two eptipoe binding sites, z'.e., a bivalent molecule. Neither the VH or VL domain is constrained to any position within the ptide chain, l'.e., restricted to the amino (N) or carboxy (C) teminus, nor are the domains restricted in their relative ons to one another, z'.e., the VL domain may be inal to the VH domain and vice-versa. The only restriction is that a complimentary ptide chain be ble in order to form functional DARTTM diabodies. Where the VL and VH domains are derived from the same antibody, the two complimentary polypeptide chains may be identical. For example, where the binding domains are derived from an antibody specific for epitope A (i.e., the binding domain is formed from a A interaction), each polypeptide will comprise a VHA and a VLA. Homodimerization of two polypeptide chains of the dy will result in the formation two VLA-VHA binding sites, resulting in a bivalent monospecific antibody. Where the VL and VH domains are derived from antibodies specific for different antigens, formation of a onal bispecific DARTTM diabody requires the interaction of two different polypeptide chains, z'.e., formation of a heterodimer. For example, for a bispecific DARTTM diabody, one ptide chain will comprise a VLA and a VLB; homodimerization of said chain will result in the formation of two VLA-VHB binding sites, either of no binding or of unpredictable binding. In st, where two differing polypeptide chains are free to interact, e.g., in a inant expression system, one comprising a VLA and a VHB and the other comprising a VLB and a VHA, two differing binding sites will form: VLA-VHA and VLB-VHB. For all DARTTM diabody polypeptide chain pairs, the possibly of misalignment or mis-binding of the two chains is a possibility, z'.e., interaction of VL- VL or VH-VH domains; however, purification of filnctional diabodies is easily managed based on the immunospecificity of the ly dimerized binding site using any affinity based method known in the art or exemplified , e.g., affinity chromatography. to form a monomer, and said monomers interact via their ed Fc domains to One or more of the polypeptide chains of the DARTTM diabody may optionally comprise an Fc domain domain or portion thereof (6.g. a CH2 domain, or CH3 domain). The PC domain or n thereof may be d from any immunoglobulin e or allotype including, but not limited to, IgA, IgD, IgG, IgE and IgM. In preferred embodiments, the Fc domain (or portion thereof) is derived from IgG. In specific embodiments, the IgG isotype is IgGl, IgG2, IgG3 or IgG4 or an allotype thereof. In one embodiment, the diabody molecule comprises an Fc domain, which Fc domain comprises a CH2 domain and CH3 domain ndently selected from any immunoglobulin isotype (i.e. an Fc domain comprising the CH2 domain derived from IgG and the CH3 domain d from IgE, or the CH2 domain derived from IgGl and the CH3 domain derived from IgG2, etc.). The PC domain may be engineered into a polypeptide chain sing the diabody molecule of the invention in any position relative to other domains or portions of said polypeptide chain (e.g., the Fc domain, or portion thereof, may be c-terminal to both the VL and VH domains of the polypeptide of the chain; may be n-terminal to both the VL and VH domains; or may be N-terminal to one domain and c-terminal to another (i.e., between two domains of the ptide chain)).

The PC domains in the polypeptide chains of the DARTTM diabody molecules preferentially dimerize, resulting in the formation of a DARTTM molecule that exhibits immunoglobulin-like properties, e.g., Fc-FcyR, interactions. Fc comprising diabodies may be dimers, e. g., comprised of two polypeptide , each comprising a VH domain, a VL domain and an Fc domain. Dimerization of said polypeptide chains results in a bivalent DARTTM diabody comprising an Fc domain, albeit with a structure distinct from that of an unmodified bivalent antibody. Such DARTTM diabody molecules will t altered phenotypes relative to a ype immunoglobulin, e.g., altered serum half-life, binding properties, etc. In other embodiments, DARTTM diabody les comprising Fc domains may be tetramers.

Such tetramers comprise two ‘heavier’ polypeptide chains, z'.e., a polypeptide chain comprising a VL, a VH and an Fc domain, and two ‘lighter’ polypeptide chains, z'.e., polypeptide chain comprising a VL and a VH. The lighter and r chains interact to form a monomer, and said monomers ct via their unpaired Fc domains to cancer, bladder cancer, kidney cancer, brain , gleoblastoma, thyroid cancer, form an Ig-like molecule. Such an Ig-like DARTTM diabody is alent and may be monospecific, bispecific or tetraspecific.

VI. Therapeutic s of Using the Anti-CD3 Antibodies of the Present invention The anti-CD3 antibodies of the present invention and their antigen-binding fragments have particular utility in the treatment of cancers ated With CD3 sion and in the treatment of autoimmune disease and other inflammatory disorders.

These uses can involve the formation of a complex between CD3 and an antibody that binds specifically to CD3. Examples of such antibodies include but are not limited to anti-CD3 monoclonal dies mAbl and mAb2 or, more preferably, their humanized derivatives. The formation of such a complex can be in vitro or in viva. Without being bound by theory, anti-CD3 antibody can bind to CD3 through the extracellular domain of CD3 and may then be internalized inside of a living normal or cancer cell.

A. Treatment of Cancer The antibodies and antigen-binding fragments of the present invention bind to CD3 present on the surface of T cells. The antigen-binding nts of the present invention can be used in the context of a bi-speciflc (or trispecific or multispecific) molecule, such as a DART or BiTE molecule, to redirect T-cells to a tumor cell. The T-cell can then kill the tumor cell. The bispecific (or trispecific or multispecific) molecules of the t invention are capable of binding to both human CD3 and the CD3 of a non-human mammal (e.g., cynomolgus monkey), and also to a second (or additional) and different antigen(s) or epitope(s). The second antigen or epitope is preferably a tumor antigen expressed on a tumor cell. Such tumor cells may be from s, for example, breast cancer, prostate , gastric cancer, lung , stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer, laryngeal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular , bladder cancer, kidney cancer, brain cancer, gleoblastoma, thyroid cancer, The antibodies and antigen-binding nts of the present invention bind to CD3 present on the surface of T cells. Using conventional methods, such lymphoma, a, and leukemia. Such The additional antigens or epitopes are preferably cell surface tumor antigens or epitopes (such as: l7-lA, A33, adult erythrocyte primary rm I antigen, alpha otein, an envelope antigen of an RNA tumor Virus, bladder tumor oncofetal n, B7-Hl, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, Burkitt's lymphoma n-38.13, CAl25, CD18, CD19, human B- lymphoma antigen-CD20, CD22, CD33, CD44, CD52, CEA, COl7-lA, CTA-l, CTLA-4, epidermal grth factor or, Ep-CAM, EphA2, fetal ocyte I antigen, fibrosarcoma antigen, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, GICA 19-9, gp IIIb/IIIa, gp72, HERl, HER-2/neu, HER3, HER4, high molecular weight melanoma antigen, HLA-DR antigen, human leukemia T cell antigen-Gp37, human lung carcinoma antigen L20, human lung carcinoma antigen L6, human milk fat globule antigen, IgE, KS l/4 pan-carcinoma antigen, LEA, lung adenocarcinoma F3 antigen, ant human lymphocyte antigen-APO-l, melanoma antigen gp75, melanoma-associated antigen p97, coprotein, nuC242, polymorphic epithelial mucin antigen, prostate ic antigen, prostate specific membrane antigen, prostatic acid phosphate, SK-l antigen, TAG-72, T-antigen, tumor antigen CAl25, tumor antigen MUCl, tumor-specific transplantation type of cell- surface n, vascular endothelial growth factor, vascular endothelial grth factor-receptor, and aVB3). Alternatively, such additional antigens or epitopes may be associated with a pathogen (such as: hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial Virus, papilloma Virus, papova Virus, cytomegalovirus, echinovirus, arbovirus, huntaVirus, coxsackie Virus, mumps Virus, measles Virus, a Virus, polio Virus, small pox, Epstein Barr Virus, human immunodeficiency Virus type I (HIV-I), human immunodeficiency Virus type II (HIV-II), Viral miningitis, Viral encephalitis, dengue, small pox; cteria rickettsia, asma, Neisserz'a, S. pneumonia, Borrelia burgdorferi, Bacillus anthracis, Streptococcus, Staphylococcus, Mycobacterz'um, tetanus, pertissus, cholera, , diptheria, chlamydia, and legionella; leishmania, kokzidioa, trypanosoma or 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 the present invention could be employed in the same way as the CD3 antibodies of antibodies may be labeled with fluorescein, as described above. When such labeled molecules are incubated in the presence of a if1c molecule (such as for example, a UDARTTM diabody having an epitope g domain that binds to the T-cell receptor and an epitope binding domain that binds to fluorescein (“TCRUDARTTM” )), they can bind to the fluorescein label and thereby localize themselves to the surface of cells that express CD3 and cause redirected killing of such cells.

In an alternative embodiment, CD19 may be used as the “second” e, such that a bispecif1c antibody, or more preferably, a DART TM diabody, recognizing CD3 and CD19 is employed to eradicate B-cell lymphoma through co-engagement of the B-cell specific antigen (CD19) and the T-cell or/CD3 complex on effector T-cells. As disclosed by Moore, RA. et al. (2011) “Application Of Dual Afiinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing 0f B-Cell Lymphoma,” Blood 2011 blood09-306449, a CD3/CD19 DARTTM diabody was used to eradicate B-cell lymphoma through co-engagement of the B-cell specific antigen CD19 and the T-cell receptor/CD3 complex on effector s. Side by side comparison with a single-chain bispecif1c antibody bearing identical CD19 and CD3 antibody Fv sequences revealed the DART to be more potent in directing B-cell lysis.

The enhanced activity with the CDl9xCD3 DART was observed on all CD19 expressing B-cell target cells evaluated using resting and pre-stimulated human PBMC or purified or T-cell populations. Characterization of a CDl9xTCR bispecif1c DART revealed equivalent potency to the CDl9xCD3 DART demonstrating flexibility of the DART architecture to t T-cell/B-cell associations for redirected T-cell killing applications. Importantly the enhanced level of g mediated by DART molecules was unaccompanied with any increase in non-specif1c T-cell activation or lysis of CD19 negative cells. Cell association studies indicate the DART ecture is well suited for maintaining cell:cell contact apparently contributing to the high level of target cell killing. y, the ability of the CDl9xTCR DART to inhibit B-cell ma in NOD/SCID mice when co- administered with human PBMC r trates the value of DART molecules for the treatment of B-cell malignancies. The cross-reactive anti-CD3 dies of the present invention could be employed in the same way as the CD3 antibodies of and/or reduce the need for other therapy (e.g., 1n the treatment or prophylax1s of Type I Aiokofon flan mofhnr‘n AP {-140 invronfinn movr 1110 {-140 Moor] ‘an‘ Avnn-onn110 1.1401111.“ Moore, PA. et al. Thus, the invention provides a therapy for cancers (especially lymphomas and leukemias) involving CD3-expressing cancer cells.

The bispecific (or trispecific or multispecific) les of the present invention are preferably administered to a t in one or more unit doses of typically 0.0001 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a 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 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight.

B. Treatment of Autoimmune Disease and Inflammation The invention also provides methods of treating, preventing, slowing the progression of and/or ameliorating the symptoms of T-cell ed diseases or disorders, including graft rejection, graft versus host disease, unwanted delayed-type hypersensitivity reactions (such as delayed-type allergic reactions), T-cell ed pulmonary diseases, and autoimmune diseases. T-cell mediated pulmonary diseases include sarcoidosis, hypersensitivity pneumonitis, acute interstitial nitis, alveolitis, pulmonary fibrosis, idiopathic pulmonary is and other diseases characterized by inflammatory lung damage. T-cell autoimmune diseases e multiple sclerosis, is, polymyositis, sis, vitiligo, Sjogren's syndrome, rheumatoid arthritis, Type 1 diabetes, autoimmune pancreatitis, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), celiac e, glomerulonephritis, scleroderma, dosis, autoimmune thyroid diseases (e. g., Hashimoto's thyroiditis and Graves’ disease), myasthenia , Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia, and systemic lupus erythematosis, lupus (particularly, cutaneous), effects from organ lantation, graft vs. host disease (GVHD), etc. Particularly, the methods of the invention are advantageous in subjects with early stage disease to slow or reduce the damage from the autoimmunity and maintain a high level of fianction and/or reduce the need for other therapy (e.g., in the treatment or prophylaxis of Type I diabetes, the methods of the invention may reduce the need for exogenous insulin ug/kg or less, 175 ug/kg or less, 150 ug/kg or less, 128 ug/kg or less, 100 ug/kg or administration in the subject). In addition, the methods of the invention may advantageously reduce the incidence of or result in no incidence of cytokine release syndrome previously associated with administration of therapeutic antibodies, and, in particular, anti-T-cell (e.g., anti-CD3 antibody or antigen-binding fragments.

In certain embodiments, the course of ent with an anti-CD3 antibody or antigen-binding fragments according to the methods of the invention is repeated at 2 month, 4 month, 6 month, 8 month, 9 month, 10 month, 12 month, 15 month, 18 month, 24 month, 30 month, or 36 month intervals. In specific embodiments efficacy of the treatment with an anti-CD3 antibody or antigen-binding nts of the invention is ined as described herein or as is 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 subsequent to the previous treatment.

In r embodiment, a subject is administered one or more unit doses of approximately 05-50 ug/kg, approximately 05-40 ug/kg , approximately 05-30 ug/kg, approximately 05-20 ug/kg, approximately 05-15 ug/kg, approximately 0.5- ug/kg, approximately 0.5-5 ug/kg, approximately 1-5 ug/kg, approximately 1-10 ug/kg, approximately 20-40 ug/kg, imately 20-30 ug/kg, approximately 22-28 ug/kg or approximately 25-26 ug/kg of one or more anti-CD3 antibody or nbinding fragments to prevent, treat or ameliorate one or more symptoms of an autoimmune er or T cell malignancy. In another embodiment, a subject is administered one or more unit doses of 200 ug/kg, 178 ug/kg, 180 ug/kg, 128 ug/kg, 100 ug/kg, 95 ug/kg, 90 ug/kg, 85 ug/kg, 80 ug/kg, 75 ug/kg, 70 ug/kg, 65 ug/kg, 60 ug/kg, 55 ug/kg, 50 ug/kg, 45 ug/kg, 40 ug/kg, 35 ug/kg, 30 ug/kg, 26 ug/kg, 25 ug/kg, 20 ug/kg, 15 ug/kg, 13 ug/kg, 10 ug/kg, 6.5 ug/kg, 5 ug/kg, 3.2 ug/kg, 3 Mg/kg,2.5 , 2 lug/kg, 1.6 lug/kg, 1.5 lug/kg, 1 lug/kg, 0.5 lug/kg, 0.25 lug/kg, 0.1 ug/kg, or 0.05 ug/kg of one or more D3 antibody or antigen-binding fragments to prevent, treat or ameliorate one or more symptoms of an autoimmune disorder or T-cell ancy.

] In a one embodiment, a subject is administered one or more doses of 200 ug/kg or less, 175 ug/kg or less, 150 ug/kg or less, 128 ug/kg or less, 100 ug/kg or u; w va VAL vv VVAx “LL“ LLVI/ wuLALLLLLut/VLLLLb uuuvu u; ULLV tJLthLLJ WLLJ u; I/LLVLWIJVMULUWLLJ effective amount of one or more anti-CD3 antibody or antigen-binding fragments on less, 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or less, 80 ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55 ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or less, 30 ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or less, 5 ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 15 ug/kg or less, 1 ug/kg or less, 0.5 ug/kg or less, 0.25 ug/kg or less, 0.1 ug/kg or less, or 0.05 ug/kg or less of one or more anti-CD3 antibody or antigen-binding fragments of the invention to prevent, treat or ameliorate one or more symptoms of an autoimmune disorder or T-cell malignancy.

In particular embodiments, a subject is administered one or more doses of about 5 - l200 ug/mz, preferably, 51 - 826 ug/mz. In another embodiment, a subject is administered one or more unit doses of 1200 ug/mz, ll50 ug/mz, 1100 ug/mz, 1050 ug/mz, 1000 ug/mz, 950 Mg/l’nz, 900 Mg/l’nz, 850 Mg/l’nz, 800 ug/m2, 750 Mg/l’nz, 700 Mg/l’nz, 650 Mg/l’nz, 600 Mg/l’nz, 550 z, 500 Mg/l’nz, 450 Mg/l’nz, 400 Mg/l’nz, 350 Mg/l’nz, 300 Mg/l’nz, 250 ug/m2, 200 Mg/l’nz, 150 Mg/l’nz, 100 Mg/l’nz, 50 Mg/l’nz, 40 Mg/l’nz, ug/m2, 20 ug/mz, l5 ug/mz, 10 ug/mz, or 5 ug/m2 of one or more anti-CD3 antibody or antigen-binding fragments to prevent, treat, slow the progression of, delay the onset of or ameliorate one or more ms of an autoimmune disorder or T-cell malignancy.

In another ment, the subject is administered a treatment regimen comprising one or more doses of a prophylactically or eutically effective amount of one or more anti-CD3 antibody or antigen-binding fragments, wherein the course of ent is administered over 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 comprises administering doses of the prophylactically or therapeutically effective amount of one or more anti-CD3 dy or antigen-binding fragments every day, every 211d day, every 3rd day or every 4th day. In n ments, the treatment regimen comprises administering doses of the prophylactically or therapeutically ive amount of one or more anti-CD3 antibody or antigen-binding fragments on Monday, Tuesday, Wednesday, Thursday of a given week and not administering doses of the prophylactically or therapeutically effective amount of one or more anti-CD3 dy or antigen-binding fragments on minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, Friday, Saturday, and Sunday of the same week until 14 doses, 13, doses, 13 doses, 12 doses, 11 doses, 10 doses, 9 doses, or 8 doses have been administered. In certain embodiments the dose stered is the same each day of the regimen. In certain embodiments, a subject is administered a treatment regimen comprising one or more doses of a prophylactically or therapeutically effective amount of one or more anti- CD3 antibody or antigen-binding fragments, wherein the prophylactically or eutically effective amount is 200 ug/kg/day, 175 ug/kg/day, 150 ug/kg/day, 125 ug/kg/day, 100 ug/kg/day, 95 ug/kg/day, 90 ug/kg/day, 85 ug/kg/day, 80 ug/kg/day, 75 ug/kg/day, 70 ug/kg/day, 65 ug/kg/day, 60 ug/kg/day, 55 ug/kg/day, 50 ug/kg/day, 45 ug/kg/day, 40 ug/kg/day, 35 day, 30 ug/kg/day, 26 ug/kg/day, ug/kg/day, 20 ug/kg/day, 15 ug/kg/day, 13 ug/kg/day, 10 ug/kg/day, 6.5 ug/kg/day, 5 day, 3.2 ug/kg/day, 3 ug/kg/day, 2.5 ug/kg/day, 2 ug/kg/day, 1.6 ug/kg/day, 1.5 ug/kg/day, 1 ug/kg/day, 0.5 ug/kg/day, 0.25 day, 0.1 day, or 0.05 day; and/or Wherein the prophylactically or therapeutically effective amount is 1200 ug/mZ/day, 1150 ug/mZ/day, 1100 ug/mZ/day, 1050 ug/mZ/day, 1000 ug/mZ/day, 950 day, 900 ug/mZ/day, 850 ug/mZ/day, 800 ug/mZ/day, 750 ug/mZ/day, 700 ug/mZ/day, 650 ug/mZ/day, 600 ug/mZ/day, 550 ug/mZ/day, 500 ug/mZ/day, 450 day, 400 ug/mZ/day, 350 ug/mZ/day, 300 ug/mZ/day, 250 ug/mZ/day, 200 ug/mZ/day, 150 ug/mZ/day, 100 ug/mZ/day, 50 ug/mZ/day, 40 ug/mZ/day, 30 ug/mZ/day, 20 ug/mZ/day, 15 day, 10 ug/mZ/day, or 5 ug/mZ/day.

In another embodiment, the intravenous dose of 1200 ug/m2 or less, 1150 ug/m2 or less, 1100 ug/m2 or less, 1050 ug/m2 or less, 1000 ug/m2 or less, 950 ug/m2 or less, 900 ug/m2 or less, 850 ug/m2 or less, 800 ug/m2 or less, 750 ug/m2 or less, 700 ug/m2 or less, 650 ug/m2 or less, 600 ug/m2 or less, 550 ug/m2 or less, 500 ug/m2 or less, 450 ug/m2 or less, 400 ug/m2 or less, 350 ug/m2 or less, 300 ug/m2 or less, 250 ug/m2 or less, 200 ug/m2 or less, 150 ug/m2 or less, 100 ug/m2 or less, 50 ug/m2 or less, 40 ug/m2 or less, 30 ug/m2 or less, 20 ug/m2 or less, 15 ug/m2 or less, 10 ug/m2 or less, or ug/m2 or less of one or more anti-CD3 antibody or antigen-binding fragments is administered over about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1.5 hours, about 1 hour, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, of 5 until the daily prophylactically or therapeutically effective amount of one or more anLr‘nq nnfil’xnr‘lv nr nnfimfinJfiinrlinn fc ic anhipvpr‘l about 5 s, about 2 minutes, about 1 minute, about 30 seconds or about 10 seconds to prevent, treat or ameliorate one or more ms of an mune disease or T-cell malignancy. The total dosage over the duration of the regimen is preferably a total of less than 9000 ug/mz, 8000 ug/mz, 7000 ug/mz, 6000 ug/mz, and may be less than 5000 ug/l’nz, 4000 ug/l’nz, 3000 ug/mz, 2000 ug/m2,01‘1000 ug/l’nz.

In specific embodiments, the total dosage administered in the n is 100 ug/m2 to 200 Mg/l’nz, 100 11ng to 500 ug/mz, 100 Mg/l’nz to 1000 Mg/l’nz, or 500 Mg/l’nz to 1000 ug/mz.

In preferred embodiments, the dose escalates over the first fourth, first half or first 2/3 ofthe doses (e.g., over the first 2, 3, 4, 5, or 6 days ofa 10, 12, 14, 16, 18 or 20 day regimen of one dose per day) of the treatment regimen until the daily prophylactically or therapeutically ive amount of one or more anti-CD3 antibody or antigen-binding fragments is achieved. In certain embodiments, a subject is administered a treatment regimen comprising one or more doses of a prophylactically or therapeutically effective amount of one or more anti-CD3 antibody or antigen-binding nts, wherein the prophylactically or therapeutically effective amount is increased by, e. g., 0.01 ug/kg, 0.02 ug/kg, 0.04 ug/kg, 0.05 ug/kg, 0.06 ug/kg, 0.08 ug/kg, 0.1 ug/kg, 0.2 ug/kg, 0.25 ug/kg, 0.5 rig/kg, 0-75 rig/kg, 1 , 1-5 lug/kg 2 rig/kg, 4 rig/kg, 5 rig/kg, 10 Mil/kg, 15 ug/kg, ug/kg, 25 ug/kg, 30 ug/kg, 35 ug/kg, 40 ug/kg, 45 ug/kg, 50 ug/kg, 55 ug/kg, 60 ug/kg, 65 ug/kg, 70 ug/kg, 75 ug/kg, 80 ug/kg, 85 ug/kg, 90 ug/kg, 95 ug/kg, 100 ug/kg, or 125 ug/kg each day; or increased by, e.g., l ug/mz, 5 ug/mz, 10 ug/mz, l5 ug/l’nz, 20 ug/l’nz, 30 ug/mz, 40 ug/l’nz, 50 ug/l’nz, 60 ug/mz, 70 ug/mz, 80 z, 90 ug/mz, 100 ug/mz, 150 Mg/l’nz, 200 Mg/l’nz, 250 Mg/l’nz, 300 Mg/l’nz, 350 ug/mz, 400 ug/mz, 450 ug/mz, 500 ug/mz, 550 ug/mz, 600 ug/mz, or 650 ug/mz, each day as treatment progresses. In certain embodiments, a subject is administered a treatment regimen comprising one or more doses of a prophylactically or therapeutically effective amount of one or more anti-CD3 dy or antigen-binding fragments, wherein the prophylactically or therapeutically ive amount is increased by a factor of 1.25, a factor of 1.5, a factor of 2, a factor of 2.25, a factor of 2.5, or a factor of 5 until the daily prophylactically or therapeutically effective amount of one or more anti-CD3 antibody or antigen-binding fragments is achieved. uuuv u; Avv rub/1x5 u; Lvuu, 1.1 rub/1x5 u; Lvuu, 1v rub/1x5 u; Lvuu, Uu rub/1x5 u; Lvuu, uv ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55 In a specific embodiment, a t is intramuscularly administered one or more doses of a 200 ug/kg or less, ably 175 ug/kg or less, 150 ug/kg or less, 125 ug/kg or less, 100 ug/kg or less, 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or less, 80 ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55 ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or less, 30 ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or less, 5 ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 1.5 ug/kg or less, 1 ug/kg or less, 0.5 ug/kg or less, or 0.5 ug/kg or less of one or more anti-CD3 antibody or antigen-binding nts to prevent, treat or ameliorate one or more symptoms of an autoimmune disorder or T-cell malignancy.

In another embodiment, a subject is subcutaneously administered one or more doses of a 200 ug/kg or less, preferably 175 ug/kg or less, 150 ug/kg or less, 125 ug/kg or less, 100 ug/kg or less, 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or less, 80 ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55 ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or less, 30 ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or less, 5 ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 1.5 ug/kg or less, 1 ug/kg or less, 0.5 ug/kg or less, or 0.5 ug/kg or less of one or more anti-CD3 antibody or antigen-binding nts to prevent, treat or ameliorate one or more symptoms of an autoimmune disorder.

In another embodiment, a subject is intravenously administered one or more doses ofa 100 ug/kg or less, preferably 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or less, 80 ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55 ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or less, 30 ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or less, 5 ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 1.5 ug/kg or less, 1 ug/kg or less, 0.5 ug/kg or less, or 0.5 ug/kg or less of one or more D3 antibody or antigen-binding fragments to prevent, treat or ameliorate one or more symptoms of an autoimmune disorder or T-cell malignancy. In another embodiment, the intravenous dose of 100 ug/kg or less, 95 ug/kg or less, 90 ug/kg or less, 85 ug/kg or less, 80 ug/kg or less, 75 ug/kg or less, 70 ug/kg or less, 65 ug/kg or less, 60 ug/kg or less, 55 ug/kg or less, 50 ug/kg or less, 45 ug/kg or less, 40 ug/kg or less, 35 ug/kg or less, 30 ug/kg or less, 25 ug/kg or less, 20 ug/kg or less, 15 ug/kg or less, 10 ug/kg or less, 5 ug/kg or less, 2.5 ug/kg or less, 2 ug/kg or less, 15 ug/kg or less, 1 ug/kg or less, 0.5 ug/kg or less, or 0.5 ug/kg or less of one or more anti-CD3 antibody or antigenbinding fragments is administered over about 6 hours, about 4 hours, about 2 hours, about 1.5 hours, about 1 hour, about 50 minutes, about 40 s, about 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 ameliorate one or more symptoms of an autoimmune disorder or T-cell ancy.

In specific embodiments in which ting doses are administered for the first days of the dosing regimen, the dose on day l of the regimen is 5 - 100 ug/mZ/day, and tes to the daily dose as recited immediately above by day 3, 4, 5, 6 or 7. For example, on day l, the t is administered a dose of approximately 51 ug/mZ/day, on day 2 approximately 103 ug/mZ/day, on day 3 approximately 207 ug/mZ/day, on day 4 approximately 413 ug/mZ/day and on subsequent days of the regimen (e.g., days 5-14) 826 ug/mZ/day.

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

In c embodiments, to reduce the possibility of cytokine release and other adverse effects, the first 1, 2, 3, or 4 doses or all the doses in the regimen are administered more slowly by intravenous administration. For example, a dose of 51 ug/mZ/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 certain embodiments, the dose is administered by slow infiJsion over a period of, e.g., 20 to 24 hours. In specific VALLuLLLb LwaLALVLLt/u ALL ULLV UMVJVUI/ “LI/VA wuLALLLLLUwwaLVLL. ALL yv; UWLLL VLALVUuLLALVLLt/U, ULLV level of free anti-CD3 antibody or antigen-binding fragments should not exceed 200 embodiments, the dose is infused in a pump, preferably increasing the concentration of dy administered as the 111filS1011 progresses.

In other embodiments, a set on of the n may be administered in escalating doses. For example, for the 51 ug/mz/day to 826 ug/mZ/day regimen described above, the fraction may be 1/10, 1/4, 1/3, 1/2, 2/3 or 3/4 ofthe daily doses. ingly, when the fraction is 1/ 10, the daily doses will be 5.1 ug/m2 on day 1, .3 ug/m2 on day 2, 20.7 ug/m2 on day 3, 41.3 ug/m2 on day 4 and 82.6 ug/m2 on days 5 to 14. When the fraction is 1/4, the doses will be 12.75 ug/m2 on day 1, 25.5 ug/m2 on day 2, 51 ug/m2 on day 3, 103 ug/m2 on day 4, and 207 ug/m2 on days 5 to 14. When the fraction is 1/3, the doses will be 17 ug/m2 on day 1, 34.3 ug/m2 on day 2, 69 ug/m2 on day 3, 137.6 ug/m2 on day 4, and 275.3 ug/m2 on days 5 to 14. When the fraction is 1/2, the doses will be 25.5 ug/m2 on day l, 51 ug/m2 on day 2, 103 ug/m2 on day 3, 207 ug/m2 on day 4, and 413 ug/m2 on days 5 to 14. When the fraction is 2/3, the doses will be 34 ug/m2 on day 1, 69 ug/m2 on day 2, 137.6 ug/m2 on day 3, 275.3 ug/m2 on day 4, and 550.1 ug/m2 on days 5 tol4. When the fraction is 3/4, the doses will be 38.3 ug/m2 on day 1, 77.3ug/m2 on day 2, 155.3 ug/m2 on day 3, 309.8 ug/m2 on day 4, and 620 ug/m2 on days 5 to 14.

In specific embodiments, the anti-CD3 antibody or antigen-binding fragments is not stered by daily doses over a number of days, but is rather administered by infusion in an uninterrupted manner over 4 hours, 6 hours, 8 hours, hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 30 hours or 36 hours. The 111filS1011 may be constant or may start out at a lower dosage for, for example, the first 1, 2, 3, 5, 6, or 8 hours of the infusion and then increase to a higher dosage thereafter.

Over the course of the infusion, the patient receives a dose equal to the amount administered in the exemplary regimens set forth above. For e, a dose of approximately 150 ug/mz, 200 ug/mz, 250 ug/mz, 500 ug/mz, 750 ug/mz, 1000 ug/mz, 1500 Mg/l’nz, 2000 Mg/l’nz, 3000 Mg/l’nz, 4000 Mg/l’nz, 5000 Mg/l’nz, 6000 Mg/l’nz, 7000 ug/mz, 8000 ug/mz, or 9000 ug/mz. In particular, the speed and on of the on is designed to minimize the level of free anti-CD3 antibody or antigenbinding fragments in the subject after administration. In certain embodiments, the level of free anti-CD3 antibody or antigen-binding fragments should not exceed 200 amount of one or more anti-CD3 antibody or n-binding fragments does not ng/ml free antibody. In on, the infusion is ed to achieve a ed T cell receptor g and modulation of at least 50%, 60%, 70%, 80%, 90%, 95% or of 100%.

] In certain embodiments, the anti-CD3 antibody or antigen-binding fragments is administered so as to achieve a certain level of combined g and modulation of T cell receptor complexes on T cells, as determined by methods well known in the art, see, e. g., Example ll of US. patent application publication US 2003/0108548, which is hereby incorporated by reference in its entirety. In ic embodiments, the dosing regimen achieves a combined T cell receptor coating and modulation of at least 50%, 60%, 70%, 80%, 90%, 95% or of 100% with, in c embodiments, little to no free anti-CD3 antibody or antigen-binding fragments detected (for example, less than 200 ng/mL of the drug is detected in the blood of the patient).

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

In a specific embodiment, the administration of one or more doses or a dosage regimen of a prophylactically or therapeutically effective amount of one or more anti-CD3 antibody or antigen-binding fragments does not induce or reduces relative to other immunosuppressive agents one or more of the following unwanted or adverse effects: vital sign abnormalities (fever, tachycardia, bardycardia, hypertension, nsion), hematological events (anemia, lymphopenia, leukopenia, thrombocytopenia), headache, chills, dizziness, nausea, asthenia, back pain, chest pain (chest pressure), diarrhea, myalgia, pain, pruritus, psoriasis, rhinitis, sweating, injection site reaction, latation, an increased risk of opportunistic ion, tion of Epstein Barr Virus, apoptosis of T cells and an increased risk of developing certain types of cancer. In another specific embodiment, the administration of one or more doses of a prophylactically or therapeutically effective amount of one or more anti-CD3 antibody or antigen-binding fragments does not antibody or antigen-binding fragments may be administered to a subject when, for example, the subject's HA 1 or HA 1 C levels at 1 month, 2 , 4 months, 6 induce or reduces relative to other immunosuppressive agents one or more of the ing unwanted or adverse effects: vital sign abnormalities (fever, tachycardia, bardycardia, hypertension, hypotension), hematological events (anemia, lymphopenia, leukopenia, ocytopenia), headache, chills, dizziness, nausea, asthenia, back pain, chest pain (chest pressure), diarrhea, myalgia, pain, pruritus, psoriasis, is, sweating, injection site reaction, vasodilatation, an sed risk of opportunistic infection, Epstein Barr Virus activation, apoptosis of T cells, and an increased risk of developing certain types of cancer.

In accordance with the invention, the dose or dosage regimen comprising a prophylactically or therapeutically effective amount of one or more anti-CD3 antibody or antigen-binding fragments for the treatment of an mune disorder may be repeated at 1 month, 2 months, 4 months, 6 months, 8 months, 12 months, 15 months, 18 months or 24 months or longer after the initial or previous dose or dosage regimen comprising a prophylactically or therapeutically effective amount of one or more anti-CD3 antibody or antigen-binding fragments. The repeat dose or dosage regimen may be administered as a matter of course, when symptoms associated with said autoimmune disorder recur after an improvement following the initial or previous dose or dosage regimen, or when symptoms ated with said autoimmune disorder do not improve after the initial dose or dosage regimen of anti-CD3 antibody or antigen-binding fragments according to methods of the invention. For example, with respect to diabetes, a repeat dose or dosage n comprising a prophylactically or therapeutically ive amount of one or more anti-CD3 antibody or antigen-binding nts may be administered to a subject when, for example, the t's e daily insulin use at 1 month, 2 , 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 nts does 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. Alternatively, with respect to diabetes, a repeat dose or a dosage regimen comprising a prophylactically or therapeutically effective amount of one or more anti-CD3 antibody or antigen-binding fragments may be administered to a subject when, for e, the subject's HA 1 or HA 1 C levels at 1 month, 2 months, 4 months, 6 w ALLALvau Lnyunnvv; u; LLALLALMLLUUMIJIJLVUULVV auLvLLu LALwJ LVL’MLI/ LL; tjvllvuu u; remission or disappearance of active disease. Immunosuppressive agents used for 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 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- ent levels. In another embodiment, with respect to diabetes, a repeat dose or dosage n comprising a prophylactically or therapeutically effective amount of one or more anti-CD3 antibody or n-binding fragments may be administered to a subject when, for example, the subject's C-peptide response at 1 month, 2 months, 4 months, 6 months, 8 months, 12 months, 15 months, 18 months or 24 months or longer after initial or us treatment with anti-CD3 antibody or antigen-binding fragments decreases by more than 5%, more than 10%, more than 20%, more than %, more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more than 90% compared to eatment levels.

Autoimmune es are non-infectious immunological es caused by immune ses that are directed to normal components of human cells, tissues and organs. mune diseases are often chronic diseases that gradually erode ed tissues and organs. Common es now classified as autoimmune diseases due to the presence of inappropriate autoimmune responses include type I insulin-dependent diabetes, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD); myasthenia gravis, celiac’s e, Sjogren's me, Grave’s disease, Crohn’s disease, autoimmune hepatitis, psoriasis, psoriatic tis, asthma, allergic rhinitis, effects from organ transplantation, or graft vs. host disease (GVHD) and us other diseases involving an inflammatory immune response.

Because autoimmune diseases are typically chronic, they generally require lifelong treatment and monitoring. Conventional therapies for autoimmune disease are therefore primarily directed to managing the consequences of inflammation caused by the disease, and only a few autoimmune diseases can be cured or made to disappear with such treatment. For some autoimmune diseases, administering one of a limited number of immunosuppressive medications may result in periods of remission or disappearance of active disease. Immunosuppressive agents used for LLLuwvv pus/LL LLALLALMLLUUMIJIJL vuuLuLL VJ L Lb tJWI/LLUbVLLLU A UVLLL’ “LL“ LLLuMVLLLb regulatory T cells (WO 2007/117600; St. Clair E.W. (2009) “Novel Targeted adjunct therapy e substances that suppress cytokine production, down-regulate or suppress self-antigen expression or mask major histocompatibility (MHC) antigens. suppressive medications include anti-inflammatory drugs (e.g., a nonsteroidal anti-inflammatory drug (“NSAID”), cyclophosphamide, bromocryptine cyclosporine A, methotrexate, steroids such as glucocorticosteroids and cytokines or cytokine receptor antagonists. Patients are rarely able to discontinue these immunosuppressive medications as their mune disease y reappears when medication is discontinued. Autoimmune disease may become refractive to treatment when immunosuppressive medications are continued long term and may require ever increasing doses of suppressive agents.

] Therapeutic antibodies directed to CD3 are believed to e fewer long- term side effects than many of the immunosuppressive chemotherapies that are presently available for autoimmune es (). However, prior antibody based therapies have been found to be problematic, particularly where repeated administration was employed. Anti-lymphocyte therapies, such as antilymphocyte globulin (ALG), and monoclonal antibodies directed to B cells, such as rituximab (Rituxin®) and alemtuzumab (CAMPATH®) reduce circulating and tissue B cell populations in treated subjects. However, these therapies also cause severe suppression, which is undesirable for the long term treatment of a chronic autoimmune disease. The principal complication of severe immune- suppressive therapy is infection. Systemic immunosuppression can also be accompanied by undesirable toxic effects and a ion in levels of ietic stem cells. In addition, patients receiving antibody therapies often develop significant levels of human anti-mouse antibodies (HAMA), human anti-chimeric antibodies (HACA) and anti-idiotypic responses, which may limit repeated treatments when a remission ends.

As discussed above, antibodies directed to ns of the T cell, such as the T-cell receptor complex (TCR), have been ted as possible therapeutics for the immunosuppression of autoimmune disease. Anti-CD3 dies are believed to induce such immunosuppression by reducing pathogenic T cells and inducing regulatory T cells (WO 2007/117600; St. Clair E.W. (2009) “Novel Targeted u UVLL “LLuLbVLL. ies for Autoimmunity,” Curr. Opin. Immunol. 21(6):648-657; Ludvigsson, J. (2009) “The Role of Immunomodulation Therapy in Autoimmune Diabetes,” J.

Diabetes Sci. l. 3(2):320-330). Anti-T cell antibodies, including anti-CD3, have therefore been used to influence logical status in a subject by suppressing, ing or redirecting T cell responses to an antigen. In particular, Teplizumab, also known as hOKT3yl(Ala-Ala) (containing an alanine at positions 234 and 235) (MacroGenics, Inc.) is an anti-CD3 antibody that had been engineered to alter the fianction of the T lymphocytes that mediate the destruction of the insulin- producing beta cells of the islets of the pancreas. Teplizumab binds to an epitope of the CD38 chain expressed on mature T cells and by doing so.

Due in part to their cross-reactivity with non-human CD3 (which permits more accurate and responsive ), the anti-CD3 antibodies of the present ion are considered to have particular utility in the treatment of autoimmune disease notwithstanding the apparent failures of the prior art.

Such antibodies and their n binding fragments may be used alone or in conjunction with other pharmacological agents. In ular, the present invention contemplates therapies involving the administration of such antibodies or antigen binding fragments in conjunction with anti-B cell antibodies (or antigen-binding fragments thereof). Anti-B cell antibodies are known in the art (see, WC 2007/117600; ; ; United States s Nos. ,500,362 and 5,736,137; US. Patent Publications Nos. 2003/0219433; 2005/0271658; 271658; 281817; 2006/024295; 2006/024300 and 2006/034835; Clark, EA. et al. (1985) “Role Of The Bp35 Cell Surface Polypeptide In Human B-Cell Activation,” Proc. Natl. Acad. Sci. (USA) 82(6):1766-1770; Press, O.W. et al. (1987) “Monoclonal Antibody IF5 (Anti-CD20) Serotherapy ofHuman B Cell Lymphomas,” Blood 69:584-591). Such conjunctive administration may be accomplished using joint administration of distinct antibodies or antigen-binding fragments thereof, or by forming bispecif1c antibodies, or more preferably, by forming DARTTM diabodies, as described above, having the ability to bind to both CD3 and a B cell antigen. combination with an dy targeted to rferon for treatment of a subject having multiple sclerosis.

Preferably the employed anti-B cell antibody or antigen-binding fragment will be directed to a B cell surface marker, such as a marker selected from CD19, CD20, CD22, CD23, CD32B, CD40, B7-l (CD80), B7-2 (CD86), CD79a, CD79b, CD38, CD27, a lymphocyte fianction-associated antigen (LFA), such as LFA-I or LFA-3, CFA-I, or another accessory molecule involved in the T cell, B cell association that leads to T cell and B cell activation in an adaptive immune response.

In a fiarther preferred embodiment, the anti-B cell antibody may be a B cell ing dy, such as an antibody directed to a marker selected from CD19, CD20, CD22, CD23, CD32B, CD40, B7-l (CD80), B7-2 (CD86), a lymphocyte function-associated antigen (LFA), such as LFA-I or LFA-3, CFA-I, or an accessory molecule involved in the T cell, B cell association.

Alternatively, such combination therapy may comprise administration of an anti-CD3 antibody or antigen-binding nt thereof, in combination with an antibody (or antigen-binding fragment thereof) that recognizes an n present on an antigen presenting cell (e.g., B7-H3). In a still further preferred embodiment, the combination therapy comprises administration of an anti-CD3 antibody (or n- binding fragment thereof) in combination with an antibody (or antigen-binding fragment f) that recognizes a polypeptide involved in B cell activation (either directly or indirectly) or an immunomodulator such as a member of TNF cytokine family, or an interferon (e.g., or, B or y interferon). As is understood by those of skill in the art, such erons are involved in the regulation of proteins that work together in antigen processing and presentation. These nes stimulate cells to increase their expression of HLA class I heavy chains. In one preferred embodiment, the ation therapy comprises administering to a t having active autoimmune disease an antibody to a T cell antigen in combination with an antibody to B—interferon. In a fiarther preferred embodiment, the combination therapy comprises administering to a subject an antibody targeted to a T cell antigen in combination with an antibody selected from rferon antibodies AVONEX®, BETASERON® and REBIF®. In a further embodiment, the combination therapy comprises stering to a subject an antibody targeted to a T cell antigen in combination with an antibody targeted to B-interferon for treatment of a subject having le sclerosis.

A subject rece1v1ng the combination therapy also may have reduced ‘l‘ cell responses to autoantigens as detected by in vitro by proliferation or cytokine production assays 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 a T cell comes in contact with its specific n on an antigen presenting cell, in particular an antigen presenting B cell. As used herein, the term “non-mitogenic T cell antibody” means an antibody that is engineered by ng the Fc receptor of the antibody such that it does not trigger the initial activation events and ensuing release of cytokines that are seen when a T cell is activated. A “reduced mitogenic T cell dy” is an antibody c for a T cell antigen that reduces the initial activation events and release of cytokines that occur when a T cell is activated. The non- mitogenic or d mitogenic antibody may be useful for preventing initial “first dose side effects” seen when an anti-lymphocyte antibody is administered to patient.

The non-mitogenic or reduced mitogenic antibody may be an ered antibody having a modified Fc nt that prevents or inhibits binding by effector cells.

C. Methods of Administration In one , embodiments of the present invention provide treated human subjects so as to achieve and maintain clinical remissions for longer periods than remissions achieved by subjects d with a conventional therapy. For example, where a conventional y achieves a remission of symptoms of an autoimmune disease for three months, the compositions of the present invention may provide a complete remission of symptoms of up to six months, up to 12 months and in some cases up to one to two years or longer. It is plated that for certain autoimmune diseases it may be possible to provide a complete remission that does not e, particularly where therapy begins shortly after the autoimmune disease is sed.

The clinical ion achieved with the ation therapy may be a complete remission, or it may be a partial remission in which significant reductions in disease ms are maintained for an extended period. For example, a subject receiving the therapy of the present invention may have d mune responses as determined by reduced levels of detectable autoantibodies in body fluids and tissues, for example in cerebrospinal fluid (CSF), serum, urine or in body tissues.

A subject receiving the combination therapy also may have reduced T cell responses to autoantigens as detected by in vitro by proliferation or cytokine production assays desired clinical response. s for determining the dosage of a therapeutic using peripherial blood mononuclear cells (PBMCs) or purified T cells when compared with subjects d with a conventional therapy.

The itions of the present invention may be administered by any suitable means, including parenteral, topical, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and/or intralesional administration. eral infilsions include intramuscular, intravenous, rterial, intraperitoneal, or subcutaneous administration. In addition, the antibody may suitably be administered by pulse infilsion, e.g., with increasing doses of the antibody. Preferably, the dosing is given intravenously or subcutaneously, and more preferably by intravenous infusion(s).

Each exposure may be provided using the same or a different administration means.

In one embodiment, each exposure is by intravenous administration. In another embodiment, each exposure is given by subcutaneous administration. In yet another embodiment, the exposures are given by both intravenous and aneous administration.

In one embodiment, the therapeutic antibodies are administered as a slow enous on which may commence at a rate hour to deliver the molecules of the invention in imately 15 minutes to 4 hours. However, if the subject is experiencing an infiJsion-related reaction, the infusion rate is preferably reduced, e. g., to half the current rate. The treated subjects may receive a prophylactic treatment of acetaminophen/paracetamol (e.g., about lg) and diphenhydramine HCl (e.g., about 50 mg or equivalent dose of similar agent) by mouth about 30 to 60 s prior to the start of an infilsion.

The therapy provided by combination compositions of the present invention (including DARTTM diabodies) may be stered to a subject using an initial dose of first antibody that is less than the amount of such antibody needed to achieve a clinical response in therapy for an autoimmune disease when administered as a single antibody therapy. A dose of a therapeutic anti-T cell antibody that is less than the dose needed to e depletion of T cells that are able to ize and respond to autoantigens in a therapy providing a single antibody may be sufficient to provide a desired clinical se. Methods for determining the dosage of a therapeutic pharmaceutical art in the form of lyophilized formulations or aqueous solutions. antibody needed to achieve a clinical response are known to those of skill in the art.

For example, a clinical response in the subject may be ed as time to disease ssion, reduction of clinical symptoms, reduction in levels of laboratory s, reduction in the need for retreatment, or by any other clinical means ized as a useful indicator of improvement in status of the autoimmune disease.

The second antibody of a combination therapy may also be administered to a subject in need of treatment as an initial dose that is less than an effective dose for achieving a clinical response when the antibody is administered alone. For example, doses of a depleting anti-B cell antibody that achieve less than 100% B-cell depletion, less than 50% B cell depletion, less that 30% depletion or even no B cell depletion may be administered together with a first anti-T cell antibody to achieve a clinical se that provides suppression of an immune response to an autoantigen equal to, or better than the clinical response achieved by administering an amount of a B cell depleting antibody that provides 100% depletion of B cells in the subject when administered alone.

In some instances, clinical response may be a response that neither the first nor the second antibody achieves when administered alone. In other instances, the clinical response may be equivalent to that ed by stration of a single antibody therapy, where the combination therapy provides less suppression of a treated t’s immune system than a single antibody therapy. In one preferred embodiment, the synergistic response provided by the ation therapy reduces or eliminates a subject’s se to an autoantigen while providing lower levels of immunosuppression. General immunosuppression is a significant m for previously available antibody therapies.

D. Pharmaceutical Formulations Therapeutic formulations of the antibodies used in embodiments of the present invention are prepared for storage, shipment and administration by mixing a composition of the present invention having a d purity with optional pharmaceutically able carriers, excipients or stabilizers recognized in the pharmaceutical art in the form of lyophilized formulations or aqueous solutions.

LJutJLLLLLuvu yuvvuv; u; vkuv; Lva vuLLVVLLuquv LL; w LLVLLLLVULUWLLJ vaLvu UULLUMLLLVL ”mun; QC on amnnnlp nr aanhpfl'p inr‘linafinn- Hap nuanfifv n‘F anfivn arr-sni- ‘Xnmarp Hop The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the US. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, nt (e.g., Freund’s adjuvant ete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such ceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, , vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline ons and aqueous dextrose and ol solutions can also be employed as liquid carriers, ularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium te, ol monostearate, talc, sodium de, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The ition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, on, tablets, pills, capsules, s, sustained-release formulations and the like.

] The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g, impure or non-sterile compositions) and pharmaceutical compositions (z'.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of a prophylactic and/or therapeutic agent sed herein or a combination of those agents and a pharmaceutically acceptable carrier. Preferably, compositions of the invention comprise a prophylactically or therapeutically effective amount of humanized antibodies of the invention and a pharmaceutically acceptable carrier.

Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infiJsion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by ion, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions where the active agents are water soluble, or dispersions or sterile powders for oraneous preparation of e injectable solutions. Compositions for use in the combination therapy may be prepared by incorporating the active antagonist or antibody in the required amount with appropriate carriers, for example water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride may be included in the composition.

The compositions of the invention can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts e, but are not limited to, those formed with anions such as those derived from hydrochloric, oric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, ne, etc.

E. Kits The ion provides a pharmaceutical pack or kit comprising one or more containers filled with humanized antibodies of the invention. onally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the ceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the cture, use or sale of pharmaceuticals or biological products, which notice reflects al by the agency of manufacture, use or sale for human administration.

The present invention provides kits that can be used in the above methods.

In one embodiment, a kit comprises one or more humanized antibodies of the invention. In another embodiment, a kit fiarther comprises one or more other prophylactic or eutic agents useful for the treatment of cancer, in one or more containers. In another embodiment, a kit fiarther comprises one or more cytotoxic antibodies that bind one or more cancer antigens associated with cancer. In certain ments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

] In one embodiment, the present invention es an article of manufacture containing antibodies to be used for the combined therapy for treatment of autoimmune disease. The article of manufacture ses a container sing a first antibody that binds an antigen present on a T cell and a pharmaceutically able carrier, excipient or diluent within the container. The article of manufacture further comprises a second container sing a second antibody directed to 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 mune disease. Where the first and second antibodies are determined to be complementary and to not adversely affect each other, the first and the second antibody may be provided in a single container containing the first and second antibody in appropriate concentrations for administration together with a package insert and instructions for administration.

Containers of the article of cture may be of any suitable material that will not react with or otherwise affect the preparation. The article of manufacture may r comprise a second or a third container comprising a pharmaceutically- acceptable diluent buffer, such as iostatic water for injection, phosphate- buffered saline, Ringer's on and dextrose solution. The article of manufacture may also include other material that may be desired from a cial and user standpoint including other buffers, diluents, filters, needles and syringes.

L/Alj1L/DD1U11. no uovu 11V1L/111, 111L/L11UUD 1U1 cuuuls u1u511UD1D 11D L11CLL L11L/DL/ 111L/L11UUD assist in making a clinical determination regarding the classification, or nature, of VII. Diagnostic Methods Using the Anti-CD3 Antibodies of the Present invention Antibodies to CD3 made by the methods sed herein may also be used to identify the presence or absence of cancerous cells, or the level thereof, which are circulating in blood after their release from the cell surface (e.g., soluble CD3). Such circulating antigen may be an intact CD3 antigen, or a fragment thereof that retains the ability to be detected according to the methods taught herein. Such detection may, for example, be effected by FACS is using standard methods commonly used in the art.

In a preferred embodiment of the diagnostic methods of this invention, the antibody bears a detectable label. Examples of labels that may be used include a radioactive agent (e.g., Scandium-47, Technetium-99m, -l l l, Iodine-l3l, Rhenium-186, Rhenium-188, Samarium-153, Holmium-166, Lutetium-l77, Copper- 64, Scandium-47, Yttrium-900), an enzyme or a fluorophore, such as phycoerythrin or fluorescein isothiocyanate (also known as fluoroisothiocyanate or FITC).

One method of using the antibodies for diagnosis is in vivo tumor imaging by linking the antibody to a radioactive or radio-opaque agent, administering the antibody to the individual and using an x-ray or other imaging machine to visualize the localization of the labeled antibody at the surface of cancer cells expressing the antigen. The antibody is administered at a concentration that promotes binding at physiological conditions.

In vitro techniques for detection of CD3 are routine in the art and include enzyme linked sorbent assays (ELISAs), precipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot is.

] The invention also provides s of aiding diagnosis of cancer characterized by cancer cells that express CD3 in an individual using any dy that binds to CD3 and any other methods that can be used determine the level of CD3 expression. As used herein, s for g sis” means that these methods assist in making a clinical determination regarding the classification, or nature, of LLLLLLWUVLJ vu n v; “Lukuv LLLULMuLLLb mnvvxwun v v vuLLuLu MAL“ VLULLLL u “Lukuv, myasthenia gravis, lupus, toid arthritis, , acute iuvenile onset diabetes, cancer, and may or may not be conclusive with respect to the definitive diagnosis.

Accordingly, a method of aiding sis of cancer can comprise the step of detecting the level of CD3 in a biological sample from the individual and /or determining the level of CD3 expression in the sample. dies recognizing the antigen or a portion thereof may also be used to create stic assays for ing antigen released or secreted from living or dying cancer cells in bodily fluids, including but not limited to, blood, saliva, urine, pulmonary fluid, or ascites fluid. The anti-CD3 antibodies made by the methods disclosed herein may be used to determine whether an individual diagnosed with cancer may be deemed a candidate for immunotherapy using antibodies directed against CD3. In one embodiment, a biopsy sample may be tested for expression of CD3, using antibodies directed against CD3. Individuals with cancer cells that express CD3 are suitable candidates for immunotherapy using antibodies directed against CD3. Staining with anti-CD3 antibody may also be used to distinguish cancerous tissues from normal tissues. s of using anti-CD3 antibodies for diagnostic purposes are useful both before and after any form of anti-cancer treatment, e.g., chemotherapy or radiation therapy, to determine which tumors are most likely to respond to a given treatment, prognosis for dual with cancer, tumor subtype or origin of metastatic disease, and progression of the disease or response to treatment.

The compositions of this invention are particularly suitable for the diagnosis of disease states other than cancer, using the methods generally described above in ation with other diseased (non-cancerous) cells. Disease states suitable for use in the methods of this invention include, but are not d to, diseases or disorders ated with inflammatory or autoimmune responses in individuals. The methods described above may be used for modulating inflammatory or autoimmune ses in individuals. Diseases and conditions resulting from inflammation and autoimmune disorders that may be subject to diagnosis and /or treatment using the compositions and methods of the invention include, by way of illustration and not of limitation, multiple sclerosis, meningitis, encephalitis, stroke, other cerebral traumas, inflammatory bowel e including ulcerative colitis and Crohn's disease, myasthenia , lupus, rheumatoid arthritis, asthma, acute juvenile onset diabetes, “Lulu ALLVL- J\U/-AVV./ Avxu, L VA A “VLvauLuLL vv v vau/ v1 Ajvul. VLLuvLLLLLu hvnnthesis, the CSCs provide a small, distinct subset of cells within each tumor that AIDS dementia, atherosclerosis, nephritis, tis, atopic dermatitis, psoriasis, myocardial ischemia and acute leukocyte-mediated lung injury.

Still other indications for diagnostic and/or therapeutic use of dies and other therapeutic agents of the ion include administration to individuals at risk of organ or graft rejection. Over recent years there has been a considerable improvement in the efficiency of surgical techniques for transplanting tissues and organs such as skin, kidney, liver, heart, lung, pancreas and bone marrow. Perhaps the principal outstanding problem is the lack of satisfactory agents for ng immunotolerance in the ent to the transplanted aft or organ. When allogeneic cells or organs are transplanted into a host (i.e., the donor and donee are different individuals from the same species), the host immune system is likely to mount an immune response to foreign antigens in the lant (host-versus-graft disease) g to destruction of the transplanted .

Monoclonal antibodies to CD3 made by the methods disclosed herein may be used to identify the presence or absence of human cancer stem cells in a variety 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. . Biol. 85(11):955-962; Gupta, P.B. et al. (2009) “Cancer Stem Cells: Mirage 0r Reality?” Nat. Med. lS(9):lOlO-lOl2; 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):127l-l278; Schatton, T. et al. (2009) “Identification And Targeting OfCancer Stem Cells,” Bioessays 31(10):lO38-1049; Mittal, S. et al. (2009) r Stem Cells: The Other Face OfJanus,” Amer. J. Med. Sci. 338(2):lO7-ll2; Alison, M.R. et al. (2009) “Stem Cells And Lung Cancer: Future Therapeutic s?” Expert Opin. Biol. Ther. 9(9):1127-1141; Charafe-Jauffret, E. et al. (2009) “Breast Cancer Stem Cells: Tools And Models To Rely 0n,” BMC Cancer 9:202; Scopelliti, A. et al. (2009) “Therapeutic Implications Of Cancer Initiating Cells,” Expert Opin.

Biol. Ther. 9(8):1005-1016; PCT Publication WO 2008/091908). Under this hypothesis, the CSCs provide a small, distinct subset of cells within each tumor that human mammalian species. Additionally, the binding of the chimeric mAbl antibody xxron nnmnornr] fn flnof AP 014 onfdlanrltr nnmnnnor‘ AP {-140 Inn/“0141.701: Y7OT‘1‘O14" 1m Ala1 T F f) are capable of indefinite self-renewal and of developing into the more adult tumor cell(s) that are relatively limited in replication capacity. It has been esized that these cancer stem cells might be more resistant to chemotherapeutic agents, radiation or other toxic conditions, and thus, persist after clinical ies and later grow into secondary , metastases or be responsible for relapse. It has been suggested that CSCs can arise either from 'normal' tissue stem cells or from more differentiated tissue progenitor cells.

Uses described in this application that recite their use for anti-CD3 antibodies also encompass the use of other CD3 agonists, antagonists and tors as bed herein for the use of identification and treatment of cancer stem cells. In such embodiments, anti-CD3 antibodies and other CD3 agonists, antagonists and modulators are used for identification, diagnosis or therapeutic treatment of cancer stem cells using similar methods described, and alterations within the scope of the ordinary skilled practitioner are made to tailor the method to the identification /diagnosis or treatment of cancer stem cells.

Having now generally described the invention, the same will be more y understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless specified. mAbl Binds to Both Human and Cynomolgus Monkey CD3 In order to assess the ability of mAbl to bind to human CD3, a capture ELISA was med. Plates were coated with l ug/ml of soluble lgus CD3 (“sCD3”) and incubated in the ce of various concentrations of a chimeric variant of mAbl dy (ch-mAbl) (containing the variable sequences of mAbl and the constant regions of a human antibody), a humanized variant (h-mAbl) and an antibody composed of the light chain of the chimeric mAbl antibody and the heavy chain of the humanized variant of mAbl. The s of this experiment are shown in Figure 1A. The experiment shows the ability of mAbl to bind to the CD3 of a non- human mammalian species. Additionally, the binding of the chimeric mAbl antibody was ed to that of an antibody composed of the humanized variant mAbl LC-2 aaccgagttt iiiiiiiiiiiiiiiiiiiiiactctgacca tttccagcct gcagcctgaa gatttcgcaa and the heavy chain of mAbl. The s of this experiment are shown in Figure 1B.

The experiment shows the ability of mAbl to bind to human CD3. Figures 1A and 1B thus reveal that the humanized mAbl was e of binding to both human CD3 and a CD3 of a non-human mammal. Humanized mAb showed binding to sCD3 and hCD3 that was r to that of the ic mAbl.

Example 2 Humanization of mAbl Humanized tives of mAbl were prepared. The amino acid sequences and encoding polynucleotide sequences of these humanized derivatives are shown below. The CDRs are shown in boldface and underlined.

Amino Acid Sequence of Humanized mAbl Variable Light Chain Variant l (SEQ ID NO:10): DIQ TQSPSS LSASVGDRVT :TCSASSSVS YMNWYQQKPG KAPKRLIY2§ SKLASGVPSR GT*E TLT SSLQPfl DEATYYCQQW SRNPPTFGGG TKVfl K Polynucleotide Sequence Encoding Humanized mAbl Variable Light Chain Variant 1 (SEQ ID NO:11): caga agtc cccc:ccagc ch c Lngggcga cagagtgaca aLcachg L ccgccachc chchchc L ggLaLcagca gaagcccggc aaggccccca agcgchgaL cLacgachc tccaagc:gg cctccggch gccceccaga ch c caccgagLLc accctgacca :ctccagcct gcagcccgag gacttcgcca chacLach ccagcagtgg :cccggaacc cccctacct: cggcggaggc accaaggtgg aaatcaag Amino Acid Sequence of Humanized mAbl Variable Light Chain Variant 2 (mAbl LC-2) (SEQ ID NO:12): DVV TQSPA" MSAhPGflKVT TCSASSSVS YMNWYQQKPG KAPKRWIY2§ SKLASGVPSR FSGSGSGTflh TLT SSLQPfl DEATYYCQQW SRNPPTFGGG TKVfl K Polynucleotide ce Encoding Humanized mAbl Variable Light Chain Variant 2 (SEQ ID NO:13): gachgnga Lgacccach aLc aLgangcLL Lcccaggcga gaaagtgacc aLLacaLch chcLLccag chLngLcc LacaegaacL ggtatcagca gaagccaggg aaagcaccca agaggeggae cLacgachc cegg chccggcg: gccaagccgg 'LchngLa ngchcaggL aaccgagLLL achLgacca t:tccagcct gcagcctgaa gatttcgcaa (h-mAb2 VL-2) (SEQ ID NO:18): ca sacLa ng Lcagcagtgg tccagaaatc cccctacatt tggcggaggg ac saaag egg aaatcaag ] Amino Acid Sequence of Humanized mAbl Variable Heavy Chain (SEQ ID NO:14): QVQLVQSGAE VKKPGASVKV SCKASGYTFT RSTMHWVRQA PGQGLnW GI.|.

INPSSAYTNY NQKFKDRVT: TADKSTSTAY MELSSLRSED TAVYYCASBQ VHYDYNGFPY WGQGTLVTVS S Polynucleotide ce Encoding Humanized mAbl Variable Heavy Chain Chain (SEQ ID NO:15): caggtgcagc sggsgcagsc ngcgccgaa gtgaagaaac ctggcgcctc cgcgaaggvg vcccgcaagg cc:ccggcta caccttcacc cggtccacca tgcactggg: gcgacaggcc ccaggccagg aatg gatcggc:ac atcaacccc: ccagcgcc:a caccaac:ac aaccagaaat tcaaggaccg cgvgaccasc accgccgaca agtccaccag caccgchac acggaacvgv csagcccgcg gagcgaggac accgccgtgt actactgcgc thcccccag gcgcacvacg acgg cLsccccsac ngggccagg tgg: gacagtgtcc tcc Example 3 Humanization of mAb2 Humanized derivatives of mAb2 were prepared. The amino acid sequences and encoding polynucleotide sequences of these humanized derivatives are shown below. The CDRs are shown in boldface and underlined.

Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant l (h-mAb2 VL-l) (SEQ ID : QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQQ KPGQAPRTL: APWT PARFSGSLLG GKAALTITGA DYYC ALWYSNLWVF GGGTKLTVLG Polynucleotide ce Encoding Humanized mAb2 Variable Light Chain Variant l (h-mAb2 VL-l) (SEQ ID NO:17): Lgachagga gccttcactg tccc caggcggaac agaL ccagcacagg cgcagtgacc acatc:aact gchcagcag aagccaggac aggcaccaag gasc acaaaagggc cccc cctgcacggs Lchngaag sgcsgggc ggaaaggccg ggca caggccgagg acgaagccga LeacLangL aLagcaaLcs gagggsgLsc gggggtggca caaaac,gac schggga Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 2 2 VL-2) (SEQ ID NO:18): ggggguggca UddddULgdU LngUnggd QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQQ KPGQAPRTL: GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF GGGTKLTVLG Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain Variant 2 (h-mAb2 VL-2) (SEQ ID NO:19): caggcdgdgg Lgachagga gccttcactg accgtgtccc caggcggaac ,g,gaccc:g acatgcagat cagg cgcagtgacc acatc:aact gngcagcag aagccaggac aggcaccaag gaccc,ga,c acaaaagggc ,ccc,ggacc cctgcacggc L,Lchgaag ,c,gc,gggc ggaaaggccg ctctgacta: ggca caggccgagg acgaagccga LLacLachL gc,c,nggc aLagcaaLcc gdgggdngc gggggtggca caaaac,gac dgdchggga Amino Acid Sequence of zed mAb2 Variable Light Chain Variant 3 (h-mAbZ VL-3) (SEQ ID NO:20): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT WFQE KPGQAPRTL: GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF GGGTKLTVLG Polynucleotide Sequence Encoding Humanized mAb2 le Light Chain Variant 3 (h-mAb2 VL-3) (SEQ ID NO:21): caggcdgdgg gga actg accgtgtccc caggcggaac ,g,gaccc:g acatgcagat ccagcacagg cgcagtgacc acatc:aact chccaggag aagccaggac aggcaccaag ga,c acaaaagggc ,ccc,ggacc cctgcacggc L,Lchgaag ,c,gc,gggc ggaaaggccg ctctgacta: ggca caggccgagg acgaagccga chL gcvcvgtggv aLagcaaLcc gvgggngvc ggca caaaac,gac dgdchggga Amino Acid Sequence of Humanized mAb2 le Light Chain Variant 4 (h-mAbZ VL-4) (SEQ ID NO:22): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQQ KPGQAPRGL: GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF GGGTKLTVLG Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain Variant 4 (h-mAb2 VL-4) (SEQ ID NO:23): Lgachagga gccttcactg tccc caggcggaac acatgcagat ccagcacagg cgcagtgacc acatc:aact chccagcag aagccaggac aggcaccaag gggccvgavc acaaaagggc ,ccc,ggacc cctgcacggc L,Lchgaag ,c,gc,gggc ggaaaggccg ctctgacta: taccggggca caggccgagg acgaagccga L,acLachL gcvcvgtggv aLagcaaLcc gdgggdngc gggggtggca ,gac dgdchggga gggggtacaa acaaaagggc tccctggacc cctgcacggt gaag gggc ggaaaggccg ctctgactat taccggggca caggccgagg Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 5 (h-mAb2 VL-S) (SEQ ID NO:24): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQE KPGQAPRTL: GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain Variant 5 (h-mAb2 VL-5) (SEQ ID NO:25): caggcvgvgg Lgachagga gccttcactg accgtgtccc caggcggaac ,g,gaccc:g acatgcagat ccagcacagg cgcagtgacc acatc:aact gngcaggag aagccaggac aggcaccaag gaccc,ga,c acaaaagggc ,ccc,ggacc cctgcacggv L,Lchgaag vcvgcvgggc ggaaaggccg ctctgacta: taccggggca caggccgagg acgaagccga LLacLaSLgL gcvcvgtggv aLcS gvgggSgch ggca caaaaCSgac SgSgCngga Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 6 (h-mAb2 VL-6) (SEQ ID NO:26): QAVVTQEPSL TVSPGGTVTL GAVT TSNYANWVQQ KPGQAPRGL: GGTNKRAPWT PARFSGSLLG GKAALTITGA QAEDEADYYC LWVF GGGTKLTVLG Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain Variant 6 (h-mAb2 VL-6) (SEQ ID NO:27): caggcvgvgg Lgachagga gccttcactg accgtgtccc caggcggaac ,g,gaccc:g acatgcagat cagg gacc aact gngcagcag aagccaggac aggcaccaag gggccvgavc acaaaagggc ,ccc,ggacc cctgcacggv L,Lchgaag vcvgcvgggc ggaaaggccg ctctgacta: taccggggca caggccgagg acgaagccga LLacLaSLgL gcvcvgtggv aLagcaaLcS gch gggggtggca Sgac SgSgCngga Amino Acid Sequence of Humanized mAb2 le Light Chain Variant 7 (h-mAb2 VL-7) (SEQ ID NO:28): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWFQE KPGQAPRGL: APWT PARFSGSLLG GKAALTITGA QAEDEADYYC ALWYSNLWVF GGGTKLTVLG Polynucleotide ce Encoding Humanized mAb2 Variable Light Chain Variant 7 (h-mAb2 VL-7) (SEQ ID NO:29): caggcvgvgg Lgachagga gccttcactg tccc caggcggaac ,g,gaccc:g acatgcagat cagg cgcagtgacc acatc:aact acgccaa,,g gLLccaggag aagccaggac aggcaccaag gggcc:gatc gggggtacaa acaaaagggc tccc:ggacc cctgcacggt tttctggaag vcvgcvgggc ggaaaggccg ctat taccggggca caggccgagg caggctgtgg tgactcagga actg accgtgtccc caggcggaac cctg acatgcagat ccagcactgg agcagtgact acctctaact acgaagccga LLacLaeLgL gc,chngL aLagcaatct gtgggtgttc gggggtggca caaaac,gac Lgechggga Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 8 (h-mAb2 VL-8) (SEQ ID NO:30): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQE KPGQAPRGL: GGTNKRAPWT PARFSGSLLG GKAALTITGA QAflDflADYYC ALWYSNLWVF GGGTKLTVLG Polynucleotide Sequence Encoding Humanized mAb2 le Light Chain Variant 8 2 VL-8) (SEQ ID NO:31): caggcegegg Lgachagga gccttcactg accgtgtccc caggcggaac ,g,gaccc:g acatgcagat ccagcacagg cgcagtgacc acatc:aact gngcaggag aagccaggac aggcaccaag gggccvgavc acaaaagggc gacc cctgcacgge L,Lchgaag vcvgcvgggc ggaaaggccg ctctgacta: taccggggca caggccgagg acgaagccga eLgL gcvcvgtggv aLagcaaLce gagggegLec gggggtggca caaaac,gac vgvchggga Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant 9 (h-mAb2 VL-9) (SEQ ID NO:32): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TSNYANWVQE KPGQAFRGL: GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF GGGTKLTVLG Polynucleotide Sequence Encoding Humanized mAb2 Variable Light Chain Variant 9 (h-mAb2 VL-9) (SEQ ID NO:33): caggcegegg Lgachagga gccttcactg accgtgtccc caggcggaac ,g,gaccc:g acatgcagat ccagcacagg cgcagtgacc acatc:aact acgccaa,,g gngcaggag aagccaggac aggca,,cag gggccvgavc gggggtacaa acaaaagggc gacc cctgcacgge L,Lchgaag vcvgcvgggc ggaaaggccg ctctgacta: taccggggca gagg acgaagccga LLacLaeLgL gcvcvgtggv aLagcaaLce gagggegLec ggca ,gac vgvchggga ] Amino Acid Sequence of Humanized mAb2 Variable Light Chain Variant lO (h-mAb2 VL-lO) (SEQ ID NO:34): EPSL TVSPGGTVTL TCRSSTGAVT WFQQ KPDHLFTGL: GGTNKRAPWT PARFSGSLLG GKAALTITGA QAfiDflADYYC ALWYSNLWVF GGGTKLTVLG ] Polynucleotide Sequence Encoding Humanized mAb2 le Light Chain Variant lO (h-mAb2 VL-lO) (SEQ ID : caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg acatgcagat ctgg agcagtgact acctctaact nUlVlULVDIVD WEI-ll. VVUQULLI V J. V00 acgctaattg gttccagcag aagcccgacc achgLLcac egggc,gaec ggcggaacca acaaaagggc ,ccc,ggacc cctgcacgg, aag tctgc:gggc ggaaaggccg ctctgacta: taccggggca caggccgagg acgaagccga L,acLaeLgL gc,c,ngg aLagcaaLce geggg,gL,c gggggtggca caaaac,gac ,g,chggga Amino Acid Sequence of Humanized mAb2 Heavy Chain t 1 (h- mAb2 VH-l) (SEQ ID NO:36): flVQLVflSGGG LVQPGGSLR; SCAASGFTFS TYAMNWVRQA PGKGLEWVGE IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT HDTAVYYCAR HGNFGNSYVS WFAYWGQGT; VTVSS Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain Variant 1 (h-mAb2 VH-l) (SEQ ID NO:37): gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag ac,g ,c g cacc, aca,acgcca tgaactgggt Ok—Qk—QFI'OO ,anggc,L ggc: ctggaaagg ggc,ggag,g gg,gggcagg atcaggtcca ag:acaacaa :atgcaacc ,ac,a,gccg tgaa gga,agaL,c acaatttccc cgacga,,c ,aaaaacag, c,g,aLc,gc aga,gaac,c gac: aagacaccg ,a L,gegcaaga cacggaaact tcggcaach ,achgecc ,gca, a,,gggg,ca gggcacac:g gtgaccgtg: ccagc Amino Acid Sequence of Humanized mAb2 Heavy Chain t 2 (h- mAb2 VH-2) (SEQ ID NO:38): flVQLVflSGGG LVQPGGSLR; SCAASGFTFN TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT HDTAVYYCAR HGNFGNSYVS WFAYWGQGT; VTVSS ] Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain Variant 2 (h-rnAb2 VH-2) (SEQ ID NO:39): gaggtgcagc aaag cggcggagga ctggtgcagc caggtggcag cctgcgac,g ,c ,gcgccg Ok—Qk—QFI'OO ,anggc,L cacc, acatacgcca tgaactgggt ggc: agg ggc,ggag,g gg,gggcagg atcaggtcca ag:acaacaa :atgcaacc ,ac,a,gccg ac:cagtgaa gga,agaL,c acaatttccc cgacga,,c ,aaaaacag, c,gc aga,gaac,c cc,gaagac: aagacaccg ,a L,gegcaaga cacggaaact tcggcaach ,achgecc ,gca, a,,gggg,ca gggcacac:g gtg: ccagc Amino Acid Sequence of Humanized mAb2 Heavy Chain Variant 3 (h- mAb2 VH-3) (SEQ ID NO:40): flVQLVflSGGG LVQPGGSLR; SCAASGFTFS TYAMNWVRQA PGKGLEWVAR NYAT YYADSVKDRF TISRDDSKNS SHKT HDTAVYYCAR HGNFGNSYVS WFAYWGQGT; VTVSS Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain Variant 3 (h-mAb2 VH-3) (SEQ ID NO:41): gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag cctgcgac,g ,c Vgcgccg ,L cacc,,Lch aca,acgcca tgaactgggt gaggcaggc: ctggaaagg ggc,ggag,g gg,ggccagg atcaggtcca acaa :atgcaacc vac,avgccg ac:cagtgaa gga,agaL,c acaatttccc cgacga,vc ,aaaaacag, cag,aLc,gc aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,gcaaga cacggaaact tcggcaach Ok—Qk—QN'OO ,achgvcc ,gng,gca, av,gggg,ca gggcacac:g gtgaccgtg: ccagc Amino Acid Sequence of Humanized mAb2 Heavy Chain Variant 4 (h- mAbZ VH-4) (SEQ ID NO:42): flVQLVflSGGG LVQPGGSLRL SCAASGFTFS TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT ?DTAVYYCVR HGNFGNSYVS WFAYWGQGT; VTVSS Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain Variant 4 (h-mAb2 VH-4) (SEQ ID NO:43): gaggtgcagc ,gg,ggaaag agga ctggtgcagc caggtggcag ac,g ,c Vgcgccg ,L cacc,,Lch aca,acgcca tgaactgggt gaggcaggc: agg ggc,ggag,g gg,gggcagg atcaggtcca ag:acaacaa :atgcaacc vac,avgccg ac:cagtgaa gga,agaL,c acaatttccc cgacga,vc ,aaaaacag, cag,a,c,gc aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,g,gaga cacggaaact tcggcaach Ok—Qk—QN'OO ,achgvcc ,gng,gca, av,gggg,ca ac:g gtgaccgtg: ccagc Amino Acid Sequence of Humanized mAb2 Heavy Chain t 5 (h- mAbZ VH-5) (SEQ ID NO:44): flVQLVflSGGG LVQPGGSLR; SCAASGFTFN VRQA WVAR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT YCAR HGNFGNSYVS WFAYWGQGT; VTVSS Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain Variant 5 (h-mAb2 VH-S) (SEQ ID NO:45): gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag cctgcgac,g dc,,gcgccg ,anggc,L cacc,,Laac gcca tgaactgggt gaggcaggc: ctggaaagg ag,g gg,ggccagg tcca acaa :atgcaacc gccg ac:cagtgaa gga,agaL,c acaatttccc Ok—Qk—QN'OO cgacga,vc ,aaaaacag, c,gc aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,gcaaga cacggaaact tcggcaach ,achgvcc ,gng,gca, av,gggg,ca gggcacac:g gtgaccgtg: ccagc cctgagactc tcctgtgcag cctctggatt caac acatacgcta tgaattgggt ccgccaggct ccagggaagg ggctggagtg ggttgcaagg Amino Acid Sequence of Humanized mAb2 Heavy Chain Variant 6 (h- mAb2 VH-6) (SEQ ID NO:46): SGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVGR IRSKYNNYAT YYADSVKDRF SKNS LYLQMNSHKT EDTAVYYCVR SYVS WFAYWGQGT; VTVSS Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain Variant 6 (h-mAb2 VH-6) (SEQ ID : gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag cctgcgac,g ,c g ,anggc,L cacc,,Laac acatacgcca tgaactgggt gaggcaggc: ctggaaagg ggc,ggag,g gg,gggcagg tcca ag:acaacaa :atgcaacc vac,avgccg ac:cagtgaa gga,agaL,c acaatttccc cgacga,vc ,aaaaacag, cag,a,c,gc aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,g,gaga aact tcggcaach QN'OO cc ,gng,gca, av,gggg,ca gggcacac:g gtgaccgtg: ccagc Amino Acid Sequence of Humanized mAb2 Heavy Chain t 7 (h- mAb2 VH-7) (SEQ ID NO:48): flVQLVflSGGG LVQPGGSLR; SCAASGFTFS TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGT; VTVSS Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain Variant 7 (h-mAb2 VH-7) (SEQ ID NO:49): gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caggtggcag cctgcgac,g ,c Vgcgccg ,anggc,L cacc,,Lch aca,acgcca tgaactgggt ggc: ctggaaagg ggc,ggag,g gg,ggccagg atcaggtcca ag:acaacaa :atgcaacc gccg ac:cagtgaa gga,agaL,c acaatttccc cgacga,vc ,aaaaacag, cag,a,c,gc aga,gaac,c cc,gaagac: aagacaccg ccg,g,ac,a L,g,g,gaga cacggaaact tcggcaach Ok—Qk—QN'OO ,achgvcc ,gng,gca, av,gggg,ca gggcacac:g gtgaccgtg: ccagc Amino Acid ce of Humanized mAb2 Heavy Chain Variant 8 (h- mAb2 VH-8) (SEQ ID NO:50): flVQLVflSGGG LVQPGGSLR; FTFN TYAMNWVRQA PGKGLEWVAR IRSKYNNYAT YYADSVKDRF TISRDDSKNS LYLQMNSHKT EDTAVYYCVR SYVS WFAYWGQGT; VTVSS Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain Variant 8 (h-mAb2 VH-8) (SEQ ID NO:51): gaggtgcagc ,gg,ggach nggggaggc ttggtccagc ctggagggtc cctgagach vcc,ngcag cctctggatt cacc:tcaac aca,acgc,a tgaattgggt ccgccaggct ccagggaagg ggctggagtg ggttgcaagg atcaggtcca agtacaacaa ,aLgcaacc eac,aegccg ac:ctgtgaa gga,agaL,c accatctcaa attc aaagaac,ca ceg,a,chc aaa:gaacag cotgaaaacc gaggacacgg ccg,g,a,,a c,g,g,gaga cacggtaact tcggcaa,Lc ,,achg,cL ,ggeL,gceL ae,ggggaca ggggacac,g ngach,gL c,Lcc Amino Acid Sequence of Humanized mAb2 Variable Heavy Chain Variant QV (h-mAb2 VL-QV) (SEQ ID NO:52): flVQLVflSGGG LVQPKGSLK; SCAASGFTFN TYAMNWVRQA PGKGLEWVA? IRSKYNNYAT YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCV? HGNFGNSYVS WFAYWGQGT; VTVSA Polynucleotide Sequence Encoding Humanized mAb2 Variable Heavy Chain Variant QV (h-mAb2 VL-QV) (SEQ ID NO:53): gaggtgcagc ,gg,ggaaag cggcggagga ctggtgcagc caaagggatc actgaaac,g ,cc,gcgccg gc:t cacc,,Laac aca,acgc,a tgaattgggt gcgacaggca cotggcaagg gcc,ggag,g gg,ggcaagg atcaggtcca agtacaacaa e,aLgcaacc gccg ac:ctgtgaa gga,agaL,c acaatcagtc gcgacga,,c ca,e c,g,a,chc agatgaacaa ,c,gaaaac: gaagacaccg cca,g,ac,a L,g,g,gcgg cacggtaact :cggcaa,,c ,cL ,ggeL,gc,, ae,ggggaca ggggacac,g ngach,ge c,Lcc mAb2 Binds to Both Human and Cynomolgus Monkey CD3 ] As discussed above, the mAb2 antibody was originally isolated based upon its y to bind human CD3. In order to assess the y of mAb2 to bind to non- human CD3, a capture ELISA was performed. Plates were coated with l ug/ml of CD3 (either human or cynomolgus ) and incubated in the presence of various concentrations of a ic variant of mAb2 dy (ch-mAb2) (containing the variable sequences of mAb2 and the constant regions of a human antibody). As a control, plates were also incubated with an antibody composed of the light chain of a humanized mAb2 antibody and the heavy chain of the chimeric antibody. The results of this experiment are shown in Figures 2A and 2B, and reveal that the chimeric mAb2 variant exhibited equivalent binding to human CD3 and to cynomolgus monkey CD3. formed and their binding assessed using a capture ELISA. Plates were coated with l ”(T/m] nf this thrm‘pllnlm‘ r‘lnmnin nf 1111an fin? filp kph? nr “thn2”\ nnr‘l Example 5 Analysis of g Characteristics of Variants of h-mAb2 Light and Heavy Chains An analysis was conducted to determine the effect of variations in the framework residues of the light chain of mAb2. Table 2 indicates the substitutions studied.

Table 2 42 43 SEQ IDN h-mAb2VL-1 F Q G Q A P R T 16 —_ 18 h—mAb2 VL-3 E - 20 h_mAb2 VL-4 22 —_ 24 —_ 26 h—mAb2 VL-7 E - 28 h_mAb2 VL-8 V E 30 —_ 32 Y V 7 A 36 - 4° V 42 — 44 hVH-6M 72 hVH-7 A V 48 hVH-8L N A E V 55 hVH-8M N A N 74 Antibodies having mAb2 light chains of SEQ ID NO:11, but containing a (Kabat numbered) tution of D4lG, H42Q, L43A, F44P, T45R, or G46T and heavy chains of chimeric mAb2 (CDRs of mAb2 with hFRl-mFR2-hFR3-4) were formed and their binding assessed using a capture ELISA. Plates were coated with l ug/ml of the extracellular domain of human CD3 (soluble hCD3 or “shCD3”) and Vdfl'dIlI VH-OIVI) are p'dITlCUl'dle preIerreu I01" pTOLIUCng 'dIlIlDOLllCS Il'dVC 'd IOWCI" affinity for CD3 than antibodies composed of hVH-l, hVH-2, hVH-3, hVH-4, hVH-S, ted in the presence of various concentrations of dy. The results (Figure 3) indicate that a substitution of T at Kabat position 46 eliminated the ability of the antibody to bind to shCD3.

Additional studies were conducted to assess the impact of variations at Kabat light chain positions 36, 38, 44 and 46. Antibodies were formed having an h-mAb2 VL-8, h-mAb2 VL-9 or h-mAb2 VL-lO light chain and the heavy chain of the mAb2 chimeric antibody and evaluated using the above-described capture ELISA. The results of this experiment are shown in Figure 4, and reveal that the binding to shCD3 by an dy having the hVL-8 light chain was similar to that of an antibody having the chimeric mAb2 light chain.

] Antibodies were also formed having an h-mAb2 VL-6, h-mAb2 VL-7 or h- mAb2 VL-8 light chain and the heavy chain of the mAb2 chimeric antibody and evaluated using the above-described capture ELISA (except that the plates were coated with 0.5 ug/ml of shCD3 in phosphate buffered saline) to determine the impact of additional substitutions at positions 36, 38 and 46. The results of this experiment are shown in Figure 5, and reveal that the Kabat substitutions F36V and T46G were sufficient to yield an antibody whose binding to shCD3 was similar to that of an antibody having the chimeric mAb2 light chain.

The impact of substitutions in the sequence of the heavy chain of mAb2 was ed by forming dies having the light chain of the chimeric mAb2 dy and heavy chain h-mAb2 VH-S, h-mAb2 VH-6 or h-mAb2 VH-7 and ting binding using the above-described capture ELISA (using a l ug/ml coating of shCD3). The results of these investigations are shown in Figure 6. Antibodies were additionally formed having the light chain of the chimeric mAb2 antibody and a humanized variant of heavy chain h-mAb2 VH-4, h-mAb2 VH-7 or h-mAb2 VH-9.

Such antibodies were evaluated for binding using the described capture ELISA.

The results of these investigations are shown in Figure 7.

Heavy chains hVH-6L (and its t, hVH-6M), and hVH-8L (and its variant VH-8M) are particularly preferred for producing antibodies have a lower affinity for CD3 than antibodies composed of hVH-l, hVH-2, hVH-3, hVH-4, hVH-S, Amino acid sequence of hVH-8M (SEQ ID NO:74): «.VOT.V«.SGGG LVOPGGSLRL SCAASGFTFN TYAMNWVROA PGKGLEWVAR hVH-6, hVH-7 or hVH-8 of Table 2. Such reduced affinity antibodies will preferably be composed of either heavy chain hVH-6L or heavy chain VH-8L in combination with any of light chain h-mAb2 VL-l, h-mAb2 VL-2, h-mAb2 VL-3, h-mAb2 VL-4, h-mAb2 VL-S, h-mAb2 VL-6, h-mAb2 VL-7, h-mAb2 VL-8, h-mAb2 VL-9, or h- mAb2 VL-lO. A particularly preferred deimmunized antibody will be composed of heavy chain hVH-6L (or its variant, hVH-6M )and light chain h-mAb2 VL-6, or heavy chain hVH-8L (or its variant, hVH-8M) and light chain h-mAb2 VL-6. The sequences of such polypeptides are presented below: Amino acid sequence of hVH-6L (SEQ ID NO:54): flVQLVflSGGG LVQPGGSHRH SCAASGFTFN VQQA PGKGLEWVG? :RNKY NYAT EYADSVKDQF TISRDDSKNS LYLQMNSHKT EDTAVYYCV? {GNFG SYVS WFAYWGQGT; VTVSS Amino acid sequence of hVH-8L (SEQ ID NO:55): SGGG LVQPGGSHRH SCAASGFTFN TYAMNWVQQA PGKGLEWVA? :RNKY NYAT EYADSVKDRF TISRDDSKNS LYLQMNSHKT YCV? {GNFG SYVS WFAYWGQGT; VTVSS Heavy chains hVH-6L and hVH-8L were further modified to produce variants possessing an asparagine at position 52a (S52aN) modification. The amino acid sequences and corresponding polynucleotide-encoding sequences of these modified heavy chains are as follows: Amino acid sequence of hVH-6M (SEQ ID NO:72): flVQLVflSGGG LVQPGGSHRH FTFN TYAMNWVRQA PGKGLEWVG? IRSKY NYAT EYAASVKDQF TISRDDSKNS LYLQMNSHKT YCV? {GNFG SYVS WFAYWGQGT; VTVSS Polynucleotide Sequence Encoding hVH-6M Variable Heavy Chain (SEQ ID NO:73): gaggtgcagc ,gg,ggach aggc ttggtccagc ctggagggtc ac,c scc,ngcag chc,gga,L cacc,,caac gc,a tgaattgggt ccgccaggct aagg ggc,ggag,g gg,,ggaagg atcaggtcca agtacaacaa s,aLgcaacc gag,a,gccg ac:ctgtgaa gga,agaL,c accatctcaa a,,c aaagaac,ca csg,a,chc aaatgaacag cotgaaaacc gaggacacgg ccg,g,a,,a c,g,g,gaga cacggtaact a,Lc ,achg,cL ngsL,gc,L as,ggggaca ggggacac,g ngach,gL c Amino acid sequence of hVH-8M (SEQ ID NO:74): «.VQLVnSGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVAR 9M di-l and light chain h-mAb2 VL-6, or heavy chain hVH-8L di-2 and light chain h- mAb2 VL-6.

NYAT EYAASVKDRF TISRDDSKNS LYLQMNSLKT EDTAVYYCVR HGNFGNSYVS WFAYWGQGTL VTVSS Polynucleotide ce Encoding hVH-8M Variable Heavy Chain (SEQ ID NO:75): gagg:gcagc ,gg,ggach ,gggggaggc ttgg':ccagc ctggaggg:c cctgagac,c occ,ngcag chc,gga,L cacc,,caac aca,acgc,a tgaa:tgggt ccgccaggct ccagggaagg ggc,ggag,g ggwgcaagg atcaggaaca agtacaacaa ,aLgcaacc gag,a,gccg ac:ctgtgaa gga,agaL,c accatctcaa gaga,ga,oc aaagaac,ca cog,a,chc aaa:gaacag cotgaaaacc gaggacacgg ccg,g,a,,a c,g,g,gaga cacggtaact tcggcaayuc ,achgfiL ngoL,gc,L awggggaca ggggacac,g gL c oLcc Heavy chains hVH-8 di-l and hVH-8 di-2 are ularly preferred for producing antibodies that are less immunogenic than dies composed of hVH-l, hVH-2, hVH-3, hVH-4, hVH-S, hVH-6, hVH7 or hVH-8 of Table 2. Such deimmunized antibodies will preferably be composed of either heavy chain hVH-8 di- 1 or heavy chain hVH-8 di-2 in combination with any of light chain h-mAb2 VL-l, h- mAb2 VL-2, h-mAb2 VL-3, h-mAb2 VL-4, h-mAb2 VL-S, h-mAb2 VL-6, h-mAb2 VL-7, h-mAb2 VL-8, h-mAb2 VL-9, or h-mAb2 VL-lO. A particularly preferred deimmunized antibody will be composed of heavy chain hVH-8 di-l and light chain h-mAb2 VL-6, or heavy chain hVH-8 di-2 and light chain h-mAb2 VL-6.

Amino acid ce of hXR32VH-8 di-l (SEQ ID NO:56): flVQLVflSGGG LVQPGGSLRL SCAASGFTFN VRQA PGKGLEWVA? TRSKA SYTT YYAASVKGRF TISRDDSKNS LYLQMNSHKT ?DTAVYYCAR {GNFG SYVS WFAYWGQGT; VTVSS Amino acid sequence of hXR32VH-8 di-2 (SEQ ID NO:57): flVQLVflSGGG LVQPGGSLRL SCAASGFTFN TYAMNWVRQA PGKGLEWVG? TRSKA SYTT YYAASVKGRF TISRDDSKNS LYLQMNSHKT ?DTAVYYCAR {GNFG SYVS QGT; VTVSS Such deimmunized antibodies will preferably be composed of either heavy chain hVH-8L di-l or heavy chain VH-8L di-2 in combination with any of light chain h-mAb2 VL-l, h-mAb2 VL-2, h-mAb2 VL-3, h-mAb2 VL-4, h-mAb2 VL-S, h-mAb2 VL-6, h-mAb2 VL-7, h-mAb2 VL-8, h-mAb2 VL-9, or h-mAb2 VL-lO. A particularly preferred deimmunized antibody will be composed of heavy chain hVH- 9M di-l and light chain h-mAb2 VL-6, or heavy chain hVH-8L di-2 and light chain h- mAb2 VL-6.

EGNFGNSYVS WFAYWGQGTL VTVSA Additional humanized variants of the mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:7) were also produced. The amino acid sequences of such variants are presented below, with changes from SEQ ID NO:7 indicated in boldface and underlining: Amino Acid Sequence of variant “a” (151T Y52cA) of humanized mAb2 murine monoclonal antibody le heavy chain (SEQ ID NO:76): flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA? EASKA.NYAT YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCVR {GNFE SYVS QGT; VTVSA Amino Acid ce of variant “b” (ISlT N54S) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:77): flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA? ERSKY §YAT YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCV? {GNFG SYVS WFAYWGQGT; VTVSA Amino Acid Sequence of variant “c” (151T A56T) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:78): flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA? ERSKY NYET YYADSVKDRF SQS" LYLQMNNHKT EDTAMYYCVR {GNFG SYVS WFAYWGQGT; VTVSA Amino Acid Sequence of variant “d” (151T Y52cA N54S) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:79): flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA? ERSK§.§YAT YYADSVKDRF TISRDDSQS" NHKT EDTAMYYCV? {GNFG SYVS WFAYWGQGT; VTVSA Amino Acid Sequence of variant “e” (151T N54S A56T) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:80): flVKLLflSGGG SHKH SCAASGFTFN TYAMNWVRQA PGKGLEWVA? ERSKY §YET KDRF TISRDDSQS" LYLQMNNHKT YCV? {GNFG SYVS WFAYWGQGT; VTVSA ] Amino Acid Sequence of variant “f” (151T Y52cA N54S A56T) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID flVKLLflSGGG LVQPKGSHKH SCAASGFTFN TYAMNWVQQA PGKGLEWVA? ERSK§.§YET YYADSVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCV? {GNFG SYVS WFAYWGQGT; VTVSA Amino Acid Sequence of variant “9” (151T D6lA) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:82): SGGG LVQPKGSHKH SCAASGFTFN VQQA WVA? ERSKY NYAT YYAESVKDRF TISRDDSQS" LYLQMNNHKT EDTAMYYCVR {GNFG SYVS WFAYWGQGT; VTVSA Amino Acid Sequence of t “h” (151T D65G) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:83): flVKLLflSGGG LVQPKGSLK; SCAASGFTFN TYAMNWVQQA PGKGLEWVA? ERSKY NYAT YYADSVKERF TISRDDSQS" LYLQMNNHKT EDTAMYYCV? {GNFG SYVS WFAYWGQGT; VTVSA Amino Acid Sequence of variant “i” (ISlT Y52cA N54S D6lA) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:84): nVKLLnSGGG LVQPKGsnKn SCAASGFTFN TYAMNWVRQA WVAR ERSK§_§YAT YYAgSVKJRF TISRDDSQS" LYLQMNNnKT ?DTAMYYCVR {GNFG syvs WFAYWGQGT; VTVSA Amino Acid Sequence of variant “j” (ISlT Y52cA N54S D65G) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:85): SGGG LVQPKGSLK; SCAASGFTFN TYAMNWVRQA PGKGLEWVAR ERSKA.§YAT YYADSVKERF TISRDDSQS" NHKT EDTAMYYCV? {GNFE SYVS WFAYWGQGT; VTVSA ] Amino Acid Sequence of variant “k” (ISlT Y52cA N54S D6lA D65G) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:86): nVKLLnSGGG LVQPKGSLK; SCAASGFTFN TYAMNWVRQA PGKGLEWVAR ERSK§_§YAT YYAgSVKgRF TISRDDSQS" LYLQMNNnKT ?DTAMYYCVR {GNFG syvs WFAYWGQGT; VTVSA Amino Acid Sequence of variant “2k” (ISlT Y52cA N54S D6lA D65G (VH8-A49G V93A)) of humanized mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:87): nVQLVnSGGG LVQPGGSLR; FTFE VRQA PGKGLEWVGR ERSK§_§YTT YYAgSVKgaF TISRDDSKNS LYLQMNsnKT YCAR {GNFG syvs WFAYWGQGT; VTVSS ALVA; AAWAAAWAA way, ‘4 v “‘4‘va vvwv I’VLAVLAAAvw. A vavv nvxv vvwvvw "AVA; A r4101 ALL; of the extracellular domain of CD3 (soluble CD3) (either human or cynomolgus Amino Acid Sequence of variant “5k” (151T Y52cA N54S D6lA D65G (VH8-V93A)) of zed mAb2 murine monoclonal antibody variable heavy chain (SEQ ID NO:88): +.VQT.V«.SGGG LVQPGGSLR; SCAASGFTFE VRQA PGKGLEWVER gRSKg §YTT YYAgSVKgaF TISRDDSKNS T.YT.QMNS'.KT '«TDTAVYYCAR {GNFG syvs WFAYWGQGT; VTVSS All such additional humanized variants of the mAb2 murine monoclonal antibody variable heavy chain may be employed to form the deimmunized dies of the present invention. Such additional deimmunized and humanized antibodies will preferably be composed of any of heavy chains: a, b, c, d, e, f, g, h, i, j, k, 2k or 5k, in ation with any of light chain: h-mAb2 VL-l, h-mAb2 VL-2, h-mAb2 VL-3, h-mAb2 VL-4, h-mAb2 VL-S, h-mAb2 VL-6, h-mAb2 VL-7, h-mAb2 VL-8, h-mAb2 VL-9, or h-mAb2 VL-lO. A particularly preferred deimmunized antibody will be composed of heavy chain 2k or 5k and light chain h-mAb2 VL-6, or heavy chain hVH-8M8L di-2 and light chain h-mAb2 VL-6. Variants 2k and 5k bind to Protein A in the variable region, thus facilitating the purification of molecules (such as diabodies) that may lack Fc regions or other domains that may be used to sequester such molecules from other molecules. ts hVH-8M, hVH-8L. hVH-6M and hVH-6L exhibit reduced immunogenicity relative to their respective parental polypeptides.

The invention particularly ns nized and humanized antibodies composed of heavy chain hVH-8 and light chain VL-6. The invention additionally particularly concerns deimmunized and humanized antibodies composed of heavy chain hVH-4 and light chain VL-6. The ion additionally particularly concerns deimmunized and humanized dies composed of heavy chain hVH-2k and light chain VL-6 Example 6 Analysis of Binding teristics of Variants of Chimeric and Humanized mAb2 Light and Heavy Chains In order to assess the ability of chimeric and humanized mAb2 to bind to non-human CD3, a capture ELISA was performed. Plates were coated with l ug/ml of the extracellular domain of CD3 (soluble CD3) (either human or cynomolgus receptor (EGFR) M diabody “ERBITUXTM-h-mAbZ”). monkey) and incubated in the presence of various concentrations of antibody. The results of this experiment are shown in s 8A and 8B, and reveal that mAb2 and its humanized variant exhibited equivalent g to soluble human CD3 and to soluble cynomolgus monkey CD3.

Example 7 Quantitation of g of mAbZ to Human and Cynomolgus Monkey CD3 In order to quantitate the extent of binding between mAb2 and human or cynomolgus monkey CD3, BIACORETM analyses were conducted. BIACORETM es measure the dissociation off-rate, kd. The binding affinity (KD) between an antibody and its target is a fimction of the kinetic constants for association (on rate, ka) and dissociation (off-rate, kd ) according to KD= kd/ka. The BIACORETM is uses surface plasmon resonance to directly measure these kinetic ters. Anti- CD3 antibody mAb2 (6.3-100 nM) was immobilized to a support using anti-EK antibodies and incubated in the presence of soluble human CD3 (shCD3) or soluble cynomolgus monkey CD3 (scCD3). The time course of dissociation was measured and a bivalent fit of the data conducted. The results of the BIACORETM es are shown in s 9A-9D. The kinetic data is summarized in Table 3.

Table 3 Antibod ka kd ch-mAb2 1.7 x 10 M" sec" 2.5 x 10" sec" -1 -1 -3 -1 n10 a d ch-mAb2 1.6 x 10 M" sec" 2.3 x 10" sec" h-mAb2 1.7 x 10 M" sec" 4.1 x 10" sec" Example 8 Bispecific Binding Data for DARTTM Diabodies Containing CDRs of h-mAb2 The CDRs of humanized mAb2 2) were used to produce a series of DARTTM diabodies having an anti-CD3 first epitope binding site and a second epitope binding site capable of binding to Her2/neu (DARTTM diabody h-mAb2”), or to CD19 (DARTTM diabody “CD19-h-mAb2”) or to the epidermal grth factor receptor (EGFR) (DARTTM diabody “ERBITUXTM-h-mAbZ”).

/ \ \ / QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TS Q KPGQAPRGLT GGTNKRAPWT SLLG GKAALTT TGA OAfl D-I.ADYYC ALWYSNITWVF eu-h-mAb2 DARTTM Diabody Amino acid sequence of the hXR32VL-Her—2VH E coil of the Her2-h-mAb2 DARTTM diabody (the linkers between the hXR32VL sequence and the Her2VH sequence and between the Her2VH ce and the E coil ce are underlined) (SEQ ID NO:58): QAVVTQITPSL TVSPGGTVTL TCRSSTGAVT TS YANWVQQ KPGQAPRGLT GGTNKRAPWT PAQFSGSH G GKAALTT TGA QA*I D.ADYYC ALWYSNITWVF GGGTKITTVLG GGGSGGGGQV QLQQSGPT IV KPGASLKLSC TASGFNT KDT YT HWVKQKP. QGIITIW G? Y PTNGYTRY DP KFQ D<ATT TA DTSSNTAYI4Q VSRLTST4 DTA VYYCSRWGG D GFYAMDYWGQ GASVTVSSGG CGGGKVAATK TKVAAI I<TKV AAI<ITKVAAII KT Amino acid sequence of the Her2VL-hXR32VH-K coil of the Her2-h-mAb2 DARTTM diabody (the linkers between the Her2VL sequence and the hXR32VH sequence and between the hXR32VH sequence and the K coil sequence are underlined) (SEQ ID NO:59): DTV TQS {KF MSTSVGDRVS TTCKASQDVN TAVAWYQQKP GHSPKLLTYS ASFQYTGVP D RFTGNRSGT D SVQA ADLAVYYCQQ HYTTPPTFGG GTKIIT. KRAG GGSGGGGTVQ LVTSGGGHVQ PGGSI I? ISCA ASGFTFNTYA MNWVRQAPG < GLTWVA? RS KY NYATYYA DSVK jQFTT S RDDSKNSLYL QMNS I<TTDT AVYYCVRTG FG SYVSWFA TVTV SSGGCGGGTVI AATI*.<*.VAAH TKTVAAI ITKT VAALTK CD19-h-mAb2 DARTTM y Amino acid sequence of the CDl9VL-hXR32VH-E coil of the CDl9-h- mAbZ DARTTM diabody (the linkers between the CDl9VL sequence and the hXR32VH sequence and between the hXR32VH sequence and the E coil sequence are underlined) (SEQ ID NO:60): QSPAS QRAT TSCKASQSVD YDGDSYL WY QQTPGQPPKT IITYDAS IVS GTPPRFSGSG SGTDhTLN { PVTKVDAATY ITDPW TFGGGT<U*I {GGGSGGGGIT VQLVTSGGGU VQPGGSIKIS CAASGFTFNT YAM WVRQAP GKGTITWVAR RSKY NYATY YADSVKDRFT TSRDDSKNST YLQ NSII<TIT DTAVYYCVRT G FG SYVSW FAYWGQGTTV TVSSGGCGGG TVAALTKTVA ALTKTVAAHT KflVAALflK Amino acid sequence of the hXR32VL-CD19VH-K coil of the CDl9-h- mAbZ DARTTM diabody (the linkers between the hXR32VL ce and the CDl9VH sequence and between the CDl9VH sequence and the K coil sequence are ined) (SEQ ID NO:61): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TS YANWVQQ KPGQAPRGLT APWT PARFSGSLLG GKAALTT TGA QA dlD-I.ADYYC ALWYSNITWVF GGGT<LTVLG GGGSGGGGQV AELV RPGSSVKISC KASGYAFSSY QQPG QGTIflW GQ W PGDGDTNYNG KFKGKATLTA DESSSTAYMQ LSSLAS_EDSA VYFCARQETT YAMD YWGQGTTVTV GGKV AATIKTKVAATI KTKVAAI<TK VAAIKT ERBITUXTM-h-mAb2 DARTTM Diabody Amino acid sequence of the hXR32VL-EGFRVH-E coil of the ERBITUXTM-h-mAbZ DARTTM diabody (the linkers between the hXR32VL sequence and the EGFRVH sequence and between the EGFRVH sequence and the E coil sequence are underlined) (SEQ ID NO:62): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TS YANWVQQ KPGQAPRGLL GGTNKRAPWT PAQFSGSLLG GKAALT: TGA QATIDIADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QL<QSGPGLV QPSQSLSL TC TVSGFSLT Y GVHWVQQSPG KGHflWTGV W SGGNTDYNTP NKD FF<M SLQS DTA__ YYCARALTYY DYEFAYWGQG TLVTVSSGGE EGGflVAALnK IVAATflKflVA AALn K Amino acid sequence of the EGFRVL-hXR32VH-K coil of the ERBITUXTM-h-mAbZ DARTTM diabody (the linkers between the EGFRVL sequence and the hXR32VH sequence and between the hXR32VH sequence and the K coil sequence are underlined) (SEQ ID NO:63): D"H'ITQSPV" HSVSPGERVS FSCRASQS G TN {WYQQRT NGSPRHL"KY ASflS SG PS KESGSGSGTD hTTIS NSVflS *ID ADYYCQQ NNNWPTTFGA GT<'IT IKGGG VQLV TSGGGTIVQPG SCAAS GFTFNTYAMN WVRQAPGKGH flWVAK RSKY NYATYYADS VKDRFTLSQD DSKNSLYLQM NSH<TTDTAV YYCVRTGNFG AYW GQGTJVTVSS GGCGGGKVAA LKT<VAALKT <VAAH<TKVA ALK_3 Such DARTTM diabodies were found to be capable of binding to cynoniolgus monkey CD3 (Figures 10A—10C).

The CDRs of humanized mAb2 (h-mAb2) were used to produce a fiarther series of DARTTM diabodies having an anti-CD3 first epitope binding site and a second e binding site capable of binding to B7-H3 (DARTTM diabody “B7-H3- b2” and B7-H3h-n1Ab2”).

B7-H3h-mAb2 DARTTM Diabody Amino acid sequence of the hBRCA69DVL-hXR32VH-E coil of the B7-H3- l-h-mAb2 DARTTM diabody (the linkers between the hBRCA69DVL sequence and LJL‘ULLLHJHHLLL y LLVvaxvLuLL‘ L.4_4_\_; ULHLHL w vaLL y VKJKJ \vavvv T.KITKVAALKT KVAALKEKVA ALKE the hXR32VH sequence and between the hXR32VH sequence and the E coil sequence are underlined) (SEQ ID NO:64): DT Q TQSPSS LSASVG DRVT TTCRASQDTS YLNWYQQKP F .TYY GVPSJ RFSGSGSGTD FTLTT SSUQP LD ATYYCQQ GNTLPPTFGG GT (UL <GGG TVQL VTSGGGLVQP GGS.¥ .SCAA SGFTFNTYAM NWVRQAPGKGI ASK Y AD SVK DRFT IISR DDSKNSLYLQ MNSH (TTDTA VYYCV {G F G SYVSWFAY WGQGTLVTVS SGGCGGIGLVA AL L<LVAAL. <. *KLV AAT. -—uLK Amino acid sequence of the hXR32VL-hBRCA69DVH-K coil of the B7- H3-l-h-n1Ab2 DARTTM diabody (the linkers between the hXR32VL sequence and the hBRCA69DVH sequence and between the hBRCA69DVH sequence and the K coil sequence are underlined) (SEQ ID NO:65): QAVVTQEPSL TVSPGGTVTL TCKSSTGAVT TS YANWVQQ KPGQAPRGLT APWT RFSGSLLG G<AAI .TIITGA QA LD LADYYC ALWYSNILWVF GGGTKLTVLG QILVQSGAILVK KPGASVKVSC FTSY WMQWVQQAPG PGDG DTRYTQ KF I TA DKSTSTAYMI*_‘4 USSTRSITDTA R .WYFDVWGQ GTTVTVSSGG CGGGKVAAL < TKVAAI. (TKV KI_‘_.

B7-H3h-mAb2 DARTTM Diabody Amino acid sequence of the hBRCA84DVL—hXR32VH-E coil of the B7-H3- 2-h-n1Ab2 DARTTM diabody (the linkers between the hBRCA84DVL sequence and the hXR32VH sequence and between the hXR32VH ce and the E coil ce are ined) (SEQ ID NO:66): DL Q' .TQSPSF LSASVG DRVT TTCKASQNV D TNVAWYQQKP I .TYS ASYQYSGVPS RFSGSGSGTD FTTTT SSTQP TDFATYYCQQ YNNYPFTFGQ GT <I.L {GGG GSGGGGITVQI. VITSGGGTVQP GGSI.¥ .SCAA SGFTFNTYAM NWVRQAPGKG ImWVA? QS< Y NYATYYA D SVK DRFT IISR DDSKNSLYLQ TTDTA VYYCVK {G F G SYVSWFAY WGQGTLVTVS SGGCGGGLVA AL L<LVAALL (LVAAH *KLV AAT.TK Amino acid sequence of the hXR32VL-hBRCA84DVH-K coil of the B7- H3h-n1Ab2 DARTTM diabody (the linkers between the hXR32VL sequence and the hBRCA84DVH sequence and between the hBRCA84DVH sequence and the K coil sequence are underlined) (SEQ ID NO:67): QAVVTQEPSL TVSPGGTVTL TCRSSTGAVT TS YANWVQQ KPGQAPRGHT GGTNKKAPWT PAQFSGSH .G G<AALTII TGA QA LD LA DYYC ALWYSNILWVF JTVLG GGGSGGGGTV QI.VITSGGGTIV QPGGSLQLSC AASGFTFSSF GM {WVQQAPG KGI: LWVAY S SDSSAIIYYAD TV {GAFTT SR DNAKNSI .Y .Q MNSLRDIL DTA VYYCGRGKLN YYGSRLDYW GQGTTVTVSS GGCGGG<VAA LKITKVAAI .KT KVAALKIL<VA AI .KT PGLICIILD lclllalll uuucr LIIC balC UL L11C11 PCIDUIIGI Pllyblblallb uuuug LIIC qulDC UL LIIC study.

Such DARTTM diabodies were found to be e of binding to soluble cynomolgus monkey CD3 (Figure 10D).

Dual Affinity Retargeting Reagents (DARTTMs) Diabodies Specific For HER2/neu and CD3 Mediate Potent Redirected T-Cell Killing Dual affinity retargeting reagent (DARTTM) diabodies c for HER2/neu and CD3 are prepared. Such DARTTM diabodies have the ability to localize a T-cell (by binding such T-cell to the nding portion of a CD3-binding DARTTM diabody) to the on of a tumor cell (by binding such cancer cell to the HER2/neu- binding portion of the DARTTM diabody). The localized T-cell can then mediate the killing of the tumor cell in a process termed herein “redirected” killing.

The dual affinity eting reagent (DARTTM) diabody specific for HER2/neu and CD3 is constructed having the anti-HER2/neu variable domains of trastuzumab and anti-CD3 variable domains of h-mab2 VH-8 and h-mab2 VL-6 (SEQ ID NOS: 58-59).

In order to demonstrate the ability of DARTTM diabodies to mediate such redirected killing of cancer cells, the above-described HER2/neu x CD3 DARTTM diabody is 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 effector resting PBMC (E:T ratio = 30:1) and cytotoxicity is determined (LDH Assay). The results of these investigations demonstrate the ability of the HER2/neu x CD3 DARTTM diabody to mediate redirected killing of tumor cells.

Example 10 Anti- TCR Monoclonal dy y for Patients with Autoimmune Diabetes Patients: Forty patients with Type 1 diabetes are recruited for participation according to the ing criteria: between 7 and 20 years of age, within 6 weeks of diagnosis according to the American Diabetes Association criteria, and confirmation of the presence of anti-GAD65, anti-ICAS 12, and/or anti-insulin autoantibodies. The patients remain under the care of their al physicians during the course of the study.

Annunnunnu ALL kuv u; LLULLLLWL wavvuv wVLvaLLVV. L wvaLqu “AV “Lvavvu uv anvv w LLULLLLWL diet, and remain under the care of their personal physician throughout the duration of ] Eligible patients are randomly assigned to a control group and a humanized anti- CD3 antibody ) (comprising h-mab2 VH-8 and h-mab2 VL-6) treatment group. After randomization, blood samples are drawn to establish baseline HAlc levels, a pretreatment ide response to a MMTT is established and a pretreatment FPIR to IGTT is performed. Patients in both groups are hospitalized to receive either a 6-day course treatment of the zed anti- CD3 monoclonal antibody (N297Q) or placebo. The antibody is administered intravenously in the following dosage: 17 ug/m2 on day 1, 34.3 ug/m2 on day 2, 69 ug/m2 on day 3, 137.6 ug/m2 on day 4, and 275.3 ug/m2 on days 5 and 6. Alternatively, antibody may be administered intravenously in the following dosage: 1.6 ug/kg/day on day 1; 3.2 ug/kg/day on day 2; 6.5 ug/kg/day on day 3; 13 ug/kg/day on day 4; and 26 ug/kg/day on days 5 through 14. In dose escalation studies, the treatment may be, e.g., 1.42 ug/kg/day on day l; 5.7 ug/kg/day on day 2; 11 ug/kg/day on day 3; 26 ug/kg/day on day 4; and 45.4 ug/kg/day on days 5 through 14. In subsequent studies, the therapy is altered to increase dosage and/or decrease the time course of treatment.

For example, in subsequent studies patients may be stered a 4 day treatment: 6.4 ug/kg/day on day l; 13 ug/kg/day on day 2, and 26 ug/kg/day on days 3 and 4.; during additional dose escalation studies, the treatment may be 8 ug/kg/day on day 1; 16 day on day 2; and 32 ug/kg/day on days 3 and 4.

During initial studies the antibody dosage on the first three days of treatment is stered via slow infusion IV over 20 hours to r for e reactions.

Subsequent studies will decrease the time of administration and/or split the dosage into 2 to 4 equal parts to be administered as bolus injections evenly distributed over the course of 12 hours. Patients in the control group undergo metabolic and immunologic tests but do not receive monoclonal dies. Patients are monitored throughout the study for immunosuppressive effects of the anti- CD3 monoclonal antibody (N297Q). ts are monitored for 18 months after the treatment. B-cell fianction is determined every 6 months in the case of ed glucose nce and every 12 months in case of normal glucose tolerance. Patients are allowed to have a normal diet, and remain under the care of their personal physician throughout the duration of the study. Immunological assays are repeated in intervals of 6 months. Insulin therapy will be given to the patients as directed by their personal physician.

B-cell filnction will be analyzed according to the changes of the C-peptide levels as measured by radioimmunoassay. After drawing samples for baseline C- peptide and glucose, the patients are given a mixed meal. The C-peptide levels are measured in samples drawn after 15, 30, 60, 90, 120, 150, 180, 210, and 240 min. The C-peptide response to the mixed-meal tolerance test (MMTT) is expressed as the total area under the response curve (AUC). A change in the se is considered to have occurred if the response differs by more than 7.5 percent from the response at study entry. The patients’ C-peptide ses to MMTT are continuously monitored 6 months, 9 months, 12 months, 15 months and 18 months after the ent.

Alternatively, the B-cell fianction is assessed by FPIR to IGTT. Serum insulin levels are measured by a modification of a double-antibody radioimmunoassay method using monoiodinated tyrosine Al4—labeled insulin ham Pharmacia). FPIR is calculated as the sum of insulin levels at l and 3 minutes after a glucose load (0.5 g/kg). Glycosylated obin levels are measured by latex-agglutination inhibition test.

Immunological Monitoring: The level of autoantibodies against GAD65, A512, and insulin are measured with radiobinding assays as known in the art (e.g., Woo et al., 2000, J. Immunol s 244:91-103). A and HLADQB genotyping are performed by direct sequencing of exon 2 polymorphisims after PCR amplification. The level of cytokines in serum after the administration of the monoclonal antibody is ed by enzyme-linked sorbent assay (ELISA).

Production of anti-idotype dies is monitored by ELISA assay using a plate bound anti-CD3 ) or by flow cytometry to measure blockade of binding of anti-CD3-FITC to the CD3 chain of TCR.

Statistical Analysis: Data analysis will be conducted on residual beta-cell function, autoantibody level, cytokine level, and glycosylated hemoglobin level. A x2 analysis will be performed to test the effect of drug treatment before and after drug ULL U1 u Lu111u1 UULL \U)’ U111u1115 ouu11 Uu11uv1 UULL LU LLLU 1.1/11.) U111u1115 PU1 L1U11 U1 administration. Comparison between the control group and the treatment group will be made with the Mann-Whitney U test.

Example 1 1 Dual Affinity Retargeting Reagents (DARTTMs) Diabodies Specific for B7H3 and CD3 Mediate Potent Redirected T-Cell Killing Dual affinity retargeting reagents (DARTTM) diabodies specific for the B7H3 antigen and CD3 were prepared. B7H3 has been histologically detected in tumor cell lines (Chapoval, A. et al. (2001) .‘ A Costimulatory Molecule For T Cell Activation and IFN—y Production,” Nature Immunol. 274; Saatian, B. et al. (2004) “Expression 0f Genes For B7-H3 And Other T Cell Ligands By Nasal lial Cells During Difi’erentiation And Activation,” Amer. J. Physiol. Lung Cell.

Mol. Physiol. 287:L217—L225; Castriconi et al. (2004) “Identification 0f 4Ig-B7-H3 As A Neuroblastoma-Associated le That Exerts A tive Role From An NK Cell-Mediated Lysis,” Proc. Natl. Acad. Sci. (USA) lOl(34):l2640-l2645); 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 t a marked increase in expression of B7-H3 protein and that this increased expression was associated with sed disease severity (Zang, X. et al. (2007) “The B7 Family And Cancer Therapy: Costimulation And Coinhibition,” Clin.

Cancer Res. 13:5271-5279), suggesting that B7-H3 is exploited by tumors as an immune evasion pathway (Hofmeyer, K. et al. (2008) “The Contrasting Role 0fB7— H3,” Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278).

The CD3 binding portion of such DARTTM diabodies was composed of the above-described light and heavy variable regions of humanized anti-CD3 mAb2 (h- mAb2 VH-8 and h-mAb2 VL-6). The B7H3 portion of such DARTTM diabodies was composed of 4D-2 Light Chain and hBRCA84D-2 Heavy Chain (SEQ ID NOS. 64-65).

Such DARTTM diabodies have the ability to localize a T-cell (by binding such T-cell to the CD3-binding portion of a CD3-binding DARTTM diabody) to the location of a tumor cell (by binding such cancer cell to the B7H3 binding portion of lU‘I- the DARTTM diabody). The localized T-cell can then mediate the killing of the tumor cell Via the s of ected” killing.

In order to demonstrate the ability of such DARTTM diabodies to mediate such redirected killing of cancer cells, the DARTTM diabody was incubated at various concentrations with target tumor cells (A498 tumor cells, RECA905021E tumor cells) and effector resting PBMC (E:T ratio = 30:1) and cytotoxicity was ined (LDH Assay). A DARTTM y (4420-h-mAb2) haVing dual specificity for CD3 (h- mAb2) and fluorescein (antibody 4420) was employed as a control. 4420-h-mAb2 DARTTM Diabody Amino acid sequence of the 4420VL-hXR32VH-E coil of the 4420-h-mAb2 DARTTM diabody (the linkers between the 4420VL ce and the hXR32VH sequence and between the hXR32VH sequence and the E coil sequence are underlined) (SEQ ID NO:68): DVVMTQTPFS LPVS_JGDQAS :SCRSSQSLV HSNGNTY_JRW YLQKPGQSPK VLIYKVS RF SGVPDQFSGS GSGTDhTLK SRV*A*IDIGV THVP WTFGGGT<Ifl KGGGSGGGG *IVQUVISGGG LVQPGGSIRU SCAASGFTFN TYAMNWVRQA PGKGHIWVAR __RS<Y NYAT YYADSVKDRF TISRDDSKNS LYLQMNSH<T YCVR {G FG SYVS WFAYWGQGTL VTVSSGGCGG GfiVAALn<flV AALfl<flVAAH flKIVAALnK Amino acid sequence of the hXR32VL-4420VH-K coil of the 4420-h-mAb2 DARTTM diabody (the s between the L sequence and the 4420VH sequence and between the 4420VH sequence and the K coil sequence are underlined) (SEQ ID NO:69): QAVVTQ?PSH TVSPGGTVT; TCQSSTGAVT TS YANWVQQ KPGQAPRGH" GGTNKRAPWT PARFSGSLLG GKAALT: TGA QA*IDflADYYC A'IWYSNHWVF TVUG GGGSGGGG?V KTIDTTGGGTIV QPGQPM<LSC VASGFTFSDY WMNWVRQSPfl KGHflWVAQ R NKPYNYETYY SDSV<GRFTI SRDDSKSSVY TIQMNN IRV*ID MG YYCTGSY YG DYWGQGT SVTVSSGGCG GGKVAAIKTK VAALKEKVAA LK?<VAAL<? ] The results of these investigations (Figures B) demonstrate the ability of the B7H3 x CD3 DARTTM diabodies to mediate redirected killing of tumor cells expressing B7H3.

RECA47VH sequence and between the RECA47VH sequence and the E coil Example 12 Dual Affinity Retargeting Reagents (DARTTMs) Diabodies Specific For A33 and CD3 e Potent Redirected T-Cell Killing Dual affinity retargeting ts (DARTTM) diabodies specific for the A33 antigen and CD3 (“A33-h-mAb2” DARTTM diabody) were prepared. A33 is a membrane antigen that is expressed in normal human colonic and small bowel epithelium and >95% of human colon cancers (Heath, J.K. et al. (1997) “The Human A33 Antigen Is A Transmembrane Glycoprotez'n And A Novel Member Of The Immunoglobulz’n Supely’amily,” Proc. Natl. Acad. Sci. (USA) 94:469-474).

Such DARTTM diabodies have the ability to localize a T-cell (by binding such T-cell to the CD3-binding portion of a CD3-binding DARTTM diabody) to the location of a tumor cell (by binding such cancer cell to the A33 binding portion of the DARTTM diabody). The localized T-cell can then mediate the g of the tumor cell Via the s of “redirected” g.

The CD3 binding n of such DARTTM diabodies was composed of the described light and heavy variable regions of humanized mAb2 (h-mAb2 VH-8 and h-mAb2 VL-6). The A33 portion of such DARTTM diabodies was composed of antibody RECA47.

A33-h-mAb2 DARTTM Diabody Amino acid ce of the RECA47VL-hXR32VH-K coil of the A33-h- mAb2 DARTTM diabody (the linkers between the VL sequence and the hXR32VH sequence and n the hXR32VH sequence and the K coil sequence are underlined) (SEQ ID NO:70): Q"VHTQSPA" SASPGERVT TCSARSSIS FMYWYQQKPG "YDT SNLASGVPV? FSGSGSGTSY SET SQMflAfl DAATYYCQQW SSYPLTFGSG TKHEL<RGGG SGGGGEVQLV ESGGGUVQPG GSUQHSCAAS GFTFNTYAMN WVRQAPGKGH flWVAR QSKY NYATYYADS VKDQFTISRD DSKNSLYLQM NSH<TEDTAV YYCVRiGNFG SYVSWFAYW GQGTLVTVSS GGCGGGKVAA LK?<VAALK? <VAAH<EKVA ALKE Amino acid sequence of the hXR32VL-RECA47VH-E coil of the A33-h- mAb2 DARTTM y (the linkers between the hXR32VL sequence and the RECA47VH sequence and between the RECA47VH sequence and the E coil sequence are underlined) (SEQ ID NO:71): Molecular Pathogenesis l o New Y herapeutic Perspectives,” Haematologica 91 :1 1-16; Jeon, H]. et al. (1998) “Establishment And Characterization Of A Mantle Cell EPSL TVSPGGTVTL GAVT TS Q RGL: APWT PARFSGSLLG GKAAHT"TGA QAflDflADYYC ALWYSNLWVF GGGTKLTVLG GGGSGGGGQV QLQQS PELV KPGASVKISC KASGYTFSGS WMNWV<QRPG QGHflW G? Y PGDGETNYNG KF<D<ATLTA DKSSTTAY E LSSLTSVDSA IYG NVYFDVWGAG TTVTVSSEEE EggflVAALn< flVAALflKflVA ALn<flVAALn K In order to demonstrate the ability of such DARTTM diabodies to mediate such redirected killing of cancer cells, the DARTTM diabody was incubated at various concentrations with target tumor cells (Colo205 tumor cells, RECA905021E tumor cells) and effector resting PBMC (E:T ratio = 30:1) and cytotoxicity was determined (LDH Assay). The results of these investigations (Figures 12A-12E) trate the ability of the A33 x CD3 DARTTM ies to mediate redirected killing of tumor cells expressing A33.

Example 13 Dual Affinity Retargeting ts (DARTTMs) ies Specific For CD3 Cause Redirected T-Cell-Mediated Killing Equivalent to That of Other Human-Specific CD3 Diabodies In order to further assess the CD3-specific dual affinity retargeting reagents (DARTTM) ies of the present invention, the capacity of the above-described CD19-h-mAb2 DARTTM diabody to cause redirected T-cell-mediated killing was compared to that of the CD19 x CD3 DART diabody of Moore, RA. et al. (2011) (“Application OfDual y Retargeting Molecules To e Optimal Redirected T-Cell g 0f B-Cell Lymphoma,” Blood 117(17):4542-4551). The CD19-h- mAb2 DARTTM diabody exhibits specificity to human as well as non-human CD3; the CD19 x CD3 DART diabody of Moore, RA. et al. (2011)) exhibits specificity only to 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):305-3l6; Amdt, R. (1984) “Demonstration 0f C3-Binding Circulating Immune Complexes Using Raji, Conglutinin And Anti-C3 Assays--A Critical Review,” Immun. Infekt. 12(1):3-11) or JeKo-l human mantle cell lymphoma cells (Salaverria, I. et al. (2006) “Mantle Cell Lymphoma: From Pathology And Molecular Pathogenesis To New Therapeutic Perspectives,” Haematologica 91 :1 1-16; Jeon, H]. et al. (1998) “Establishment And Characterization Of A Mantle Cell cytolysis only in the presence of cynolmolgus monkey s.

Lymphoma Cell Line,” Br. J. Haematol. 102(5):1323-1326) were incubated in the presence of a DARTTM y and resting eral blood mononuclear cells (PBMC) (E:T = 30:1). The results of this experiment (Figures 13A and 13B) revealed that the CD19-h-mAb2 DARTTM y of the present invention cause redirected T-cell-mediated killing that was equivalent to that observed using a CD19 x CD3 DART diabody specific for human CD3. Thus the extension of specificity to non-human CD3 holologs did not impair the y of the DARTTM diabody to e redirected killing.

Example 14 Redirected Cytolysis by Cynomolgus Monkey Cross Reactive Dual Affinity Retargeting Reagents (DARTTMs) ies Specific For CD3 The ability of the above-described CD19-h-mAb2 DARTTM diabody to cause redirected -mediated killing in the presence of either human or cynolmolgus monkey was investigated.

HT-29 human colon cancer cells (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 cynolmolgus monkey T-cells (E:T ratio = 30:1) and either the above-described CD19-h-mAb2 DARTTM diabody or a CD19 x CD3 DART diabody whose CD3 sequences were derived from antibody FN- 18. Antibody FN—18 exhibits specificity only to cynolmolgus monkey CD3 (Nooij, F.J. et al. (1986) “Difi’erentiation Antigens 0n Rhesus Monkey Lymphocytes. I.

Identification Of T Cells Bearing CD3 And CD8, And OfA Subset 0f CD8-Bearing Cells,” Eur. J. Immunol. 16(8):975-979; Meng, G. et al. (1998) “The Efi’ect OfAnti- munotoxin 0n T Lymphocyte Function in vitro,” Transpl. Immunol. 3- 59). The resultant percent cytotoxicity as a function of diabody concentration was measured. The results (Figures 14A and 14B) show that the CD19-h-mAb2 DARTTM diabody was able to mediate cytolysis in the ce of either human or non-human T-cell effector cells. In contrast, the FN-18 diabody was capable of mediating cytolysis only in the presence of cynolmolgus monkey T-cells.

U1 “11 Llwll Uxx _1_\_/\/11 L\UUV1}LU1 1J1 x1\1 uluuuu)’ \Uutjuulv UL Ulllullls LU LUL 1\ and the T-cell receptor). The ERBITUXTM-FNlS CD3 DARTTM y (capable of Example 15 Dual Affinity eting Reagents (DARTTMs) Diabodies Require Target Cell Engagement In order to demonstrate that the observed redirected g mediated by the CD3 DARTTM diabodies of the present invention was specific, the extent of g in the presence and absence of target cells was determined.

Human PMBCs were ted in the presence of the above-described ERBITUXTM-h-rnAbZ DARTTM y, an ERBITUXTM-T-Cell Receptor DARTTM diabody (capable of binding to EGFR (Epidermal Growth Factor Receptor) and the T- cell receptor), or an ERBITUXTM-FNlS CD3 DARTTM diabody (capable of binding to EGFR and to cynolmolgus monkey CD3). The incubations were conducted in the presence or absence of A498 kidney cancer target cells (Giard, DJ. et al. (1973) “in vitro Cultivation Of Human Tumors.‘ Establishment 0f 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 0n Kidney, Testis And Bladder Tumors,” Natl. Cancer Inst. Monogr. 49:5- The CD69 glycoprotein is an early activation antigen of T and B lymphocytes that is expressed on cells of most hematopoietic lineages, ing phils after stimulation (Atzenia, F. et al. (2002) “Induction 0fCD69 tion Molecule 0n Human Neutrophils by GM-CSF, IFN-y, and IFN-a,” Cellular Immunol. 220(1): 20-29). The CD69 Mean Fluorescent Intensity (MFI) was therefore measured (as a function of diabody concentration) as a means for assessing immune system activation (see, e. g., Ampel, N.M. et al. (2002) “In Vitro Whole-Blood Analysis of Cellular Immunity in Patients with Active Coccidioidomycosis by Using the Antigen Preparation T2 7K,” Clinical Diagnostic Laboratory Immunology 9(5): 1039-1043).

] The results es 15A and 15B) show that immune system activation (as measured by the MFI of CD69) increased only when CD4+ or CD8+ T cells were incubated with the ERBITUXTM-h-mAbZ DARTTM diabody of the present ion or an ERBITUXTM-T-Cell Receptor DARTTM diabody (capable of binding to EGFR and the T-cell receptor). The ERBITUXTM-FNlS CD3 DARTTM diabody (capable of adaptations 01 I116 111V611I1011 IOllOWlng, 111 general, I116 1311116113168 01 I116 111V611I1011 8.110, including such departures from the present disclosure as come within known or binding to EGFR and to cynolmolgus monkey CD3) failed to induce an increase in the CD69 MFI.

Example 16 Redirected g by Humanized Cynomolgus Monkey / Human Cross-Reactive DARTTM Diabodies To further demonstrate the ability of the DARTTM diabodies of the present invention to e redirected killing, A498 kidney cancer target cells or A431 moid carcinoma cells (Lee, C.M. et al. (2010) “The Distribution Of The Therapeutic Monoclonal Antibodies Cetuximab And Trastuzumab Within Solid Tumors,” BMC Cancer 10:25.5; pages l-l 1; Bryant, J.A. et al. (2004) “EGF tes Intracellular And ellular Calcium Signaling By Distinct Pathways In Tumor Cells,” Cancer Biol. Ther. 3(12):1243-1249) and the extent of redirected g mediated by various DARTTM diabodies in the presence of PMBC effector cells (E:T = 30:1) was determined.

Cells were incubated in the presence of either ERBITUXTM-h-mAb2 DARTTM diabody, ERBITUXTM-m-mAb2 DARTTM y or 4420-h-mAb2 DARTTM diabody (negative control) or a l secondary dy. g to target cells was determined by measuring MFI. Redirected killing was assessed by measuring the % cytotoxicity.

The results of this investigation are shown in Figures 16A-16D. Diabodies haVing specificity for CD3 and EGFR were found to be able to bind to A498 or A431 cells (Figure 16A and 16C, respectively), and to mediate redirected killing of these cells e 16B and 16D, respectively).

All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each indiVidual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention ing, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice Within the art to which the invention pertains and as may be applied to the ial features hereinbefore set forth.

Claims (18)

What Is Claimed
1. Claim 1. A CD3-binding molecule comprising an antigen-binding fragment of an antibody, wherein said antigen-binding fragment comprises an dy CD3-specific VL domain and an antibody CD3-specific VH domain, n said ecific VL domain and said CD3-specific VH domain form an antigen-binding domain capable of immunospecifically binding to both an epitope of human CD3 and to an epitope of the CD3 of a non-human mammal, wherein: (I) said CD3-specific VL domain is selected from the group consisting of h-mab2 VL-1 (SEQ ID NO:16), h-mab2 VL-2 (SEQ ID , h-mab2 VL-3 (SEQ ID NO:20), h-mab2 VL-4 (SEQ ID NO:22), h-mab2 VL-5 (SEQ ID NO:24), hmab2 VL-6 (SEQ ID NO:26), 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), and (II) said CD3-specific VH domain is selected from the group consisting 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), h-mab2 VH-4 (SEQ ID NO:42), h-mab2 VH-5 (SEQ ID NO:44), hmab2 VH-6 (SEQ ID NO:46), h-mab2 VH-6L (SEQ ID NO:54), h-mab2 VH-7 (SEQ ID NO:48), h-mab2 VH-8 (SEQ ID NO:50), 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), hmab2 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).
2. Claim 2. The CD3-binding molecule of claim 1, wherein said CD3-specific VL domain is h-mab2 VL-6 (SEQ ID NO:26).
3. Claim 3. The CD3-binding molecule of any of claims 1-2, n said CD3- specific VH domain is h-mab2 VH-8 (SEQ ID , h-mab2 VH-4 (SEQ ID NO:42), or h-mab2 VH-2k (SEQ ID NO:87).
4. Claim 4. The nding molecule of any one of claims 1-3, wherein said molecule is an antibody.
5. Claim 5 The CD3-binding antibody of claim 4, wherein said antibody lacks an Fc region or comprises an Fc region that: (A) lacks effector function or has d effector function; or (B) impairs the y of the Fc region of said antibody to bind to an Fc receptor; 2176189v1 wherein said reduction in effector function and said impairment of binding ability is relative to that of a wild-type Fc receptor.
6. Claim 6. The CD3-binding molecule of any of claims 1-3, n said le is a CD3-binding y that comprises a first polypeptide chain and a second polypeptide chain, said chains being covalently bonded to one r, wherein: (I) said first polypeptide chain comprises an amino terminus and a carboxy terminus and from N-terminus to C-terminus: (i) a domain (A) sing said CD3-specific VL domain; (ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2); and (iii) a domain (C); wherein said s (A) and (B) do not associate with one another to form an epitope binding site; (II) said second polypeptide chain comprises an amino terminus and a carboxy terminus and from N-terminus to inus: (i) a domain (D) comprising a binding region of a light chain variable domain of said second immunoglobulin (VL2); (ii) a domain (E) comprising said CD3-specific VH domain; (iii) a domain (F); wherein said domains (D) and (E) do not associate with one another to form an epitope binding site; and wherein: (1) said domains (A) and (E) associate to form said antigen-binding domain that is capable of immunospecifically binding to both human CD3 and to the CD3 of a non-human mammal; (2) said domains (B) and (D) associate to form a binding site that immunospecifically binds to a second epitope, said second epitope being different from the CD3 epitope bound by the antigen-binding domain formed from said association of said domains (A) and (E); and (3) said domains (C) and (F) are covalently associated together.
7. Claim 7. The CD3-binding diabody of claim 6, wherein said second e is not an e of CD3. 2176189v1
8. Claim 8. The CD3-binding diabody of claim 6, wherein said second epitope is an epitope of CD3 that is different from the CD3 epitope bound by the antigen-binding domain formed from said association of said domains (A) and (E).
9. Claim 9. The CD3-binding molecule of any one of claims 1-3, the antibody of any of claims 4-5, or the diabody of any of claims 6-8 which is humanized.
10. Claim 10. The CD3-binding molecule of any one of claims 1-3 or 9 or the diabody of any one of claims 6-9 which is capable of immunospecifically binding to CD3 and to scein.
11. Claim 11. The CD3-binding le of any one of claims 1-3 or 9 or the y of any of claims 6-9, which is e of immunospecifically binding to both: (i) CD3 and (ii)(a) a tumor antigen, or (ii)(b) a cell surface antigen, receptor or receptor ligand.
12. Claim 12. The nding molecule or diabody of claim 11, wherein said molecule or diabody is capable of immunospecifically binding to CD3 and to a tumor antigen sed on a tumor cell, wherein said tumor cell is a tumor cell from a cancer selected from the group consisting of: breast cancer, prostate cancer, gastric cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, ovarian cancer, oral cavity cancer, pharyngeal cancer, esophageal cancer, eal cancer, bone cancer, skin cancer, melanoma, uterine cancer, testicular cancer, r cancer, kidney cancer, brain cancer, astoma, thyroid cancer, lymphoma, myeloma, and leukemia.
13. Claim 13. The CD3-binding molecule or diabody of claim 11, wherein said molecule or diabody is capable of immunospecifically binding to CD3 and to a cell surface antigen, or or receptor ligand, wherein said cell surface antigen, receptor or receptor ligand is HER2/neu, B7-H3, CD20, PSMA, IGF-1R., Ep-CAM, or is a molecule ed in a T cell – B cell association that leads to T cell or B cell activation in an ve immune response.
14. Claim 14. The CD3-binding molecule or diabody of claim 13, wherein said molecule or diabody is capable of immunospecifically binding to CD3 and to a molecule involved in said T cell – B cell association and said molecule involved in said T cell – B cell association is selected from the group consisting of CD19, CD20, CD22, CD23, CD27, CD32B, CD38, CD40, CD79a, CD79b, CD80, CD86, LFA-I, LFA-3 and CFAI. 2176189v1
15. Claim 15. A pharmaceutical composition comprising the CD3-binding molecule of any of claims 1-3 or 9-14, the antibody of any one of claims 4-5 , or the diabody of any one of claims 6-14 , and a pharmaceutically acceptable carrier, excipient or diluent.
16. Claim 16. The pharmaceutical composition of claim 15, for use in the treatment of cancer or an autoimmune or matory e.
17. Claim 17. The pharmaceutical composition of claim 16, for use in the treatment of an autoimmune or inflammatory disease selected from the group consisting of: type I n-dependent diabetes, rheumatoid tis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, myasthenia gravis, celiac’s disease, Sjogren's syndrome, Grave’s disease, Crohn’s disease, autoimmune hepatitis, psoriasis, tic arthritis, asthma, ic rhinitis, effects from organ transplantation, or graft vs. host disease (GVHD).
18. Claim 18. The pharmaceutical composition of claim 16, for use in the treatment of type I insulin-dependent diabetes.