Optimized Anti-CD3 Bispecific Antibodies and Uses Thereof

Abstract

The present invention provides antibodies that bind to CD3 with weak or no detectable binding affinity and methods of using the same. According to certain embodiments, the antibodies of the invention bind human CD3 with low affinity and induce human T cell proliferation and hence induce T cell-mediated killing of tumor cells with high efficacy. According to certain embodiments, the present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3 with weak or no detectable binding affinity in an in vitro assay, and a second antigen-binding molecule that specifically binds human tumor-associated antigen. In certain embodiments, the bispecific antigen-binding molecules of the present invention are capable of inhibiting the growth of tumors expressing target antigen, such as PSMA. The antibodies and bispecific antigen-binding molecules of the invention are useful for the treatment of diseases and disorders in which an upregulated or induced targeted immune response is desired and/or therapeutically beneficial. For example, the antibodies of the invention are useful for the treatment of various cancers or other diseases where immunotherapy, i.e. effector cell immunomodulation is warranted.

Description

REFERENCE TO A SEQUENCE LISTING

[0001] This application incorporates by reference the Sequence Listing submitted in Computer Readable Form as file 10151WO01_ST25.txt, created on Sep. 22, 2016 and containing 264,418 bytes.

FIELD OF THE INVENTION

[0002] The invention related to bispecific antibodies, targeting an effector antigen, such as CD3 antigen, and a tumor associated antigen, and methods of tumor killing. The invention relates to methods of reducing or eliminating tumor burden and controlling the toxic side effects that may be associated with tumor immunotherapy. The present invention provides bispecific antibodies comprising an effector arm which binds to an effector antigen with weak affinity or with no detectable binding affinity, for example, an anti-CD3 antigen-binding arm which binds to CD3 with a K.sub.D of greater than about 500 nM, in an in vitro affinity binding assay.

BACKGROUND

[0003] The promise of therapeutic bispecific antibodies (bsAbs), particularly in cancer immunotherapies, aims to bridge multiple antigen targets in order to elicit a more robust innate immune response to the unwanted target-bearing cells or organism.

[0004] It is now well established that to mediate redirected lysis, a bsAb must cluster a target cell directly to a triggering molecule on an effector cell, such as a T cell. There are many factors to consider in bsAb design, for example, size and composition will affect biodistribution and stability in vivo (Segal, DM, Weiner, G J, and Weiner, LM. Current Opinion in Immunol 1999, 11: 558-562; Chames, P. and Baty, D. MAbs. 2009, 1(6): 539-547). Differential outcomes are difficult to predict depending on the T cell subset being triggered to respond, as well as the state of the T cell being stimulated. It is well-known that bsAbs do not give consistent results (Manzke O, et al. Cancer Immunol Immunother. 1997, 45:198-202). For example, without adequate cytokine production, CD3 crosslinking can induce an apoptotic response in the T cell (Noel P J, Boise L H, Thompson C B: Regulation of T cell activation by CD28 and CTLA4. Adv Exp Med Biol 1996, 406:209-217). The subset of T cells and differentiation state of such recruited T cells, e.g. naive T cells, are important for efficacy, since naive T cells cannot lyse target cells without preactiviation (such as crosslinking with TCR in the presence of IL-2).

[0005] Certain bispecific therapies have been successful, yet, as with many cancer therapies, it comes with a price. Toxicity is the leading cause of failure among cancer therapeutics. It is well known that toxicity of so-called chemotherapeutic drugs is the leading cause of patient side effects and secondary maladies. The act of "cell killing" itself is wrought with trouble for the patient. A plethora of cytotoxic responses can be induced by activation of effector cells e.g. T cells, and a cancer target cell, yet which type of response is most beneficial in tumor immunotherapy remains to be seen. A method of identifying anti-CD3 antibodies for use in a bispecific therapy having reduced side effects while maintaining efficacy and desirable pharmacokinetic (PK) properties would be advantageous.

[0006] Techniques such as affinity maturation have been described which, based on structure/activity relationship (SAR), utilize mutagenesis to optimize antibodies to have increased and improved binding specificity or affinity for a target antigen compared to the starting antibody (see, e.g. WO2011056997, published May 12, 2011). Modified OKT3 antibodies capable of binding to and interacting with CD3 with varying degrees of affinity while still exhibiting moderate to high T cell activation have been described (U.S. Pat. No. 7,820,166). However, methods of reducing binding affinity of antibody molecules to near or beyond the detectable level of binding have not been described, nor shown to have the requisite efficacy for tumor reduction or suppression.

[0007] Thus, there exists a need for alternative bispecific antigen-binding molecules having controlled cytotoxicity and better PK properties. Such cancer therapies would be quite useful in therapeutic settings.

BRIEF SUMMARY OF THE INVENTION

[0008] In a first aspect, the present invention provides antibodies and antigen-binding fragments thereof that bind human CD3 having weak or no detectable affinity for human and/or cynomolgus CD3. The antibodies according to this aspect of the invention are useful, inter alia, for targeting T cells expressing CD3, and for stimulating T cell activation, e.g., under circumstances where T cell-mediated killing is beneficial or desirable. The anti-CD3 antibodies of the invention, or antigen-binding portions thereof, may be included as part of a bispecific antibody that directs CD3-mediated T cell activation to specific cell types such as tumor cells or infectious agents.

[0009] Exemplary anti-CD3 antibodies of the present invention are listed in Tables 2 and 3 herein. Table 2 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVRs), as well as heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3). Table 3 sets forth the sequence identifiers of the nucleic acid molecules encoding the HCVRs, HCDR1, HCDR2 and HCDR3 regions of the exemplary anti-CD3 antibodies. Tables 4 and 5 set forth light chain variable regions (LCVRs), as well as complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the exemplary anti-CD3 antibodies.

[0010] The present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 2, or a substantially similar sequence thereof having having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0011] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 2 paired with any of the LCVR amino acid sequences listed in Table 4, or a common light chain derived from the cognate light chain of the anti-TAA heavy chain, or derived from a known or public domain light chain variable region derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-CD3 antibodies listed in Table 2 paired with exemplary light chain variable regions listed in Table 4. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 10/162 (e.g., CD3-VH-G2); 18/162 (e.g., CD3-VH-G3); 26/162 (e.g., CD3-VH-G4); 34/162 (e.g., CD3-VH-G5); 42/162 (e.g., CD3-VH-G8); 50/162 (e.g., CD3-VH-G9); 58/162 (e.g., CD3-VH-G10); 66/162 (e.g., CD3-VH-G11); 74/162 (e.g., CD3-VH-G12); 82/162 (e.g., CD3-VH-G13); 90/162 (e.g., CD3-VH-G14); 98/162 (e.g., CD3-VH-G15); 106/162 (e.g., CD3-VH-G16); 114/162 (e.g., CD3-VH-G17); 122/162 (e.g., CD3-VH-G18); 130/162 (e.g., CD3-VH-G19); 138/162 (e.g., CD3-VH-G20); and 146/162 (e.g., CD3-VH-G21).

[0012] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 2 or a substantially similar sequence thereof having at least 95%, at least 98% or at least 99% sequence identity. The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 178.

[0013] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 2 or a substantially similar sequence thereof having at least 95%, at least 98% or at least 99% sequence identity. The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 179.

[0014] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 2 or a substantially similar sequence thereof having at least 95%, at least 98% or at least 99% sequence identity. The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 180.

[0015] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 4 or a substantially similar sequence thereof having at least 95%, at least 98% or at least 99% sequence identity. The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR1 (LCDR1) derived from a cognate light chain of the anti-TAA heavy chain, or derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain.

[0016] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 4 or a substantially similar sequence thereof having at least 95%, at least 98% or at least 99% sequence identity. The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR2 (LCDR2) derived from a cognate light chain of the anti-TAA heavy chain, or derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain.

[0017] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 4 or a substantially similar sequence thereof having at least 95%, at least 98% or at least 99% sequence identity. The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) derived from a cognate light chain of the anti-TAA heavy chain, or derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain.

[0018] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 2 paired with any of the LCDR3 amino acid sequences listed in Table 4. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-CD3 antibodies listed in Table 2. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 16/168 (e.g., CD3-VH-G2); 24/168 (e.g., CD3-VH-G3); 32/168 (e.g., CD3-VH-G4); 40/168 (e.g., CD3-VH-G5); 48/168 (e.g., CD3-VH-G8); 56/168 (e.g., CD3-VH-G9); 64/168 (e.g., CD3-VH-G10); 72/168 (e.g., CD3-VH-G11); 80/168 (e.g., CD3-VH-G12); 88/168 (e.g., CD3-VH-G13); 96/168 (e.g., CD3-VH-G14); 104/168 (e.g., CD3-VH-G15); 112/168 (e.g., CD3-VH-G16); 120/168 (e.g., CD3-VH-G17); 128/168 (e.g., CD3-VH-G18); 136/168 (e.g., CD3-VH-G19); 144/168 (e.g., CD3-VH-G20); and 152/168 (e.g., CD3-VH-G21).

[0019] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-CD3 antibodies listed in Tables 2 and 4. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set is selected from the group consisting of SEQ ID NOs: 12-14-16-164-166-168 (e.g., CD3-VH-G2); 20-22-24-164-166-168 (e.g., CD3-VH-G3); 28-30-32-164-166-168 (e.g., CD3-VH-G4); 36-38-40-164-166-168 (e.g., CD3-VH-G5); 44-46-48-164-166-168 (e.g., CD3-VH-G8); 52-54-56-164-166-168 (e.g., CD3-VH-G9); 60-62-64-164-166-168 (e.g., CD3-VH-G10); 68-70-72-164-166-168 (e.g., CD3-VH-G11); 76-78-80-164-166-168 (e.g., CD3-VH-G12); 84-86-88-164-166-168 (e.g., CD3-VH-G13); 92-94-96-164-166-168 (e.g., CD3-VH-G14); 100-102-104-164-166-168 (e.g., CD3-VH-G15); 108-110-112-164-166-168 (e.g., CD3-VH-G16); 116-118-120-164-166-168 (e.g., CD3-VH-G17); 124-126-128-164-166-168 (e.g., CD3-VH-G18); 132-134-136-164-166-168 (e.g., CD3-VH-G19); 140-142-144-164-166-168 (e.g., CD3-VH-G20); and 148-150-152-164-166-168 (e.g., CD3-VH-G21).

[0020] In a related embodiment, the present invention provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-CD3 antibodies listed in Tables 2 and 4. For example, the present invention includes antibodies, or antigen-binding fragments thereof, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 10/162 (e.g., CD3-VH-G2); 18/162 (e.g., CD3-VH-G3); 26/162 (e.g., CD3-VH-G4); 34/162 (e.g., CD3-VH-G5); 42/162 (e.g., CD3-VH-G8); 50/162 (e.g., CD3-VH-G9); 58/162 (e.g., CD3-VH-G10); 66/162 (e.g., CD3-VH-G11); 74/162 (e.g., CD3-VH-G12); 82/162 (e.g., CD3-VH-G13); 90/162 (e.g., CD3-VH-G14); 98/162 (e.g., CD3-VH-G15); 106/162 (e.g., CD3-VH-G16); 114/162 (e.g., CD3-VH-G17); 122/162 (e.g., CD3-VH-G18); 130/162 (e.g., CD3-VH-G19); 138/162 (e.g., CD3-VH-G20); and 146/162 (e.g., CD3-VH-G21).

[0021] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.

[0022] The present invention also provides nucleic acid molecules encoding anti-CD3 antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 3; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0023] The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 4; or an LCVR derived from a cognate light chain of the anti-TAA heavy chain, or derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0024] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 2; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0025] The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 2; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0026] The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 2; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 3, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0027] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 4; or an LCDR1 derived from a cognate light chain of the anti-TAA heavy chain, or derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0028] The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 4; or an LCDR2 derived from a cognate light chain of the anti-TAA heavy chain, or derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0029] The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 4; or an LCDR3 derived from a cognate light chain of the anti-TAA heavy chain, or derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain. In certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0030] The present invention also provides nucleic acid molecules encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary anti-CD3 antibodies listed in Table 2.

[0031] The present invention also provides nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary universal light chain antibodies listed in Table 4; or the LCDR1-LCDR2-LCDR3 is derived from a cognate light chain of the anti-TAA heavy chain, or derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain.

[0032] The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 2, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 4; or the LCVR is derived from a cognate light chain of the anti-TAA heavy chain, or derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In some embodiments, the nucleic acid molecules encoding both an HCVR and an LCVR are fully human sequences or derived from human germline immunoglobulin sequences.

[0033] The present invention also provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-CD3 antibody. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDR sequences as set forth in Table 2 or 4. Also included within the scope of the present invention are host cells into which such vectors have been introduced, as well as methods of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

[0034] The present invention includes anti-CD3 antibodies and/or anti-TAA antibodies, as well as bispecific anti-CD3/anti-TAA antibodies having a modified glycosylation pattern. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

[0035] In one aspect, the invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule that i) is not capable of specifically binding to an effector cell, and ii) specifically binds a target tumor cell, wherein specific binding is measured in an in vitro FACS binding assay or an in vitro surface plasmon resonance binding assay. In certain embodiments, the invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule that exhibits no detectable binding to an effector cell, and that specifically binds to a target tumor cell with a measurable binding affinity, wherein the binding affinity value is measured in an in vitro FACS binding assay or an in vitro surface plasmon resonance binding assay.

[0036] In other embodiments, the invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule comprising i) a first antigen-binding fragment (Fab1) that exhibits no detectable binding to CD3, and ii) a second antigen-binding fragment (Fab2) that specifically binds to a target tumor cell with a measurable binding affinity, wherein the binding affinity value is measured in an in vitro FACS binding assay or an in vitro surface plasmon resonance binding assay. In some cases, the binding affinity is monovalent binding affinity (e.g., in a bispecific antibody construct).

[0037] In another aspect the invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule that specifically binds an effector cell with a weak binding affinity, for example exhibiting an EC.sub.50 value of about or greater than about 100 nM, and that specifically binds a target tumor cell with an appreciable EC.sub.50 value, or a high affinity EC.sub.50 value such as less than 50 nM, wherein the EC.sub.50 binding affinity value is measured in an in vitro FACS binding assay. In certain embodiments, the invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule that specifically binds an effector cell with an EC.sub.50 value of greater than about 500 nM, and that specifically binds a target tumor cell with an appreciable EC.sub.50 value, or a high affinity EC.sub.50 value such as less than 50 nM, wherein the EC.sub.50 binding affinity value is measured in an in vitro FACS binding assay.

[0038] In some examples, the bispecific antigen-binding molecule includes a Fab1 that specifically binds human CD3 with an EC.sub.50 value of greater than about 40 nM, or greater than about 100 nM, greater than about 200 nM, greater than about 300 nM, greater than about 400 nM, greater than about 500 nM, or greater than about 1 .mu.M (e.g. in a monovalent binding context). In some embodiments, the bispecific antigen-binding molecule includes a Fab2 derived from a second antibody that specifically binds the target tumor cell with high affinity, e.g. an EC.sub.50 value of less than less than about 50 nM, less than about 40 nM, less than about 20 nM, less than about 10 nM or less than about 6 nM (e.g., in a monovalent binding context). In some cases, the Fab1 specifically binds each of human CD3 and cynomolgus CD3 with an EC.sub.50 value of greater than about 40 nM, or greater than about 100 nM, greater than about 200 nM, or greater than about 1 .mu.M. In some cases, the Fab1 specifically binds each of human CD3 and cynomolgus CD3 with weak or no measurable affinity.

[0039] In some embodiments, the target tumor cell is a human tumor cell. In some embodiments, the Fab1 (or the bispecific antigen-binding molecule) induces T cell-mediated tumor cell killing with an EC.sub.50 value of less than about 1.3 nM, as measured in an in vitro T cell-mediated tumor cell killing assay.

[0040] In some applications, the Fab1 or the bispecific antigen-binding molecule specifically binds human CD3 with an K.sub.D value of greater than about 11 nM, as measured in an in vitro surface plasmon resonance binding assay. In other instances, the Fab1 or the bispecific antigen-binding molecule specifically binds each of human CD3 and cynomolgus CD3 with an K.sub.D value of greater than about 15 nM, or greater than about 30 nM, greater than about 60 nM, greater than about 120 nM, or greater than about 300 nM, as measured in an in vitro surface plasmon resonance binding assay. In still some applications, the Fab1 or the bispecific antigen-binding molecule i) exhibits no detectable binding to human CD3 as measured in each of an in vitro surface plasmon resonance binding assay and a FACS binding assay, and ii) induces T cell-mediated tumor cell killing, as measured in an in vitro T cell-mediated tumor cell killing assay.

[0041] In some applications, the bispecific antigen-binding molecule comprises a first heavy chain comprising a HCDR1 region comprising an amino acid sequence set forth in SEQ ID NO: 12 or 20. In some embodiments, the first heavy chain comprises a HCDR2 region comprising an amino acid sequence set forth in SEQ ID NO: 14 or 54. In other embodiments, the first heavy chain comprises a HCDR3 region comprising an amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, or SEQ ID NO: 152. In other applications, the first heavy chain comprises a HCVR comprising HCDR1-HCDR2-HCDR3 having the amino acid sequences of SEQ ID NOs: 178-179-180. In other embodiments, a first heavy chain comprises a CDR1 comprising amino acid residues 1-7 of SEQ ID NO:178, a CDR2 comprising amino acid residues 1-7 of SEQ ID NO:179, a CDR3 comprising amino acid residues 4-11 of SEQ ID NO:180.

[0042] In more embodiments, the first heavy chain comprises variable domain framework regions having an amino acid sequence selected from FR1 (SEQ ID NO: 174), FR2 (SEQ ID NO: 175), FR3 (SEQ ID NO: 176), and FR4 (SEQ ID NO: 177).

[0043] The present invention provides bispecific antigen-binding molecules comprising a Fab1 HCVR and LCVR amino acid sequence pair (HCVR/LCVR) selected from the group consisting of: SEQ ID NOs: 10/162; 18/162; 26/162; 34/162; 42/162; 50/162; 58/162; 66/162; 74/162; 82/162; 90/162; 98/162; 106/162; 114/162; 122/162; 130/162; 138/162; 146/162.

[0044] The antibodies, and antigen-binding fragments and bispecific antibodies thereof were made by replacing amino acid residues of a parental in a stepwise manner based on differences between the germline sequence and the parental antibody sequence. The present invention provides a method of making a cytotoxic composition comprising (a) identifying the amino acid sequence of the first heavy chain derived from a first antibody that specifically binds CD3 with high affinity, for example exhibits a binding affinity EC.sub.50 value of less than about 40 nM, (b) modifying selected amino acid residues in the heavy chain variable region of the first antibody to produce a modified antibody, (c) pairing the modified antibody with a second heavy chain derived from a second antibody that specifically binds a target tumor antigen to produce a bispecific antibody, (d) testing the bispecific antibody in a binding affinity assay, and if the binding affinity to CD3 has an EC.sub.50 value of greater than about 40 nM, or greater than 100 nM or greater than 300 nM or greater than 500 nM, or no detectable binding, then (e) preparing a composition comprising the bispecific antibody and a pharmaceutically acceptable carrier or diluent. In addition to modifying the heavy chain variable region of selected antibodies to engineer antigen-binding arms having weak or no affinity for, yet specifically target an effector cell, the invention provides methods herein for modifying the heavy chain constant region (e.g. C.sub.H3 domain) of each binding arm to prepare and isolate bispecific antibodies.

[0045] An exemplary method provides a method of producing a cytotoxic bispecific antibody, comprising: (a) identifying a first human antibody or antigen-binding fragment thereof that interacts with an effector cell antigen from multiple species; (b) identifying the germline amino acid residues of the heavy chain variable region (HCVR) of the human antibody; (c) comparing the amino acid sequence of the HCVR of the first human antibody to the amino acid sequence of the corresponding germline HCVR; (d) identifying amino acids within a modified region of the HCVR of the first antibody, whereby a modified region in the first antibody displays at least one amino acid modification by substitution, deletion or addition of a single amino acid residue compared to the same region in the germline HCVR; (e) producing a plurality of modified antibodies each comprising at least one modified region of the HCVR; (f) screening each of the plurality of modified antibodies for monovalent affinity to the effector cell antigen; (g) selecting those modified antibodies that exhibit weaker binding affinity or no detectable binding affinity to the effector cell antigen compared to the first antibody; and (h) pairing the selected first antibody with a second antibody that interacts with a tumor-associated antigen to produce a cytotoxic bispecific antibody.

[0046] In another aspect, the invention provides a pharmaceutical composition comprising a recombinant human bispecific antibody or fragment thereof which specifically binds CD3 and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition which is a combination of an anti-CD3 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-CD3 antibody. Exemplary agents that may be advantageously combined with an anti-CD3 antibody include, without limitation, other agents that bind and/or activate CD3 signaling (including other antibodies or antigen-binding fragments thereof, etc.) and/or agents which do not directly bind CD3 but nonetheless activate or stimulate immune cell activation. Additional combination therapies and co-formulations involving the anti-CD3 antibodies of the present invention are disclosed elsewhere herein.

[0047] In yet another aspect, the invention provides therapeutic methods for stimulating T cell activation using an anti-CD3 antibody or antigen-binding portion of an antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a bispecific antibody of the invention, or antigen-binding fragment thereof, to a subject in need thereof. The disorder treated is any disease or condition, which is improved, ameliorated, inhibited or prevented by cytotoxic therapy targeted to a tumor-associated antigen, such as cancer.

[0048] According to another aspect, the present invention provides bispecific antigen-binding molecules that bind CD3 and a target antigen, especially a tumor-associated antigen (TAA).

[0049] The present invention also includes the use of an anti-CD3/anti-TAA bispecific antigen-binding molecule of the invention in the manufacture of a medicament for the treatment of a disease or disorder related to or caused by TAA expression. The present invention also provides use of an anti-CD3/anti-TAA bispecific antigen-binding molecule, exhibiting weak affinity to CD3-expressing effector cells and reduced clearance, in the manufacture of a medicament for the treatment of a disease or disorder related to or caused by TAA expression, compared to an anti-CD3/anti-TAA bispecific antigen-binding molecule exhibiting high affinity to CD3-expressing effector cells.

[0050] Other embodiments will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0051] FIG. 1 shows the amino acid alignment of the following antibody heavy chain variable region (HCVR) sequences: germline hIgHV (SEQ ID NO:181); CD3-VH-P (SEQ ID NO:154); CD3-VH-G (SEQ ID NO:2); CD3-VH-G2 (SEQ ID NO:10); CD3-VH-G3 (SEQ ID NO:18); CD3-VH-G4 (SEQ ID NO:26); CD3-VH-G5 (SEQ ID NO: 34); CD3-VH-G8 (SEQ ID NO:42); CD3-VH-G9 (SEQ ID NO:50); CD3-VH-G10 (SEQ ID NO:58); CD3-VH-G11 (SEQ ID NO:66); CD3-VH-G12 (SEQ ID NO:74); CD3-VH-G13 (SEQ ID NO:82); CD3-VH-G14 (SEQ ID NO:90); CD3-VH-G15 (SEQ ID NO:98); CD3-VH-G16 (SEQ ID NO:106); CD3-VH-G17 (SEQ ID NO:114); CD3-VH-G18 (SEQ ID NO:122); CD3-VH-G19 (SEQ ID NO:130); CD3-VH-G20 (SEQ ID NO:138); and CD3-VH-G21 (SEQ ID NO:146). Each derivative HCVR is compared to the parent antibody and germline amino acid residues, with rectangular boxes denoting mutations in the CDRs.

[0052] FIGS. 2A, 2B and 2C illustrate mean concentrations of total IgG in serum following a single 0.4 mg/kg intra-peritoneal injection of BSMUC16/CD3-001, BSMUC16/CD3-005 and isotype control antibodies in wild-type mice (FIG. 2A), humanized CD3 mice (FIG. 2B) and humanized MUC16.times.CD3 mice (FIG. 2C).

DETAILED DESCRIPTION

[0053] Before the present invention is described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0055] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Definitions

[0056] The expression "CD3" refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. Human CD3-epsilon (hCD3E) comprises the amino acid sequence as set forth in SEQ ID NO:169 (UniProtKB/Swiss-Prot: P07766.2). Human CD3-delta (hCD3.delta.) comprises the amino acid sequence as set forth in SEQ ID NO:170 (UniProtKB/Swiss-Prot: P04234.1). All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, the expression "CD3" means human CD3 unless specified as being from a non-human species, e.g., "mouse CD3," "monkey CD3," etc.

[0057] The phrase "an antibody that binds CD3" or an "anti-CD3 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize and associate with a single CD3 subunit (e.g., epsilon, delta, gamma or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize and associate with a dimeric complex of two CD3 subunits (e.g., epsilon/delta, epsilon/gamma, and zeta/zeta CD3 dimers). The antibodies and antigen-binding fragments of the present invention may bind soluble CD3, bound CD3 and/or cell surface expressed CD3. Soluble CD3 includes natural CD3 proteins as well as recombinant CD3 protein variants such as, e.g., monomeric and dimeric CD3 constructs, that lack a transmembrane domain or are otherwise unassociated with a cell membrane. The present invention provides antibodies that bind and activate human and cynomolgus CD3 with weak or no detectable binding affinity. "Binding to CD3 with no detectable binding affinity" means that the antibody and or antigen-binding fragment interaction with the CD3 target may not be measurable or detectable with a known assay for detection, such as a FACS (cell-based) binding assay as described herein or a surface plasmon resonance binding assay as described herein and well-known in the art. Other binding assays are well-known in the art. The antibody and or antigen-binding fragment may recognize the CD3 target by very weak protein-protein biochemical interaction, however a determination of specific KD or EC.sub.50 value cannot be measured since the interaction is beyond the detection limit of the assay, e.g. no measurement can be determined. In another instance, "no detectable binding affinity" is determined if the affinity of an antibody corresponding to a K.sub.D value is equal to or less than ten-fold lower than a non-specific antigen such as, BSA, casein, or the like. "Binding to CD3 with weak binding affinity" includes interactions where binding affinity measurement is at or slightly above the detection limit of the assay, or equivalent to the binding affinity to a non-specific antigen.

[0058] The expression "cell surface-expressed CD3" means one or more CD3 protein(s) that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. "Cell surface-expressed CD3" includes CD3 proteins contained within the context of a functional T cell receptor in the membrane of a cell. The expression "cell surface-expressed CD3" includes CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., delta/epsilon, gamma/epsilon, and zeta/zeta CD3 dimers). The expression, "cell surface-expressed CD3" also includes a CD3 chain (e.g., CD3-delta, CD3-epsilon or CD3-gamma) that is expressed by itself, without other CD3 chain types, on the surface of a cell. A "cell surface-expressed CD3" can comprise or consist of a CD3 protein expressed on the surface of a cell which normally expresses CD3 protein. Alternatively, "cell surface-expressed CD3" can comprise or consist of CD3 protein expressed on the surface of a cell that normally does not express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0059] Effector cells include effector T cells (T lymphocytes), for example CD4+ T cells, CD8+ T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells may also include natural killer (NK) cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes). It is understood that therapies may mediate a plethora of cell-mediated immune responses, or effector functions, through Ig interaction with effector cell surface receptors, such as CD3 (T cell surface receptor), CD28 (T cells), Fc.gamma. receptors (Fc.gamma.Rs) (NK cells, activated macrophages and the like). Effector functions such as cell killing, complement activation, phagocytosis and opsonisation are subsequently triggered through these interactions. Binding to an effector cell and a tumor target cell allows for a valuable and effective immunotherapy design that propagates tumor cell killing and induces endogenous immune functions to fight the tumor or cancer.

[0060] The expression "anti-CD3 antibody" includes both monovalent antibodies with a single specificity, as well as bispecific antibodies comprising a first arm that binds CD3 and a second arm that binds a second (target) antigen, wherein the anti-CD3 arm comprises any of the HCVR/LCVR or CDR sequences as set forth in Tables 2, 3, 4 and/or 5 herein. Examples of anti-CD3 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, e.g., bispecific antibodies.

[0061] The term "antibody" includes any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD3). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V.sub.L) and a light chain constant region. The light chain constant region comprises one domain (C.sub.L1). The V.sub.H and V.sub.L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-CD3 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0062] The term "antibody" also includes antigen-binding fragments of full antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0063] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0064] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V.sub.H domain associated with a V.sub.L domain, the V.sub.H and V.sub.L domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V.sub.H-V.sub.H, V.sub.H-V.sub.L or V.sub.L-V.sub.L dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V.sub.H or V.sub.L domain.

[0065] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) V.sub.H-C.sub.H1; (ii) V.sub.H-C.sub.H2; (iii) V.sub.H-C.sub.H3; (iv) V.sub.H-C.sub.H1-C.sub.H2; (v) V.sub.H-C.sub.H1-C.sub.H2-C.sub.H3; (vi) V.sub.H-C.sub.H2-C.sub.H3; (vii) V.sub.H-C.sub.L; (viii) V.sub.L-C.sub.H1; (ix) V.sub.L-C.sub.H2; (x) V.sub.L-C.sub.H3; (xi) V.sub.L-CH1-C.sub.H2; (xii) V.sub.L-C.sub.H1-C.sub.H2-C.sub.H3; (xiii) V.sub.L-C.sub.H2-C.sub.H3; and (xiv) V.sub.L-C.sub.L. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in noncovalent association with one another and/or with one or more monomeric V.sub.H or V.sub.L domain (e.g., by disulfide bond(s)).

[0066] As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

[0067] The antibodies of the present invention may function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to lysis of antigen-expressing cells by an antibody of the invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby lead to lysis of the target cell. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity.

[0068] In certain embodiments of the invention, the anti-CD3 antibodies of the invention (monospecific or bispecific) are human antibodies. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0069] The term "recombinant human antibody" is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V.sub.H and V.sub.L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V.sub.H and V.sub.L sequences, do not necessarily naturally exist within the human antibody germline repertoire in vivo.

[0070] Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via interchain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

[0071] The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge. The instant invention encompasses antibodies having one or more mutations in the hinge, C.sub.H2 or C.sub.H3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.

[0072] The antibodies of the invention may be isolated antibodies. An "isolated antibody," as used herein, means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[0073] The present invention also includes one-arm antibodies that bind CD3. The phrase "one-arm antibody" means an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-arm antibodies of the present invention may comprise any of the HCVR/LCVR or CDR amino acid sequences as set forth in Table 2 herein.

[0074] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0075] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0076] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0077] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

Germline Mutations

[0078] The anti-CD3 antibodies disclosed herein comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived.

[0079] The present invention also includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"), and having weak or no detectable binding to a CD3 antigen. Several such exemplary antibodies that recognize CD3 are described in Table 2 herein.

[0080] Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be tested for one or more desired properties such as, improved binding specificity, weak or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner given the guidance of the present disclosure are encompassed within the present invention.

[0081] The present invention also includes anti-CD3 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-CD3 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences set forth in Table 2 herein. The antibodies and bispecific antigen-binding molecules of the present invention comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived, while maintaining or improving the desired weak-to-no detectable binding to CD3 antigen. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein, i.e. the amino acid substitution maintains or improves the desired weak to no detectable binding affinity in the case of anti-CD3 binding molecules. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0082] The present invention also includes antigen-binding molecules comprising an antigen-binding domain with an HCVR and/or CDR amino acid sequence that is substantially identical to any of the HCVR and/or CDR amino acid sequences disclosed herein, while maintaining or improving the desired weak affinity to CD3 antigen. The term "substantial identity" or "substantially identical," when referring to an amino acid sequence means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331.

[0083] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0084] Once obtained, antigen-binding domains that contain one or more germline mutations were tested for decreased binding affinity utilizing one or more in vitro assays. Although antibodies that recognize a particular antigen are typically screened for their purpose by testing for high (i.e. strong) binding affinity to the antigen, the antibodies of the present invention exhibit weak binding or no detectable binding. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are also encompassed within the present invention and were found to be advantageous as avidity-driven tumor therapies.

[0085] Unexpected benefits, for example, improved pharmacokinetic properties and low toxicity to the patient may be realized from the methods described herein.

Binding Properties of the Antibodies

[0086] As used herein, the term "binding" in the context of the binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to either, e.g., a predetermined antigen, such as a cell surface protein or fragment thereof, typically refers to an interaction or association between a minimum of two entities or molecular structures, such as an antibody-antigen interaction.

[0087] For instance, binding affinity typically corresponds to a K.sub.D value of about 10.sup.-7 M or less, such as about 10.sup.-8 M or less, such as about 10.sup.-9 M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand). Cell-based binding strategies, such as fluorescent-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlates well with other methods such as radioligand competition binding and SPR (Benedict, C A, J Immunol Methods. 1997, 201(2):223-31; Geuijen, C A, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0088] Accordingly, the antibody or antigen-binding protein of the invention may bind to the predetermined antigen or cell surface molecule (receptor such as CD3) having an affinity corresponding to a K.sub.D value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). According to the present invention, if the affinity of an antibody corresponding to a K.sub.D value is equal to or less than ten-fold lower than a non-specific antigen, this may be considered non-detectable binding, however such an antibody may be paired with a second antigen binding arm for the production of a bispecific antibody of the invention.

[0089] The term "K.sub.D" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. There is an inverse relationship between K.sub.D and binding affinity, therefore the smaller the K.sub.D value, the higher, i.e. stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" relate to a higher ability to form an interaction and therefore a smaller K.sub.D value, and conversely the terms "lower affinity" or "weaker affinity" relate to a lower ability to form an interaction and therefore a larger K.sub.D value. In some circumstances, a higher binding affinity (or K.sub.D of a particular molecule (e.g. antibody) to its interactive partner molecule (e.g. antigen X) compared to the binding affinity of the molecule (e.g. antibody) to another interactive partner molecule (e.g. antigen Y) may be expressed as a binding ratio determined by dividing the larger K.sub.D value (lower, or weaker, affinity) by the smaller K.sub.D (higher, or stronger, affinity), for example expressed as 5-fold or 10-fold greater binding affinity, as the case may be. For example, "low affinity" refers to less strong binding interaction. In some embodiments, the low binding affinity corresponds to greater than about 1 nM K.sub.D, greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or greater than about 40 nM K.sub.D, wherein such K.sub.D binding affinity value is measured in an in vitro surface plasmon resonance binding assay, or equivalent biomolecular interaction sensing assay. In some embodiments, the low binding affinity corresponds to greater than about 10 nM EC.sub.50, greater than about 15 nM EC.sub.50, 20 nM EC.sub.50, greater than about 25 nM EC.sub.50, 30 nM EC.sub.50, greater than about 35 nM EC.sub.50, or greater than about 40 nM EC.sub.50, wherein such EC.sub.50 binding affinity value is measured in an in vitro FACS binding assay, or equivalent cell-based binding assay. "Weak affinity" refers to weak binding interaction. In some embodiments, the weak binding affinity corresponds to greater than about 100 nM K.sub.D or EC.sub.50, greater than about 200, 300, or greater than about 500 nM K.sub.D or EC.sub.50, wherein such K.sub.D binding affinity value is measured in an in vitro surface plasmon resonance binding assay, or equivalent biomolecular interaction sensing assay, and such EC.sub.50 binding affinity value is measured in an in vitro FACS binding assay, or equivalent cell-based interaction detecting assay to detect monovalent biding. No detectable binding means that the affinity between the two biomolecules, for example, especially between the monovalent antibody binding arm and its target antigen, is beyond the detection limit of the assay being used.

[0090] The term "k.sub.d" (sec -1 or 1/s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. Said value is also referred to as the k.sub.off value.

[0091] The term "k.sub.a" (M-1.times.sec-1 or 1/M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[0092] The term "K.sub.A" (M-1 or 1/M) refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is obtained by dividing the k.sub.a by the k.sub.d.

[0093] The term "EC.sub.50" or "EC.sub.50" refers to the half maximal effective concentration, which includes the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time. The EC.sub.50 essentially represents the concentration of an antibody where 50% of its maximal effect is observed. In certain embodiments, the EC.sub.50 value equals the concentration of an antibody of the invention that gives half-maximal binding to cells expressing CD3 or tumor-associated antigen, as determined by e.g. a FACS binding assay. Thus, reduced or weaker binding is observed with an increased EC.sub.50 value, or half maximal effective concentration value such that 500 nM EC.sub.50 is indicative of a weaker binding affinity than 50 nM EC.sub.50.

[0094] In one embodiment, decreased binding can be defined as an increased EC.sub.50 antibody concentration which enables binding to the half-maximal amount of target cells.

[0095] In other experimental measurements, the EC.sub.50 value represents the concentration of an antibody of the invention that elicits half-maximal depletion of target cells by T cell cytotoxic activity. Thus, increased cytotoxic activity (e.g. T cell-mediated tumor cell killing) is observed with a decreased EC.sub.50, or half maximal effective concentration value.

Bispecific Antigen-Binding Molecules

[0096] The antibodies of the present invention may be bi-specific, or multispecific. Multispecific antibodies may be specific for one effector molecule, such as CD3, in combination with different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-CD3 antibodies of the present invention can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second binding specificity.

[0097] Use of the expression "anti-CD3 antibody" herein is intended to include both monospecific anti-CD3 antibodies as well as bispecific antibodies comprising a CD3-binding arm and a second arm that binds a target antigen. Thus, the present invention includes bispecific antibodies wherein one arm of an immunoglobulin binds human CD3, and the other arm of the immunoglobulin is specific for a target antigen. The target antigen that the other arm of the CD3 bispecific antibody binds can be any antigen expressed on or in the vicinity of a cell, tissue, organ, microorganism or virus, against which a targeted immune response is desired. The CD3-binding arm can comprise any of the HCVR or CDR amino acid sequences as set forth in Table 2 herein. In certain embodiments, the CD3-binding arm binds weakly to human CD3 and induces human T cell activation. In other embodiments, the CD3-binding arm binds weakly to human CD3 and induces tumor-associated antigen-expressing cell killing in the context of a bispecific or multispecific antibody. In other embodiments, the CD3-binding arm binds or associated weakly with human and cynomolgus (monkey) CD3, yet the binding interaction is not detectable by in vitro assays known in the art. In some embodiments of the invention, the CD3-binding arm does not bind or associate with human and cynomolgus (monkey) CD3, yet the bispecific molecule still elicits tumor-associated cell killing.

[0098] In the context of bispecific antibodies of the present invention wherein one arm of the antibody binds CD3 and the other arm binds a target antigen, the target antigen can be a tumor-associated antigen (TAA). Non-limiting examples of specific tumor-associated antigens include, e.g., AFP, ALK, BAGE proteins, BIRC5 (survivin), BIRC7, .beta.-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-.beta., TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0099] The inventors envision that the present invention includes numerous examples of bispecific antibodies having a weak anti-CD3 binding arm made in accordance with the invention.

[0100] According to certain exemplary embodiments, the present invention includes bispecific antigen-binding molecules that specifically bind CD3 and PSMA. Such molecules may be referred to herein as, e.g., "anti-CD3/anti-PSMA," or "anti-CD3.times.PSMA" or "CD3.times.PSMA" bispecific molecules, and so forth. The term "PSMA," as used herein, refers to the human PSMA protein unless specified as being from a non-human species (e.g., "mouse PSMA," "monkey PSMA," etc.).

[0101] The term "PSMA" refers to prostate-specific membrane antigen, also known as folate hydrolase 1 (FOLH1) (UniProtKB/Swiss-Prot. No. Q04609; SEQ ID NO: 171). PSMA is an integral, non-shed membrane glycoprotein that is highly expressed in prostate epithelial cells and is a cell-surface marker for prostate cancer.

[0102] According to other exemplary embodiments, the present invention includes bispecific antigen-binding molecules that specifically bind CD3 and EGFRvIII. Such molecules may be referred to herein as, e.g., "anti-CD3/anti-EGFRvIII" or "anti-CD3.times. EGFRvIII" or "CD3.times. EGFRvIII" bispecific molecules, and so forth. The term "EGFRvIII" refers to the human EGFRvIII protein unless specified as being from a non-human species (e.g., "mouse EGFRvIII," "monkey EGFRvIII," etc.).

[0103] The term "EGFRvIII" refers to the class III variant of the epidermal growth factor receptor (EGFRvIII; SEQ ID NO: 172) which is the most frequently found EGFR variant in glioblastoma (Bigner et al., 1990, Cancer Res 50:8017-8022; Humphrey et al., 1990, Proc Natl Acad Sci USA 87:4207-4211; Yamazaki et al., 1990, Jap J Cancer Res 81:773-779; Ekstrand et al., 1992, Proc Natl Acad Sci USA 89:4309-4313; Wikstrand et al., 1995, Cancer Res 55:3140-3148; and Frederick et al., 2000, Cancer Res 60:1383-1387). EGFRvIII is characterized by a deletion of exons 2-7 of the EGFR gene, resulting in an in-frame deletion of 801 base pairs of the coding region, i.e., deletion of 6-273 amino acid residues (based on the residue numbers of mature EGFR; see UniProtKB/Swiss-Prot. No. P00533), as well as the generation of a new glycine at the fusion junction (Humphrey et al., 1988, Cancer Res 48:2231-2238; Yamazaki et al., 1990, supra). EGFRvIII has been shown to have a ligand-independent, weak but constitutively active kinase activity as well as enhanced tumorigenicity (Nishikawa et al., 1994, Proc Natl Acad Sci USA 91:7727-7731; and Batra et al., 1995, Cell Growth and Differentiation 6:1251-1259). In addition to gliomas, EGFRvIII has been detected in ductal and intraductal breast carcinoma (Wikstrand et al., 1995, Cancer Res 55:3140-3148), non-small cell lung carcinomas (Garcia de Palazzo et al., 1993, Cancer Res 53:3217-3220), ovarian carcinomas (Moscatello et al., 1995, Cancer Res 55:5536-5539), prostate cancer (Olapade-Olaopa et al., 2000, British J Cancer 82:186-194), and squamous cell carcinoma of the head and neck (Tinhofer et al., 2011, Clin Cancer Res 17(15):5197-5204).

[0104] In still other exemplary embodiments, the present invention includes bispecific antigen-binding molecules that specifically bind CD3 and MUC16. Such molecules may be referred to herein as, e.g., "anti-CD3/anti-MUC16" or "anti-CD3.times.MUC16" or "CD3.times.MUC16" bispecific molecules, and so forth. The term "MUC16" refers to the human MUC16 protein unless specified as being from a non-human species (e.g., "mouse MUC16," "monkey MUC16," etc.).

[0105] Mucin 16 (MUC16; NCBI Reference Sequence: NP_078966.2, SEQ ID NO: 173), otherwise known as cancer antigen 125 (CA-125), is a mucin encoded by MUC16 gene in humans. The family of mucin proteins are known to protect the body from infection by pathogen binding to oligosaccharides in the extracellular domain, preventing the pathogen form reaching the cell surface. For many years, overexpression of MUC16/CA125 has been used as a prognostic and diagnostic marker for ovarian cancer (Yin and Lloyd, 2001, J. Biol. Chem. 276 (29), 27371-27375; O'Brien, T J, et al, 2001, Tumour Biol. 22 (6), 348-366; Leggieri, C. et al., 2014, Eur. J. Gynaecol. Oncol. 35 (4), 438-441). MUC16 has been shown to protect tumor cells from the immune system with its heavily glycosylated tandem repeat domain which can bind to galectin-1 (an immunosuppressive protein) (Seelenmeyer, C., et al., 2003, J. Cell. Sci. 116 (Pt 7): 1305-18; O'Brien, T J, et al., 2002, Tumour Biol. 23 (3), 154-169). Natural killer cells and monocytes are unable to attack tumor cells expressing high levels of MUC16. In its normal physiologic role, MUC16-galactin interaction serves as a barrier for bacterial and viral infection, however MUC16 is believed to be immunoprotective in the context of tumor cells, thereby preventing cancer cell cytolysis (Felder, M. et al., 2014, Molecular Cancer, 13:129). MUC16 is therefore a desirable target for immunotherapeutic bispecific antibody molecules administered to treat ovarian cancer by activating immune effector cells.

[0106] In still other exemplary embodiments, the present invention includes bispecific antigen-binding molecules that specifically bind CD3 and STEAP2. Such molecules may be referred to herein as, e.g., "anti-CD3/anti-STEAP2" or "anti-CD3.times.STEAP2" or "CD3.times.STEAP2" bispecific molecules, and so forth. The term "STEAP2" refers to the human STEAP2 protein unless specified as being from a non-human species (e.g., "mouse STEAP2," "monkey STEAP2," etc.). Six transmembrane epithelial antigen of the prostate 2 (STEAP2; UniProtKB/Swiss-Prot: Q8NFT2.3) is a 490-amino acid protein encoded by STEAP2 gene located at the chromosomal region 7q21 in humans.

[0107] The aforementioned bispecific antigen-binding molecules that specifically bind tumor-associated antigen comprise an anti-CD3 antigen-binding molecule which binds to CD3 with a weak or no detectable binding affinity such as exhibiting a K.sub.D of greater than about 100 nM, 300 nM or 500 nM, as measured by an in vitro affinity binding assay.

[0108] As used herein, the expression "antigen-binding molecule" means a protein, polypeptide or molecular complex comprising or consisting of at least one complementarity determining region (CDR) that alone, or in combination with one or more additional CDRs and/or framework regions (FRs), specifically binds to a particular antigen. In certain embodiments, an antigen-binding molecule is an antibody or a fragment of an antibody, as those terms are defined elsewhere herein.

[0109] As used herein, the expression "bispecific antigen-binding molecule" means a protein, polypeptide or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within the bispecific antigen-binding molecule comprises at least one CDR that alone, or in combination with one or more additional CDRs and/or FRs, specifically binds to a particular antigen. In the context of the present invention, the first antigen-binding domain specifically binds a first antigen (e.g., CD3), and the second antigen-binding domain specifically binds a second, distinct antigen (e.g., PSMA, MUC16, EGFRvIII or STEAP2).

[0110] In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule comprising a first and a second antigen-binding domain (e.g., a bispecific antibody), the CDRs of the first antigen-binding domain may be designated with the prefix "A1" and the CDRs of the second antigen-binding domain may be designated with the prefix "A2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

[0111] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate multimerizing domain. The association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerizing domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution. For example, a multimerizing domain may be a polypeptide comprising an immunoglobulin C.sub.H3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a C.sub.H2-C.sub.H3 domain), e.g., an Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0112] Bispecific antigen-binding molecules of the present invention will typically comprise two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

[0113] In certain embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residues. In other embodiments, the multimerizing domain is a cysteine residue, or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0114] Any bispecific antibody format or technology may be used to make the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab.sup.2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats).

[0115] In the context of bispecific antigen-binding molecules of the present invention, the multimerizing domains, e.g., Fc domains, may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wild-type, naturally occurring version of the Fc domain. For example, the invention includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a modification in a C.sub.H2 or a C.sub.H3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P).

[0116] The present invention also includes bispecific antigen-binding molecules comprising a first C.sub.H3 domain and a second Ig C.sub.H3 domain, wherein the first and second Ig C.sub.H3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig C.sub.H3 domain binds Protein A and the second Ig C.sub.H3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C.sub.H3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C.sub.H3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N, V397M, and V4221 by EU) in the case of IgG1 antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V4221 by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V821 (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221 by EU) in the case of IgG4 antibodies.

[0117] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise part or all of a C.sub.H2 sequence derived from a human IgG1, human IgG2 or human IgG4 C.sub.H2 region, and part or all of a C.sub.H3 sequence derived from a human IgG1, human IgG2 or human IgG4. A chimeric Fc domain can also contain a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" sequence, derived from a human IgG1, a human IgG2 or a human IgG4 hinge region, combined with a "lower hinge" sequence, derived from a human IgG1, a human IgG2 or a human IgG4 hinge region. A particular example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [IgG4 C.sub.H1]- [IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 C.sub.H2]- [IgG4 C.sub.H3]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [IgG1 C.sub.H1]- [IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 C.sub.H2]- [IgG1 C.sub.H3]. These and other examples of chimeric Fc domains that can be included in any of the antigen-binding molecules of the present invention are described in PCT International Publication No. WO2014/121087 A1, published Aug. 7, 2014, which is herein incorporated by reference in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, can have altered Fc receptor binding, which in turn affects Fc effector function.

[0118] In certain embodiments, the invention provides an antibody heavy chain wherein the heavy chain constant region (C.sub.H) region comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190 or SEQ ID NO: 191. In some embodiments, the heavy chain constant region (C.sub.H) region comprises an amino acid sequence selected form the group consisting of SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190 and SEQ ID NO: 191.

[0119] In other embodiments, the invention provides an antibody heavy chain wherein the Fc domain comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200 or SEQ ID NO: 201. In some embodiments, the Fc domain comprises an amino acid sequence selected form the group consisting of SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200 and SEQ ID NO: 201.

Other Fc Variants

[0120] According to certain embodiments of the present invention, anti-CD3 antibodies, and anti-CD3/anti-TAA bispecific antigen-binding molecules, are provided comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present invention includes antibodies comprising a mutation in the C.sub.H2 or a C.sub.H3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P).

[0121] For example, the present invention includes anti-CD3 antibodies, and anti-CD3/anti-TAA bispecific antigen-binding molecules, comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present invention.

Biological Characteristics of the Antibodies and Bispecific Antigen-Binding Molecules

[0122] The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies) which are capable of simultaneously binding to human CD3 and a human TAA. According to certain embodiments, the bispecific antigen-binding molecules of the invention specifically interact with cells that express CD3 and/or TAA, such as PSMA, EGFRvIII or MUC16. The binding arm that interacts with cells that express CD3 may have weak to no detectable binding as measured in a suitable in vitro binding assay. The extent to which a bispecific antigen-binding molecule binds cells that express CD3 and/or TAA can be assessed by fluorescence activated cell sorting (FACS), as illustrated in Example 4 herein.

[0123] For example, the present invention includes antibodies, antigen-binding fragments, and bispecific antibodies thereof which specifically bind human T-cell lines which express CD3 but not the TAA (e.g., Jurkat), primate T-cells (e.g., cynomolgus peripheral blood mononuclear cells [PBMCs]), and/or TAA-expressing cells. The present invention includes bispecific antigen-binding molecules which bind any of the aforementioned T cells and T cell lines with an EC.sub.50 value of from about 1.8.times.10.sup.-9 (18 nM) to about 2.1.times.10.sup.-7 (210 nM), or more (i.e. weaker affinity), and includes bispecific antibodies for which EC.sub.50 is undetectable, as determined using a FACS binding assay as set forth in Example 4 or a substantially similar assay. In certain embodiments, the antibodies, antigen-binding fragments, and bispecific antibodies of the present invention bind CD3 with an EC.sub.50 of greater than about 30 nM, greater than about 40 nM, greater than about 45 nM, greater than about 50 nM, greater than about 55 nM, greater than about 60 nM, greater than about 65 nM, greater than about 70 nM, greater than about 75 nM, at least 80 nM, greater than about 90 nM, greater than about 100 nM, greater than about 110 nM, at least 120 nM, greater than about 130 nM, greater than about 140 nM, greater than about 150 nM, at least 160 nM, greater than about 170 nM, greater than about 180 nM, greater than about 190 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 500 nM, greater than about 1 .mu.M, greater than about 2 .mu.M, or greater than about 3 .mu.M, or no detectable binding affinity, as measured by FACS binding, e.g., using an assay format as defined in Example 4 herein, or a substantially similar assay.

[0124] The present invention also includes antibodies, antigen-binding fragments, and bispecific antibodies thereof which bind to TAA-expressing cells and cell lines, such as PSMA-, EGFRvIII-, STEAP2- and MUC16-expressing cell lines, with an EC.sub.50 value of less than about 100 nM, or even less concentration necessary for binding (i.e. stronger affinity) such as less than 5.6 nM (5.6.times.10.sup.-9), as determined using a FACS binding assay as set forth in Example 4 or a substantially similar cell-based assay. The present invention includes bispecific antigen-binding molecules which bind any of the aforementioned tumor cell lines with an EC.sub.50 value of less than about 50 nM, less than about 45 nM, less than less 40 nM, less than about 35 nM, less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 6 nM, less than about 5 nM, or less than about 1 nM, e.g. using the aforementioned assay.

[0125] The present invention includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind human CD3 with low, weak or even no detectable affinity. According to certain embodiments, the present invention includes antibodies and antigen-binding fragments of antibodies that bind human CD3 (e.g., at 37.degree. C.) with a K.sub.D of greater than about 11 nM, includes antibodies that bind CD3 with a K.sub.D of greater than about 100 nM or 500 nM, and also includes antibodies having no detectable binding affinity, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 5 herein. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind CD3 with a K.sub.D of greater than about 15 nM, greater than about 20 nM, greater than about 25 nM, greater than about 30 nM, greater than about 35 nM, greater than about 40 nM, greater than about 45 nM, greater than about 50 nM, greater than about 55 nM, greater than about 60 nM, greater than about 65 nM, greater than about 70 nM, greater than about 75 nM, at least 80 nM, greater than about 90 nM, greater than about 100 nM, greater than about 110 nM, at least 120 nM, greater than about 130 nM, greater than about 140 nM, greater than about 150 nM, at least 160 nM, greater than about 170 nM, greater than about 180 nM, greater than about 190 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 1 .mu.M, greater than about 2 .mu.M, or greater than about 3 .mu.M, or no detectable affinity, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 5 herein (e.g., mAb-capture or antigen-capture format), or a substantially similar assay.

[0126] The present invention includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind monkey (i.e. cynomolgus) CD3 with low, weak, or even no detectable affinity. According to certain embodiments, the present invention includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind human CD3 (e.g., at 37.degree. C.) with a K.sub.D of greater than about 10 nM, includes antibodies that bind CD3 with a K.sub.D of greater than about 100 nM or 500 nM, and also includes antibodies having no detectable binding affinity, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 5 herein. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind CD3 with a K.sub.D of greater than about 15 nM, greater than about 20 nM, greater than about 25 nM, greater than about 30 nM, greater than about 35 nM, greater than about 40 nM, greater than about 45 nM, greater than about 50 nM, greater than about 55 nM, greater than about 60 nM, greater than about 65 nM, greater than about 70 nM, greater than about 75 nM, at least 80 nM, greater than about 90 nM, greater than about 100 nM, greater than about 110 nM, at least 120 nM, greater than about 130 nM, greater than about 140 nM, greater than about 150 nM, at least 160 nM, greater than about 170 nM, greater than about 180 nM, greater than about 190 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 1 .mu.M, greater than about 2 .mu.M, or greater than about 3 .mu.M, or no detectable affinity, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 5 herein (e.g., mAb-capture or antigen-capture format), or a substantially similar assay.

[0127] The present invention includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind human CD3 and induce T cell activation. For example, the present invention includes anti-CD3 antibodies that induce human T cell activation with an EC.sub.50 value of less than about 113 pM, as measured by an in vitro T cell activation assay, e.g., using the assay format as defined in Example 6 herein [e.g., assessing the percent activated (CD69+) cells out of total T cells (CD2+) in the presence of anti-CD3 antibodies], or a substantially similar assay that assesses T cell in their activated state. In certain embodiments, the antibodies or antigen-binding fragments of the present invention induce human T cell activation [e.g., percent activated (CD69+) T cells] with an EC.sub.50 value of less than about 100 pM, less than about 50 pM, less than about 20 pM, less than about 19 pM, less than about 18 pM, less than about 17 pM, less than about 16 pM, less than about 15 pM, less than about 14 pM, less than about 13 pM, less than about 12 pM, less than about 11 pM, less than about 10 pM, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM, or less than about 1 pM, as measured by an in vitro T cell activation assay, e.g., using the assay format as defined in Example 6 herein, or a substantially similar assay. Anti-CD3 antibodies that have weak or no detectable binding to CD3 have the ability to induce T cell activation with high potency (i.e. pM range), despite having weak or no detectable binding affinity to CD3, as exemplified in Example 6 herein.

[0128] The present invention also includes antibodies, antigen-binding fragments, and bispecific antibodies that bind human CD3 and induce T cell-mediated killing of tumor antigen-expressing cells. For example, the present invention includes anti-CD3 antibodies that induce T cell-mediated killing of tumor cells with an EC.sub.50 of less than about 1.3 nM, as measured in an in vitro T cell-mediated tumor cell killing assay, e.g., using the assay format as defined in Example 6 herein (e.g., assessing the extent of tumor antigen-expressing cells, such as PSMA-expressing, EGFRvIII-expressing or MUC16-expressing cell killing by human PBMCs in the presence of anti-CD3 antibodies), or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention induce T cell-mediated tumor cell killing (e.g., PBMC-mediated killing of OVCAR3 cells) with an EC.sub.50 value of less than about 1 nM, less than about 400 pM, less than about 250 pM, less than about 100 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM, or less than about 1 pM, as measured by an in vitro T cell-mediated tumor cell killing assay, e.g., using the assay format as defined in Example 6 herein, or a substantially similar assay.

[0129] The present invention also includes antibodies, antigen-binding fragments, and bispecific antibodies that bind CD3 with a dissociative half-life (t1/2) of less than about 10 minutes as measured by surface plasmon resonance at 25.degree. C. or 37.degree. C., e.g., using an assay format as defined in Example 5 herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind CD3 with a t1/2 of less than about 9 minutes, of less than about 8 minutes, of less than about 7 minutes, of less than about 6 minutes, of less than about 5 minutes, of less than about 4 minutes, of less than about 3 minutes, of less than about 2 minutes, of less than about 1.9 minutes, or less than about 1.8 minutes, or exhibit very weak or no detectable binding as measured by surface plasmon resonance at 25.degree. C. or 37.degree. C., e.g., using an assay format as defined in Example 5 herein (e.g., mAb-capture or antigen-capture format), or a substantially similar assay.

[0130] The anti-CD3/anti-TAA bispecific antigen-binding molecules of the present invention may additionally exhibit one or more characteristics selected from the group consisting of: (a) inducing PBMC proliferation in vitro; (b) activating T-cells via inducing IFN-gamma release and CD25 up-regulation in human whole blood; and (c) inducing T-cell mediated cytotoxicity on anti-TAA-resistant cell lines.

[0131] The present invention includes anti-CD3/anti-TAA bispecific antigen-binding molecules which are capable of depleting tumor antigen-expressing cells in a subject (see, e.g., Example 7). For example, according to certain embodiments, anti-CD3/anti-PSMA, anti-CD3/anti-MUC16, or anti-CD3/anti-STEAP2 bispecific antigen-binding molecules are provided, wherein a single administration of 1 .mu.g, or 10 .mu.g, or 100 .mu.g of the bispecific antigen-binding molecule to a subject (e.g., at a dose of about 0.1 mg/kg, about 0.08 mg/kg, about 0.06 mg/kg about 0.04 mg/kg, about 0.04 mg/kg, about 0.02 mg/kg, about 0.01 mg/kg, or less) causes a reduction in the number of tumor antigen-expressing cells in the subject (e.g., tumor growth in the subject is suppressed or inhibited) below detectable levels. In certain embodiments, a single administration of the anti-CD3/anti-PSMA bispecific antigen-binding molecule at a dose of about 0.4 mg/kg causes a reduction in tumor growth in the subject below detectable levels by about day 7, about day 6, about day 5, about day 4, about day 3, about day 2, or about day 1 after administration of the bispecific antigen-binding molecule to the subject. According to certain embodiments, a single administration of an anti-CD3/anti-PSMA bispecific antigen-binding molecule of the invention, at a dose of at least about 0.01 mg/kg, causes the number of PSMA-expressing tumor cells to remain below detectable levels until at least about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days or more, following the administration. As used herein, the expression "below detectable levels" means that no tumor cells can be directly or indirectly detected growing subcutaneously in a subject using standard caliper measurement methods, e.g., as set forth in Example 7, herein. In certain embodiments, a single administration of the anti-CD3/anti-MUC16 bispecific antigen-binding molecule at a dose of about 10 .mu.g causes a suppression of tumor growth in the subject at about day 6, and maintains tumor suppression until at least day 26 after administration of the bispecific antigen-binding molecule to the subject. In subjects receiving a single administration of the anti-CD3/anti-MUC16 bispecific antigen-binding molecule at a dose of about 10 .mu.g at least 7 days after tumor implantation, the bispecific antigen-binding molecule exhibits efficacy in suppression of established tumors from further growth in the subject at about day 26 after tumor implantation in the subject. According to certain embodiments, a single administration of an anti-CD3/anti-MUC16 bispecific antigen-binding molecule of the invention, at a dose of at least about 0.1 mg/kg, inhibits growth of MUC16-expressing tumor cells for at least about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or more, following administration of the bispecific molecule. See, e.g. Example 8.

[0132] In certain embodiments, a single administration of the anti-CD3/anti-STEAP2 bispecific antigen-binding molecule at a dose of about 0.1 mg/kg or 0.01 mg/kg maintains a suppression of tumor growth until at least day 46 after administration of the bispecific antigen-binding molecule and tumor to the subject. According to certain embodiments, a single administration of an anti-CD3/anti-STEAP2 bispecific antigen-binding molecule of the invention, at a dose of at least about 0.1 mg/kg, about 0.08 mg/kg, about 0.06 mg/kg, about 0.05 mg/kg, about 0.04 mg/kg, about 0.03 mg/kg, about 0.02 mg/kg, about 0.01 mg/kg, or less inhibits growth of STEAP2-expressing tumor cells for at least about 20 days, 30 days, 35 days, 40 days, 45 days or more, following administration of the bispecific molecule. See, e.g. Example 10.

[0133] In other embodiments, anti-CD3/anti-TAA bispecific antigen-binding molecules having a CD3 targeted binding arm having weak binding affinity to effector cells exhibit reduced drug elimination rates compared to the bispecific antibodies comprising the same anti-TAA binding arm and a strong CD3 binding arm administered in an in vivo pharmacokinetic study. The results suggest that the bispecific molecules comprising weaker binding of the CD3 targeting arm may exhibit beneficial drug exposure levels (AUC.sub.last) and drug elimination profiles (antibody clearance). See, e.g., Example 9.

[0134] The present invention provides anti-CD3/anti-PSMA, anti-CD3/anti-MUC16 and anti-CD3/anti-STEAP2 bispecific antigen-binding molecules (i.e. anti-CD3/anti-TAA bispecific antigen-binding molecules) which exhibit one or more characteristics selected from the group consisting of: (a) inhibiting tumor growth in immunocompromised mice bearing human prostate cancer xenografts; (b) inhibiting tumor growth in immunocompetent mice bearing human prostate cancer xenografts; (c) suppressing tumor growth of established tumors in immunocompromised mice bearing human prostate cancer xenografts; and (d) reducing tumor growth of established tumors in immunocompetent mice bearing human prostate cancer xenografts (see, e.g., Examples 7, 8 and 10). The present invention also provides anti-CD3/anti-PSMA, anti-CD3/anti-MUC16 and anti-CD3/anti-STEAP2 bispecific antibodies (i.e. anti-CD3/anti-TAA bispecific antibodies) comprising a first heavy chain directed to an effector T cell (i.e. CD3), and ii) a second heavy chain directed to a target tumor cell, wherein the bispecific antibodies exhibit weak binding or no detectable binding to the effector cells, and exhibit tumor growth suppression and reduced antibody clearance (i.e. elimination) from the body compared to bispecific antibodies that exhibit strong binding to effector cells.

Epitope Mapping and Related Technologies

[0135] The epitope on CD3 to which the antigen-binding molecules of the present invention bind may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a CD3 protein. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of CD3. The antibodies of the invention may interact with amino acids contained within a single CD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma), or may interact with amino acids on two or more different CD3 chains. The term "epitope," as used herein, refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0136] Various techniques known to persons of ordinary skill in the art can be used to determine whether an antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, e.g., routine cross-blocking assay such as that described Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutational analysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding domain of an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of the antigen/antibody complex may also be used for epitope mapping purposes.

[0137] The present invention further includes anti-PSMA antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g. antibodies comprising any of the amino acid sequences as set forth in Table 6 herein). Likewise, the present invention also includes anti-PSMA antibodies that compete for binding to PSMA with any of the specific exemplary antibodies described herein (e.g. antibodies comprising any of the amino acid sequences as set forth in Table 6 herein). Anti-PSMA antibodies disclosed in U.S. application Ser. No. 15/223,434 are herein incorporated by reference into this application.

[0138] The present invention also includes bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3 and/or cynomolgus CD3 with low or detectable binding affinity, and a second antigen binding domain that specifically binds human tumor-associated antigen (TAA), wherein the first antigen-binding domain binds to the same epitope on CD3 as any of the specific exemplary CD3-specific antigen-binding domains described herein.

[0139] Likewise, the present invention also includes bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3 and/or cynomolgus CD3 with low or detectable binding affinity, and a second antigen binding domain that specifically binds human tumor-associated antigen (TAA), wherein the first antigen-binding domain competes for binding to CD3 with any of the specific exemplary CD3-specific antigen-binding domains described herein.

[0140] One can easily determine whether a particular antigen-binding molecule (e.g., antibody) or antigen-binding domain thereof binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule of the present invention by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope on CD3 (or TAA) as a reference bispecific antigen-binding molecule of the present invention, the reference bispecific molecule is first allowed to bind to a CD3 protein (or TAA protein). Next, the ability of a test antibody to bind to the CD3 molecule is assessed. If the test antibody is able to bind to CD3 (or TAA) following saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope of CD3 (or TAA) than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody is not able to bind to the CD3 (or TAA) molecule following saturation binding with the reference bispecific antigen-binding molecule, then the test antibody may bind to the same epitope of CD3 (or TAA) as the epitope bound by the reference bispecific antigen-binding molecule of the invention. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference bispecific antigen-binding molecule or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. If the reference antibody is one that has no measurable binding as exemplified herein, then the reference antibody may be mutated back to germline sequence in order to determine binding to the CD3 for purpose of comparing epitope interaction, or comparing its binding properties to the test antibody as described herein. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. In accordance with certain embodiments of the present invention, two antigen-binding proteins bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antigen-binding protein inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen-binding proteins are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other. Two antigen-binding proteins are deemed to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other.

[0141] To determine if an antibody or antigen-binding domain thereof competes for binding with a reference antigen-binding molecule, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antigen-binding molecule is allowed to bind to a CD3 protein (or TAA protein) under saturating conditions followed by assessment of binding of the test antibody to the CD3 (or TAA) molecule. In a second orientation, the test antibody is allowed to bind to a CD3 (or TAA) molecule under saturating conditions followed by assessment of binding of the reference antigen-binding molecule to the CD3 (or TAA) molecule. If, in both orientations, only the first (saturating) antigen-binding molecule is capable of binding to the CD3 (or TAA) molecule, then it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to CD3 (or TAA). As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope. If the reference antibody is one that has no measurable binding as exemplified herein, then the reference antibody may be mutated back to germline sequence in order to determine binding to the CD3 for purpose of comparing epitope interaction, or comparing its binding properties or blocking interaction with the test antibody as described herein.

Preparation of Antigen-Binding Domains and Construction of Bispecific Molecules

[0142] Antigen-binding domains specific for particular antigens can be prepared by any antibody generating technology known in the art. Once obtained, two different antigen-binding domains, specific for two different antigens (e.g., CD3 and TAA), can be appropriately arranged relative to one another to produce a bispecific antigen-binding molecule of the present invention using routine methods. (A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the present invention is provided elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules of the invention are derived from chimeric, humanized or fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more of the heavy and/or light chains of the bispecific antigen-binding molecules of the present invention can be prepared using VELOCIMMUNE.TM. technology. Using VELOCIMMUNE.TM. technology (or any other human antibody generating technology), high affinity chimeric antibodies to a particular antigen (e.g., CD3 or TAA) are initially isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate fully human heavy and/or light chains that can be incorporated into the bispecific antigen-binding molecules of the present invention.

[0143] Genetically engineered animals may be used to make human bispecific antigen-binding molecules. For example, a genetically modified mouse can be used which is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa (.kappa.) constant gene at the endogenous mouse kappa (.kappa.) locus. Such genetically modified mice can be used to produce fully human bispecific antigen-binding molecules comprising two different heavy chains that associate with an identical light chain that comprises a variable domain derived from one of two different human light chain variable region gene segments. (See, e.g., US 2011/0195454 for a detailed discussion of such engineered mice and the use thereof to produce bispecific antigen-binding molecules). Antibodies of the invention may comprise immunoglobulin heavy chains associated with a common light chain. The common light chain may be derived from a cognate light chain of the anti-TAA heavy chain, or derived from a known or public domain light chain variable region derived from a light chain exhibiting promiscuity or ability to pair with a wide variety of non-cognate heavy chains, i.e. a universal or common light chain. Antibodies of the invention may comprise immunoglobulin heavy chains associated with a single rearranged light chain. In some embodiments, the light chain a variable domain derived from a human V.kappa.1-39 gene segment or a V.kappa.3-20 gene segment. In other embodiments, the light chain comprises a variable domain derived from a human V.kappa.1-39 gene segment rearranged with a human J.kappa.5 or a human J.kappa.1 gene segment, or a V.kappa.3-20 gene segment rearranged with a human J.kappa.1 gene segment, or a V.kappa.1-39 gene segment rearranged with a human J.kappa.1 gene segment.

Bioequivalents

[0144] The present invention encompasses antigen-binding molecules having amino acid sequences that vary from those of the exemplary molecules disclosed herein but that retain the ability to bind or interact with CD3 and/or TAA. Such variant molecules may comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described bispecific antigen-binding molecules.

[0145] The present invention includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules set forth herein. Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antigen-binding proteins will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

[0146] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0147] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[0148] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[0149] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.

[0150] Bioequivalent variants of the exemplary bispecific antigen-binding molecules set forth herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antigen-binding proteins may include variants of the exemplary bispecific antigen-binding molecules set forth herein comprising amino acid changes which modify the glycosylation characteristics of the molecules, e.g., mutations which eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity

[0151] According to certain embodiments of the invention, antigen-binding molecules are provided which display weak or no interaction with human CD3 and weak or no interaction with CD3 from other species, such as cynomolgous monkey CD3. Also provided are antigen-binding molecules which bind to human TAA but not to TAA from other species. The present invention also includes antigen-binding molecules that bind to human CD3 and to CD3 from one or more non-human species; and/or antigen-binding molecules that bind to human TAA and to TAA from one or more non-human species.

[0152] According to certain exemplary embodiments of the invention, antigen-binding molecules are provided which bind weakly to human CD3 and/or human TAA and may bind or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus or chimpanzee CD3 and/or TAA. For example, in certain exemplary embodiments of the present invention bispecific antigen-binding molecules are provided comprising a first antigen-binding domain that weakly binds human CD3 and cynomolgus CD3, and a second antigen-binding domain that specifically binds human PSMA, MUC16, EGFRvIII or STEAP2.

Immunoconjugates

[0153] The present invention encompasses antigen-binding molecules conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxic agents include any agent that is detrimental to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immunoconjugates are known in the art, (see for example, WO 05/103081).

Therapeutic Formulation and Administration

[0154] The present invention provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN.TM., Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0155] The dose of antigen-binding molecule administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When a bispecific antigen-binding molecule of the present invention is used for therapeutic purposes in an adult patient, it may be advantageous to intravenously administer the bispecific antigen-binding molecule of the present invention normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering a bispecific antigen-binding molecule may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0156] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[0157] A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0158] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to AUTOPEN.TM. (Owen Mumford, Inc., Woodstock, UK), DISETRONIC.TM. pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25.TM. pen, HUMALOG.TM. pen, HUMALIN 70/30.TM. pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN.TM. I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR.TM. (Novo Nordisk, Copenhagen, Denmark), BD.TM. pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN.TM., OPTIPEN PRO.TM., OPTIPEN STARLET.TM., and OPTICLIK.TM. (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to the SOLOSTAR.TM. pen (sanofi-aventis), the FLEXPEN.TM. (Novo Nordisk), and the KWIKPEN.TM. (Eli Lilly), the SURECLICK.TM. Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET.TM. (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA.TM. Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

[0159] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0160] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0161] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antigen-Binding Molecules

[0162] The present invention includes methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-tumor antibody or antigen-binding fragment thereof, or a bispecific antigen-binding molecule that specifically binds weakly or has no detectable binding to CD3 and binds a tumor-associated antigen. The therapeutic composition can comprise any of the antibodies or bispecific antigen-binding molecules as disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "a subject in need thereof" means a human or non-human animal that exhibits one or more symptoms or indicia of cancer (e.g., a subject expressing a tumor or suffering from any of the cancers mentioned herein below), or who otherwise would benefit from an inhibition or reduction in tumor activity or a depletion of tumor cells (e.g., PSMA++ prostate cancer cells).

[0163] The antibodies and bispecific antigen-binding molecules of the invention (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which stimulation, activation and/or targeting of an immune response would be beneficial. In particular, the bispecific antigen-binding molecules of the present invention may be used for the treatment, prevention and/or amelioration of any disease or disorder associated with or mediated by a cell expressing a TAA, e.g. PSMA expression or activity or the proliferation of PSMA+ cells. The mechanism of action by which the therapeutic methods of the invention are achieved include killing of the cells expressing tumor-associated antigens, in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. Cells expressing tumor-associated antigens, such as PSMA, MUC16, STEAP2 or EGFRvIII, which can be inhibited or killed using the bispecific antigen-binding molecules of the invention include, for example, prostate tumor cells.

[0164] The antigen-binding molecules of the present invention may be used to treat, e.g., primary and/or metastatic tumors arising in the brain and meninges, head and neck, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, kidney, bladder and/or special sensory organs such as the eye. In certain embodiments, the bispecific antigen-binding molecules of the invention are used to treat one or more of, but not limited to, the following cancers: pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanomaglioma, breast cancer (e.g. ductal or intraductal breast carcinoma, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, (renal) oncocytoma, (renal) transitional cell carcinoma, urothelial carcinoma, (bladder) adenocarcinoma, or (bladder) small cell carcinoma. According to certain embodiments of the present invention, the bispecific antibodies are useful for treating a patient afflicted with a refractory or treatment-resistant cancer, e.g. castrate-resistant prostate cancer. According to exemplary embodiments of the invention, methods are provided comprising administering an anti-CD3/anti-PSMA bispecific antigen-binding molecule as disclosed herein to a patient who is afflicted with a castrate-resistant prostate cancer. Analytic/diagnostic methods known in the art, such as tumor scanning, etc., may be used to ascertain whether a patient harbors a tumor that is castrate-resistant.

[0165] The present invention also includes methods for treating residual cancer in a subject. As used herein, the term "residual cancer" means the existence or persistence of one or more cancerous cells in a subject following treatment with an anti-cancer therapy, such as a first-line or standard therapy.

[0166] According to certain aspects, the present invention provides methods for treating a cancer associated with TAA expression (e.g., prostate cancer associated with PSMA expression or STEAP2 expression, glioblastoma associated with EGFRvIII expression, or ovarian cancer associated with MUC16 expression) comprising administering one or more of the bispecific antigen-binding molecules described elsewhere herein to a subject after the subject has been determined to have the cancer. For example, the present invention includes methods for treating prostate cancer comprising administering an anti-CD3/anti-TAA bispecific antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received a previous therapy.

Combination Therapies and Formulations

[0167] The present invention provides methods which comprise administering a pharmaceutical composition comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined with or administered in combination with an antigen-binding molecule of the present invention include, e.g., an anti-Programmed Cell Death 1 antibody (e.g. an anti-PD1 antibody as described in U.S. Pat. Appln. Pub. No. US2015/0203579A1), an anti-Programmed Cell Death Ligand-1 (e.g. an anti-PD-L1 antibody as described in U.S. Pat. Appln. Pub. No. US2015/0203580A1), an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member such as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvIII (e.g., an antibody that specifically binds EGFRvIII), a cMET anagonist (e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-.alpha. inhibitor (e.g., an anti-PDGFR-.alpha. antibody), a PDGFR-.beta. inhibitor (e.g., an anti-PDGFR-.beta. antibody), a VEGF antagonist (e.g., a VEGF-Trap, see, e.g., U.S. Pat. No. 7,087,411 (also referred to herein as a "VEGF-inhibiting fusion protein"), anti-VEGF antibody (e.g., bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2 antibody disclosed in US 2011/0027286 such as H1H685P), a FOLH1 (PSMA) antagonist, a PRLR antagonist (e.g., an anti-PRLR antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-STEAP1 antibody or an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin antibody), etc. Other agents that may be beneficially administered in combination with the antigen-binding molecules of the invention include cytokine inhibitors, including small-molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or to their respective receptors. The pharmaceutical compositions of the present invention (e.g., pharmaceutical compositions comprising an anti-CD3/anti-PSMA bispecific antigen-binding molecule as disclosed herein) may also be administered as part of a therapeutic regimen comprising one or more therapeutic combinations selected from "ICE": ifosfamide (e.g., Ifex.RTM.), carboplatin (e.g., Paraplatin.RTM.), etoposide (e.g., Etopophos.RTM., Toposar.RTM., VePesid.RTM., VP-16); "DHAP": dexamethasone (e.g., Decadron.RTM.), cytarabine (e.g., Cytosar-U.RTM., cytosine arabinoside, ara-C), cisplatin (e.g., Platinol.RTM.-AQ); and "ESHAP": etoposide (e.g., Etopophos.RTM., Toposar.RTM., VePesid.RTM., VP-16), methylprednisolone (e.g., Medrol.RTM.), high-dose cytarabine, cisplatin (e.g., Platinol.RTM.-AQ).

[0168] The present invention also includes therapeutic combinations comprising any of the antigen-binding molecules mentioned herein and an inhibitor of one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-.alpha., PDGFR-.beta., FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, an siRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies and minimal recognition units). The antigen-binding molecules of the invention may also be administered and/or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs. The antigen-binding molecules of the invention may also be administered as part of a treatment regimen that also includes radiation treatment and/or conventional chemotherapy.

[0169] The additional therapeutically active component(s) may be administered just prior to, concurrent with, or shortly after the administration of an antigen-binding molecule of the present invention; (for purposes of the present disclosure, such administration regimens are considered the administration of an antigen-binding molecule "in combination with" an additional therapeutically active component).

[0170] The present invention includes pharmaceutical compositions in which an antigen-binding molecule of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.

Administration Regimens

[0171] According to certain embodiments of the present invention, multiple doses of the bispecifc antigen-binding molecule (e.g., an anti-TAA bispecific antigen-binding molecule) may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an antigen-binding molecule of the invention. As used herein, "sequentially administering" means that each dose of an antigen-binding molecule is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present invention includes methods which comprise sequentially administering to the patient a single initial dose of an antigen-binding molecule, followed by one or more secondary doses of the antigen-binding molecule, and optionally followed by one or more tertiary doses of the antigen-binding molecule.

[0172] The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of administration of the antigen-binding molecule of the invention. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses" are the doses which are administered after the initial dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of the antigen-binding molecule, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of an antigen-binding molecule contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").

[0173] In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 11/2, 2, 21/2, 3, 31/2, 4, 41/2, 5, 51/2, 6, 61/2, 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2, 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22, 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of antigen-binding molecule which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses.

[0174] The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and/or tertiary doses of an antigen-binding molecule (e.g., an anti-TAA bispecific antigen-binding molecule). For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0175] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

EXAMPLES

[0176] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1: Generation of Anti-CD3 Antibodies

[0177] The following procedures were aimed at identifying antibodies that specifically recognized CD3 (T cell co-receptor) as an antigen.

[0178] A pool of anti-CD3 antibodies were derived by immunizing genetically modified mice. Briefly, mice genetically engineered to express reverse chimeric (human variable, mouse constant) and immunoglobulin heavy chains associated with a single rearranged light chain (e.g., a V.sub.K1-39/J or a V.sub.K3-20/J), were immunized with a CD3 antigen and generated B cells that comprised a diversity of human VH rearrangements in order to express a diverse repertoire of high-affinity antigen-specific antibodies. Certain exemplified antibodies described in the subject application have been made recombinantly and express the same light chain sequence of V.sub.K1-39J.kappa.5 (LCVR set forth in SEQ ID NO: 162), while other antibodies made recombinantly express a cognate light chain of one of the heavy chain arms (e.g. the tumor target arm).

[0179] Generated antibodies were tested for affinity to human and cynomolgus monkey CD3 antigen in an in vitro binding assay, and e.g. one CD3 antibody: designated CD3-VH-P (HCVR set forth in SEQ ID NO: 154) was identified, amongst a few others, that were found to bind to both human and cyno CD3 having an EC.sub.50 between 1 and 40 nM affinity (+++), as determined in a FACS titration of Jurkat cells and cynomolgus T cells, respectively. See, e.g. FACS binding experiments outlined in Example 4 herein below.

[0180] The germline amino acid residues of CD3-VH-P were subsequently identified and an antibody designated "CD3-VH-G" was engineered to contain only germline frameworks. Other antibody derivatives were engineered by well-known molecular cloning techniques to replace amino acid residues in a stepwise manner based on differences between the germline sequence and the CD3-VH-P sequence. Each antibody derivative is given a "CD3-VH-G" number designation. See Table 1 and FIG. 1.

[0181] Bispecific antibodies, comprising a first binding arm derived from the engineered anti-CD3 antibodies with the designations and descriptions shown in Table 1, and a second binding arm derived from anti-TAA antibodies, were prepared and tested for monovalent affinity to CD3-bearing cells in a FACS assay (as described in Example 4). The monovalent binding affinity results of these bispecific antibodies are shown in the two right columns of Table 1. In specific examples, bispecific antibodies having a TAA-binding arm and a CD3-binding arm with designations "CD3-VH-G," "CD3-VH-G5," and "CD3-VH-G20," respectively, bound Jurkat cells with an EC.sub.50 of 2.7E-08, no detectable binding, and 5.5E-07, respectively.

TABLE-US-00001 TABLE 1 Mutations to CDRs Based on the Germline Sequence and Corresponding FACS Binding Affinity for Each Engineered Antibody Antibody CD3-VH Description of Mutations compared cyno T Designation to antibody CD3-VH-G* JURKAT cells CD3-VH-G Germline (GL) only framework (+ + +) (+ + +) regions (FRs); CD3-VHP CDRs CD3-VH-G2 All GL (FRs and CDRs). (-) (-) CD3-VH-G3 All GL (FRs and CDRs). (-) (-) Add back A33S. CD3-VH-G4 All GL (FRs and CDRs). (-) (-) Add back Y105K CD3-VH-G5 All GL (FRs and CDRs). (-) (-) Add back A33S and Y105K. CD3-VH-G8 Germline frameworks. (+ + +) (+) Add back K58I CD3-VH-G9 Germline frameworks. (+) (-) Add backY99D CD3-VH-G10 Germline frameworks. (+) (-) Add back H108Y CD3-VH-G11 Germline frameworks. (+ + +) (+) Add back L111M CD3-VH-G12 Germline frameworks. (+ +) (+/-) Add back K58I, Y99D CD3-VH-G13 Germline frameworks. (+ +) (+) Add back K58I, H108Y CD3-VH-G14 Germline frameworks. (+ + +) (+ +) Add back K58I, L111M CD3-VH-G15 Germline frameworks. (+) (-) Add back Y99D, -H108Y CD3-VH-G16 Germline frameworks. (+ +) (+/-) Add back Y99D, L111M CD3-VH-G17 Germline frameworks. (+/-) (+/-) Add back H108Y, L111M CD3-VH-G18 Germline frameworks. (+/-) (-) Add back K58I, Y99D, H108Y CD3-VH-G19 Germline frameworks. (+/-) (-) Add back K58I, Y99D, L111M CD3-VH-G20 Germline frameworks. (+/-) (+/-) Add back K58I, H108Y, L111M CD3-VH-G21 Germline frameworks. (+/-) (-) Add back Y99D, H108Y, -L111M *Sequential numbering based on 7221G (CD3-VH-G) mature protein

[0182] While CD3-VH-G and some other engineered antibodies retained their binding affinity as seen in the FACS assays, several anti-CD3 antibodies bound to human or cyno CD3 in vitro with weak (+/-) to no (-) measurable affinity. Binding affinities, binding kinetics, and other biological properties to elucidate toxicity and pharmacokinetic (pK) profiles were subsequently investigated for bispecific antibodies comprising the exemplary anti-CD3 antibodies generated in accordance with the methods of this Example, and are described in detail in the Examples set forth below.

Example 2: Heavy and Light Chain Variable Regions (Amino Acid and Nucleic Acid Sequences of the CDRs)

[0183] Amino acid and nucleic acid sequences were determined for each antibody heavy chain sequence. Each antibody heavy chain, as a derivative of the germline sequence IGHV3-9*01/D5-12*01/J6*02 (SEQ ID NO: 181) was assigned a "G" number designation for consistent nomenclature. Table 2 sets forth the amino acid sequence identifiers of the heavy chain variable regions and CDRs of the engineered anti-CD3 antibodies of the invention. The corresponding nucleic acid sequence identifiers are set forth in Table 3. The amino acid and nucleic acid sequence identifiers of the light chain variable region and CDRs to construct each recombinant antibody are also identified below in Tables 4 and 5, respectively.

TABLE-US-00002 TABLE 2 Heavy Chain Amino Acid Sequence Identifiers Antibody CD3-VH SEQ ID NOs: Designation HCVR CDR1 CDR2 CDR3 CD3-VH-G 2 4 6 8 CD3-VH-G2 10 12 14 16 CD3-VH-G3 18 20 22 24 CD3-VH-G4 26 28 30 32 CD3-VH-G5 34 36 38 40 CD3-VH-G8 42 44 46 48 CD3-VH-G9 50 52 54 56 CD3-VH-G10 58 60 62 64 CD3-VH-G11 66 68 70 72 CD3-VH-G12 74 76 78 80 CD3-VH-G13 82 84 86 88 CD3-VH-G14 90 92 94 96 CD3-VH-G15 98 100 102 104 CD3-VH-G16 106 108 110 112 CD3-VH-G17 114 116 118 120 CD3-VH-G18 122 124 126 128 CD3-VH-G19 130 132 134 136 CD3-VH-G20 138 140 142 144 CD3-VH-G21 146 148 150 152 CD3-VH-P 154 156 158 160

TABLE-US-00003 TABLE 3 Heavy Chain Nucleic Acid Sequence Identifiers Antibody CD3-VH SEQ ID NOs: Designation HCVR CDR1 CDR2 CDR3 CD3-VH-G 1 3 5 7 CD3-VH-G2 9 11 13 15 CD3-VH-G3 17 19 21 23 CD3-VH-G4 25 27 29 31 CD3-VH-G5 33 35 37 39 CD3-VH-G8 41 43 45 47 CD3-VH-G9 49 51 53 55 CD3-VH-G10 57 59 61 63 CD3-VH-G11 65 67 69 71 CD3-VH-G12 73 75 77 79 CD3-VH-G13 81 83 85 87 CD3-VH-G14 89 91 93 95 CD3-VH-G15 97 99 101 103 CD3-VH-G16 105 107 109 111 CD3-VH-G17 113 115 117 119 CD3-VH-G18 121 123 125 127 CD3-VH-G19 129 131 133 135 CD3-VH-G20 137 139 141 143 CD3-VH-G21 145 147 149 151 CD3-VH-P 153 155 157 159

TABLE-US-00004 TABLE 4 Light Chain Amino Acid Sequence Identifiers Antibody ULC SEQ ID NOs: Designation LCVR CDR1 CDR2 CDR3 VK1-39JK5 162 164 166 168

TABLE-US-00005 TABLE 5 Light Chain Nucleic Acid Sequence Identifiers Antibody ULC SEQ ID NOs: Designation LCVR CDR1 CDR2 CDR3 VK1-39JK5 161 163 165 167

[0184] Control 1 antibody designated "CD3-L2K" was constructed based on a known anti-CD3 antibody (i.e., the anti-CD3 antibody "L2K" as set forth in WO2004/106380).

[0185] Isotype Control Antibody, referred to in the Examples hereinbelow, is an isotype matched (modified IgG4) antibody that interacts with an irrelevant antigen, i.e. FelD1 antigen.

Example 3: Generation of ULC Bispecific Antibodies that Bind CD3 and Tumor-Associated Antigens (TAA)

[0186] Bispecific antibodies comprising an anti-CD3-specific binding domain and an anti-TAA-specific binding domain, such as PSMA, EGFRvIII, MUC16, or STEAP2, were constructed using standard molecular biology methodologies utilizing a heavy chain from an anti-CD3 antibody described herein, a heavy chain from an anti-TAA antibody and a common light chain or a universal light chain (ULC). The anti-TAA antibodies used to construct the bispecific antibodies of this invention were obtained by immunizing genetically modified mice.

[0187] A summary of the component parts of the antigen-binding domains of the various bispecific antibodies made in accordance with this Example is set forth below in Tables 6, 7 and 8. All bispecific antibodies were manufactured having a modified (chimeric) IgG4 Fc domain as set forth in US Patent Application Publication No. US20140243504A1, published on Aug. 28, 2014. Exemplary EGFRvIIIxCD3 bispecific antibodies can be prepared using any of the heavy chain and light chain variable regions (or CDRs) of any of the EGFRvIII antibodies discussed in US Patent Application Publication NO. US20150259423, which is hereby incorporated by reference in its entirety, in combination with the variable regions or CDRs of any of the anti-CD3 antibodies discussed herein.

TABLE-US-00006 TABLE 6 Construction of PSMAxCD3 Bispecific Antibodies Anti-PSMA Antigen- Binding Anti-CD3 Common Domain Antigen-Binding Light Heavy Chain Domain Chain Bispecific Antibody Variable Heavy Chain Variable Identifier Region Variable Region Region BSPSMA/CD3-003 PSMA-VH-B CD3-VH-G VK1-39JK5 BSPSMA/CD3-200 CD3-VH-G2 BSPSMA/CD3-300 CD3-VH-G3 BSPSMA/CD3-400 CD3-VH-G4 BSPSMA/CD3-004 CD3-VH-G5 BSPSMA/CD3-800 CD3-VH-G8 BSPSMA/CD3-900 CD3-VH-G9 BSPSMA/CD3-1000 CD3-VH-G10 BSPSMA/CD3-1100 CD3-VH-G11 BSPSMA/CD3-1200 CD3-VH-G12 BSPSMA/CD3-1300 CD3-VH-G13 BSPSMA/CD3-1400 CD3-VH-G14 BSPSMA/CD3-1500 CD3-VH-G15 BSPSMA/CD3-1600 CD3-VH-G16 BSPSMA/CD3-1700 CD3-VH-G17 BSPSMA/CD3-1800 CD3-VH-G18 BSPSMA/CD3-1900 CD3-VH-G19 BSPSMA/CD3-005 CD3-VH-G20 BSPSMA/CD3-2100 CD3-VH-G21

TABLE-US-00007 TABLE 7 Construction of EGFRvIII xCD3 Bispecific Antibodies Anti-EGFRvIII Anti-CD3 Antigen-Binding Antigen-Binding Bispecific Domain Domain Common Light Antibody Heavy Chain Heavy Chain Chain Variable Identifier Variable Region Variable Region Region BSV3/CD3-001 EGFRvIII-VH-A CD3-VH-G EGFRvIII- BSV3/CD3-002 CD3-VH-G5 VL-A BSV3/CD3-003 CD3-VH-G9 BSV3/CD3-004 CD3-VH-G10

TABLE-US-00008 TABLE 8 Construction of MUC16xCD3 Bispecific Antibodies Anti-MUC16 Anti-CD3 Common Antigen-Binding Antigen-Binding Light Domain Domain Chain Bispecific Antibody Heavy Chain Heavy Chain Variable Identifier Variable Region Variable Region Region BSMUC16/CD3-001 MUC16-VH-A CD3-VH-G MUC16- BSMUC16/CD3-002 CD3-VH-G5 VL-A BSMUC16/CD3-003 CD3-VH-G9 BSMUC16/CD3-004 CD3-VH-G10 BSMUC16/CD3-005 CD3-VH-G20

TABLE-US-00009 TABLE 9 Construction of STEAP2xCD3 Bispecific Antibodies Anti-STEAP2 Anti-CD3 Common Antigen-Binding Antigen-Binding Light Domain Domain Chain Bispecific Heavy Chain Heavy Chain Variable Antibody Identifier Variable Region Variable Region Region BSSTEAP2/CD3-001 STEAP2-VH-A CD3-VH-G STEAP2- BSSTEAP2/CD3-002 CD3-VH-G5 VL-A BSSTEAP2/CD3-003 CD3-VH-G20

[0188] Each of the exemplary bispecific antibodies were tested in various bioassays as described herein below.

Example 4: Binding Affinities of Exemplified Bispecific Antibodies as Measured by FACS Analysis

[0189] In this example, the ability of CD3.times.TAA bispecific antibodies to bind to human and cynomolgus CD3-expressing cell lines via FACS was determined. Additionally, the ability of these bispecific antibodies to bind to target-specific (TAA-specific) cell lines was also confirmed. As described above, the various bispecific antibodies of this invention utilized a single TAA-specific binding arm (PSMA, EGFRvIII, MUC16, or STEAP2; see Example 3, Tables 6, 7 and 8) paired with one of a panel of anti-CD3 binding arms (see Examples 1 and 2 hereinabove) and a common light chain. As is also shown in Example 5, the CD3.times.TAA bispecific antibodies displayed a range of affinities to human soluble heterodimeric hCD3.epsilon./.delta..mFc protein via surface plasmon resonance.

[0190] Briefly, 2.times.10.sup.5 cells/well of human CD3-expressing Jurkat, cynomolgus T or TAA-specific expressing cells were incubated with a serial dilution of bispecific antibodies for 30 min at 4.degree. C. After incubation, cells were washed and a goat F(ab').sub.2 anti-human Fc.gamma. PE labeled secondary (Jackson Immunolabs) was added to the cells for an additional 30 min. Next, cells were washed, re-suspended in cold PBS+1% BSA and analyzed via flow cytometry on a BD FACS Canto II.

[0191] For FACS analysis, cells were gated by forward scatter height vs. forward scatter area for single events selection, followed by side and forward scatters. The EC.sub.50 for cell binding titration was determined using PRISM.TM. software (GraphPad Software, Inc., La Jolla, Calif.). Values were calculated using 4-parameter non-linear regression analysis.

TABLE-US-00010 TABLE 10A FACS Binding on CD3 and PSMA-Specific Cell lines Anti-CD3- Bispecific Binding Jurkat Cyno T-cells B16F10.9/PSMA Antibody Identifier Arm EC.sub.50 [M] EC.sub.50 [M] EC.sub.50 [M] BSPSMA/CD3-003 CD3-VH-G 1.65E-08 1.42E-08 2.26E-09 BSPSMA/CD3-200 CD3-VH-G2 NB NB 1.88E-09 BSPSMA/CD3-300 CD3-VH-G3 NB NB 1.90E-09 BSPSMA/CD3-400 CD3-VH-G4 NB NB 1.72E-09 BSPSMA/CD3-004 CD3-VH-G5 Very weak NB 1.31E-09 BSPSMA/CD3-800 CD3-VH-G8 1.93E-08 1.96E-08 1.31E-09 BSPSMA/CD3-900 CD3-VH-G9 2.74E-07 NB 1.43E-09 BSPSMA/CD3-1000 CD3-VH-G10 2.77E-07 NB 1.19E-09 BSPSMA/CD3-1100 CD3-VH-G11 1.83E-08 8.90E-07 1.03E-09 BSPSMA/CD3-1200 CD3-VH-G12 4.72E-08 NB 1.16E-09 BSPSMA/CD3-1300 CD3-VH-G13 1.02E-07 2.17E-06 1.25E-09 BSPSMA/CD3-1400 CD3-VH-G14 3.19E-08 1.70E-07 1.30E-09 BSPSMA/CD3-1500 CD3-VH-G15 9.30E-08 NB 1.21E-09 BSPSMA/CD3-1600 CD3-VH-G16 5.68E-08 NB 1.03E-09 BSPSMA/CD3-1700 CD3-VH-G17 2.00E-07 3.35E-06 1.34E-09 BSPSMA/CD3-1800 CD3-VH-G18 1.26E-07 NB 2.16E-09 BSPSMA/CD3-1900 CD3-VH-G19 6.07E-08 NB 1.35E-09 BSPSMA/CD3-005 CD3-VH-G20 2.10E-07 6.14E-06 2.09E-09 BSPSMA/CD3-2100 CD3-VH-G21 1.06E-07 NB 1.14E-09

TABLE-US-00011 TABLE 10B FACS Binding on CD3 and EGFRvIII-Specific Cell lines Anti-CD3 Cyno T- U87/ Bispecific Antibody Binding Jurkat Cells EGFRvIII Identifier Arm EC.sub.50 [M] EC.sub.50 [M] EC.sub.50 [M] BSV3/CD3-001 CD3-VH-G 1.46E-09 NT 2.40E-09 BSV3/CD3-002 CD3-VH-G5 Very weak NT 5.60E-09

TABLE-US-00012 TABLE 10C FACS Binding on CD3 and MUC16-Specific Cell lines Cyno T- OVCAR3 Bispecific Antibody Anti-CD3 Jurkat cells (MUC16+) Identifier Binding Arm EC.sub.50 [M] EC.sub.50 [M] EC.sub.50 [M] BSMUC16/ CD3-VH-G 3.21E-09 NT 1.20E-09 CD3-001 BSMUC16/ CD3-VH-G5 Very weak NT 2.69E-09 CD3-002

[0192] As shown in Table 10A, the CD3 binding arms of each CD3.times.PSMA bispecific antibody displayed a range of cell binding affinity to human CD3 expressing Jurkat cells (15 to 300 nM EC.sub.50 range). Importantly, the CD3 arms that showed weak-to-no binding to human CD3 heterodimeric protein via surface plasmon resonance (see Table 11 hereinbelow) also correlated with weak to no observable binding on Jurkat cells (i.e. CD3-VH-G2, CD3-VH-G3, CD3-VH-G5). Non-detectable binding, or no detectable binding, in the FACS assay or equivalent assay means that the affinity between the antibody and its target antigen is beyond the detection limit of the assay (e.g. >1 .mu.M). Several CD3-binding arms also displayed cross reactivity to cynomolgus T-cells. All tested bispecific antibodies displayed similar cell binding on respective PSMA, EGFRvIII and MUC16-expressing cell lines, confirming that bispecific pairing with individual CD3 arms did not affect or diminish TAA-specific binding (TAA-specific binding was less than or equal to 5.6 nM (high affinity) in all examples tested).

[0193] Antibodies exhibiting weak-to-no detectable binding to human CD3, and also exhibiting weak-to-no binding to cynomolgus CD3, are considered advantageous for avidity-driven bispecific pairing in accordance with the present invention, and were further tested for cytotoxicity in in vitro and in vivo assays.

Example 5: Binding Affinities of Exemplified Antibodies as Measured by a Surface Plasmon Resonance Binding Assay

[0194] Binding affinities and kinetic constants of anti-TAA.times.anti-CD3 bispecific antibodies to soluble heterodimeric hCD3.epsilon./.delta..mFc protein (hCD3E=UniProtKB/Swiss-Prot: P07766.2; SEQ_ID NO: 169; hCD3.delta.=UniProtKB/Swiss-Prot: P04234.1, SEQ ID NO: 170) were determined by surface plasmon resonance at 37.degree. C. using either an antigen-capture format (Table 11) or an antibody-capture format (data not shown). In this example, BSPSMA/CD3 bispecific antibodies were utilized as these pairings represented the use of a wider panel of antibodies for the CD3 binding arm. Measurements were conducted on a Sierra Sensors MASS-1 instrument.

[0195] In the antigen-capture format, the MASS-1 high-density amine sensor surface was derivatized with a goat anti-mouse IgG2a polyclonal antibody (Southern Biotech). Soluble heterodimeric CD3 protein was captured and the respective antibodies were injected over the captured antigen.

[0196] Kinetic association (k.sub.a) and dissociation (k.sub.d) rate constants were determined by processing and fitting the data to a 1:1 binding model using MASS-1 AnalyserR2 curve fitting software. Binding dissociation equilibrium constants (K.sub.D) and dissociative half-lives (t.sub.1/2) were calculated from the kinetic rate constants as: K.sub.D (M)=k.sub.d/k.sub.a; and t.sub.1/2 (min)=(In2/(60*k.sub.d).

TABLE-US-00013 TABLE 11 Affinities of anti-CD3 Bispecific Antibodies to Soluble Human CD3 Binding at 37.degree. C./Antigen-Capture Format Corresponding anti-CD3 Antigen- Bispecific Binding HCVR Antibody Identifier Identifier ka (Ms.sup.-1) kd (s.sup.-1) K.sub.D (M) T1/2 (min) BSPSMA/CD3-003 CD3-VH-G 1.32E+05 7.62E-04 5.78E-09 15.2 BSPSMA/CD3-200 CD3-VH-G2 NB NB NB NB BSPSMA/CD3-300 CD3-VH-G3 NB NB NB NB BSPSMA/CD3-400 CD3-VH-G4 NB NB NB NB BSPSMA/CD3-004 CD3-VH-G5 NB NB NB NB BSPSMA/CD3-800 CD3-VH-G8 5.95E+04 1.15E-03 1.94E-08 10.0 BSPSMA/CD3-900 CD3-VH-G9 4.38E+04 4.95E-03 1.13E-07 2.3 BSPSMA/CD3-1000 CD3-VH-G10 3.44E+04 6.37E-03 1.85E-07 1.8 BSPSMA/CD3-1100 CD3-VH-G11 9.21E+04 1.02E-03 1.11E-08 11.3 BSPSMA/CD3-1200 CD3-VH-G12 3.85E+04 2.47E-03 6.42E-08 4.7 BSPSMA/CD3-1300 CD3-VH-G13 2.03E+04 2.48E-03 1.22E-07 4.7 BSPSMA/CD3-1400 CD3-VH-G14 6.21E+04 3.31E-03 5.33E-08 3.5 BSPSMA/CD3-1500 CD3-VH-G15 7.36E+04 6.11E-03 8.29E-08 1.9 BSPSMA/CD3-1600 CD3-VH-G16 6.43E+04 2.43E-03 3.78E-08 4.7 BSPSMA/CD3-1700 CD3-VH-G17 4.70E+04 3.07E-03 6.52E-08 3.8 BSPSMA/CD3-1800 CD3-VH-G18 NB NB NB NB BSPSMA/CD3-1900 CD3-VH-G19 4.43E+04 5.09E-03 1.15E-07 2.3 BSPSMA/CD3-005 CD3-VH-G20 1.73E+04 5.77E-03 3.34E-07 2.0 BSPSMA/CD3-2100 CD3-VH-G21 3.02E+04 2.34E-03 7.75E-08 4.9 Control 1 CD3-L2K 3.68E+05 2.66E-03 7.22E-09 4.3 NB: No binding detected

[0197] As shown in Table 11, all of the derived anti-CD3.times.anti-PSMA bispecific antibodies maintained very weak binding to soluble CD3 in the surface plasmon resonance binding assay, e.g. having a K.sub.D value greater than 11 nM up to 334 nM which is weaker than that of the bispecific anti-CD3 arm derived from germline frameworks, CD3-VH-G.

[0198] Several bispecific antibodies exhibited greater than 50 nM K.sub.D values, and some were greater than 100 nM (>1.times.10.sup.-7) K.sub.D values (i.e. BSPSMA/CD3-900, BSPSMA/CD3-1000, BSPSMA/CD3-1900), greater than 300 nM (>3.times.10.sup.-7) K.sub.D values (i.e. BSPSMA/CD3-005) and even beyond the detection limit of the assay (>500 nM; >5.times.10.sup.-7), i.e. showed no detectable binding to soluble human CD3 (i.e. BSPSMA/CD3-200, BSPSMA/CD3-300, BSPSMA/CD3-400, BSPSMA/CD3-004 and BSPSMA/CD3-1800).

Example 6: T Cell Activation and Tumor-Specific Cytotoxicity Exhibited by Bispecific Antibodies of the Invention as Measured In Vitro

[0199] In this example, the specific killing of PSMA, EGFRvIII or MUC16-expressing TAA target cells in the presence of CD3-based bispecific antibodies was monitored via flow cytometry. As reported previously, the bispecific antibodies displayed a range of affinity to CD3 protein and CD3-expressing cell lines (i.e. weak, moderate and strong binding). This same panel of bispecific antibodies was tested for the ability to induce naive human T-cells to re-direct killing toward target-expressing cells.

[0200] Briefly, PSMA-expressing (C4-2, 22Rv1 and TRAMPC2_PSMA), EGFRvIII-expressing (U87/EGFRvIII) or MUC16-expressing (OVCAR3) cell lines were labeled with 1 .mu.M of the fluorescent tracking dye Violet Cell Tracker. After labeling, cells were plated overnight at 37.degree. C. Separately, human PBMCs were plated in supplemented RPMI media at 1.times.10.sup.6 cells/mL and incubated overnight at 37.degree. C. in order to enrich for lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The next day, target cells were co-incubated with adherent cell-depleted naive PBMC (Effector/Target cell 4:1 ratio) and a serial dilution of relevant bispecific antibodies or Isotype control (concentration range: 66.7 nM to 0.25 pM) for 48 hours at 37.degree. C. Cells were removed from cell culture plates using an enzyme-free cell dissociation buffer, and analyzed by FACS.

[0201] For FACS analysis, cells were stained with a dead/live far red cell tracker (Invitrogen). 5.times.10.sup.5 counting beads were added to each well immediately before FACS analysis. 1.times.10.sup.4 beads were collected for each sample. For the assessment of specificity of killing, cells were gated on live Violet labeled populations. Percent of live population was recorded and used for the calculation of normalized survival.

[0202] T cell activation was assessed by incubating cells with directly conjugated antibodies to CD2 and CD69, and by reporting the percent of activated (CD69+) T cells out of total T cells (CD2+).

[0203] As the results in Tables 12A-12C show, depletion of TAA-expressing cells was observed with anti-PSMA, EGFRvIII or MUC16.times.CD3 bispecifics. Most of the tested bispecific antibodies activated and directed human T cells to deplete the target cells with EC.sub.50s in picomolar range. Additionally, the observed target-cell lysis (depletion) was associated with an up-regulation of CD69 cells on CD2+ T cells, with picomolar (pM) EC.sub.50s.

[0204] Importantly, the results of this example demonstrate that several bispecifics which utilized a CD3 binding arm that displayed weak-to-non-observable binding to CD3 protein or CD3-expressing cells (i.e. CD3-VH-G5) still retained the ability to activate T-cells and exhibited potent cytotoxicity of tumor antigen-expressing cells.

TABLE-US-00014 TABLE 12A Cytotoxicity and T-cell activation properties of selected PSMAxCD3 Bispecific Antibodies Bispecific Anti-CD3 C4-2 Cell 22RV1 TrampC2.PSMA T cell Antibody Binding depletion Cell depletion Cell depletion activation Identifier Arm EC.sub.50 [M] EC.sub.50 [M] EC.sub.50 [M] EC.sub.50 [M] BSPSMA/ CD3-VH-G 1.03E-11 NT 6.43E-12 1.23E-12 CD3-003 BSPSMA/ CD3-VH-G2 NT No activity NT No activity CD3-200 BSPSMA/ CD3-VH-G3 NT Very weak NT 1.85E-11 CD3-300 BSPSMA/ CD3-VH-G4 NT Very weak NT Very weak CD3-400 BSPSMA/ CD3-VH-G5 2.15E-11 6.31E-12 1.15E-11 1.34E-11 CD3-004 BSPSMA/ CD3-VH-G8 NT NT 9.27E-12 1.76E-12 CD3-800 BSPSMA/ CD3-VH-G9 NT NT 3.50E-12 1.12E-12 CD3-900 BSPSMA/ CD3-VH- NT NT 5.97E-12 1.28E-12 CD3-1000 G10 BSPSMA/ CD3-VH- NT NT 3.86E-12 1.11E-12 CD3-1100 G11 BSPSMA/ CD3-VH- 8.74E-12 NT NT 2.31E-12 CD3-1300 G13 BSPSMA/ CD3-VH- 7.37E-12 2.07E-12 NT 3.89E-12 CD3-1700 G17 BSPSMA/ CD3-VH- 1.39E-11 8.32E-12 NT 6.11E-12 CD3-005 G20 NT = not tested

TABLE-US-00015 TABLE 12B Cytotoxicity and T-cell activation properties of selected EGFRvIIIxCD3 Bispecific Antibodies U87_EGFRvIII T cell Bispecific Antibody Anti-CD3 Binding cell depletion activation Identifier Arm EC.sub.50 [M] EC.sub.50 [M] BSV3/CD3-001 CD3-VH-G 3.64E-10 3.33E-11 BSV3/CD3-002 CD3-VH-G5 1.30E-09 1.13E-10

TABLE-US-00016 TABLE 12C Cytotoxicity and T-cell activation properties of selected MUC16xCD3 Bispecific Antibodies OVCAR3 T cell Bispecific Antibody Anti-CD3 Binding cell depletion activation Identifier Arm EC.sub.50 [M] EC.sub.50 [M] BSV3/CD3-001 CD3-VH-G 2.24E-11 5.88E-12 BSV3/CD3-002 CD3-VH-G5 3.06E-11 1.01E-11

Example 7: Anti-PSMA/Anti-CD3 Bispecific Antibodies Display Potent Anti-Tumor Efficacy In Vivo

[0205] To determine the in vivo efficacy of exemplary anti-PSMA/anti-CD3 bispecific antibodies identified as having weak or no detectable binding affinity to human and cynomolgus CD3, studies were performed in immunocompromised mice bearing human prostate cancer xenografts. Additional studies were also carried out in immunocompetent mice bearing mouse prostate cancer xenografts engineered to express human PSMA.

Efficacy of Anti-PSMA/Anti-CD3 Bispecific Antibodies in Human Tumor Xenograft Models

[0206] To assess the in vivo efficacy of the anti-PSMA/anti-CD3 bispecifics in human tumor xenograft studies, NOD scid gamma (NSG) mice (Jackson Laboratories, Bar Harbor, Me.) were co-implanted with human peripheral blood mononuclear cells (PBMCs) along with 22Rv1 or C4-2 human prostate tumor cells which endogenously express PSMA.

[0207] Briefly, 4.times.10.sup.6 22 Rv1 or 5.times.10.sup.6 C4-2 cells (MD Anderson, TX) cells were co-implanted s.c. with 1.times.10.sup.6 human PBMCs (ReachBio, LLC., Seattle, Wash.) in a 50:50 mix of matrigel matrix (BD Biosciences) into the right flank of male NSG mice. In the C4-2 study, mice were treated i.p. on days 0, 4, and 7 post tumor implantation with 0.1 mg/kg BSPSMA/CD3-003 or BSPSMA/CD3-005.

[0208] In an additional xenogenic model, anti-PSMA/anti-CD3 bispecifics were tested in mice engrafted with human hematopoietic CD34+ stem cells. Briefly, newborn SIRP.alpha. BALB/c-Rag2-IL2r.gamma.-(BRG) pups were engrafted with hCD34+ fetal liver cells. 3-6 months later hCD34-engrafted SIRP.alpha. BRG mice were then implanted with C4-2 cells (5.times.10.sup.6 s.c. in matrigel). 8 days later, mice were treated with 10 .mu.g of BSPSMA/CD3-004 or an isotype control antibody, followed by 2.times./week doses throughout the study.

[0209] In all studies, tumor size was measured 2.times./week using calipers and tumor volume calculated as Volume=(length.times.width.sup.2)/2.

[0210] As the results in Table 13 show, the bispecific antibodies tested in the xenogenic models described above were all effective at suppressing tumor growth compared to treatment with the isotype control.

Efficacy of Anti-PSMA/Anti-CD3 Bispecific Antibodies in Immune-Competent Tumor Model

[0211] Additionally, anti-PSMA/anti-CD3 bispecifics were assessed for anti-tumor activity in an immune-competent model (U.S. Provisional Application No. 62/083,653, filed Nov. 24, 2014). Mice humanized for the three chains (Sys) of CD3 were also humanized for PSMA and implanted with a variant murine prostate cancer cell line TRAMP-C2 transfected with human PSMA.

[0212] Prior to study initiation, the tumorigenic cell line variant TRAMP-C2_hPSMAv#1 was generated. Briefly, 7.5.times.10.sup.6 TRAMP-C2_hPSMA cells were implanted s.c. into the right flank of male mice humanized for CD3 and PSMA. A tumor was excised and cut into 3 mm fragments and subsequently implanted into the right flank of new male humanized mice. A tumor arising from the implanted tumor fragments was then harvested and disaggregated into a single cell suspension. These cells (TRAMP-C2_hPSMAv#1) were then cultured in vitro under G418 selection. 4.times.10.sup.6 cells of this variant cell line were then implanted into the right flank of male PSMA/CD3 humanized mice for the bispecific antibody efficacy studies.

[0213] Humanized PSMA/CD3 mice implanted with TRAMPC2_hPSMAv#1 were treated with 100 .mu.g or 10 .mu.g of anti-PSMA/anti-CD3 bispecific antibody BSPSMA/CD3-004 or an isotype control 2.times./week starting from the day of tumor implantation. Serum cytokine levels 4 h post-injection were also examined, as well as spleen T-cell levels. Study was terminated at Day 27.

[0214] As the results in Table 14 show, the anti-PSMA/anti-CD3 bispecific molecule tested, BSPSMA/CD3-004, showed efficacy in significantly delaying tumor growth across treatment groups. Minimal cytokine release was observed after administration of BSPSMA/CD3-004, possibly due to the weak binding of the anti-CD3. Both antibodies tested showed anti-tumor efficacy without depleting T cells in the spleen.

[0215] In summary, the anti-PSMA/anti-CD3 bispecific antibodies of this invention display potent anti-tumor efficacy in both immune-compromised and immune-competent tumor models, despite having low to no detectable binding to CD3 antigen.

Example 8: Anti-MUC16/Anti-CD3 Bispecific Antibodies Display Potent Anti-Tumor Efficacy In Vivo

[0216] To determine the in vivo efficacy of exemplary anti-MUC16/anti-CD3 bispecific antibodies identified as having weak or no detectable binding affinity to human and cynomolgus CD3, studies were performed in immunocompromised mice bearing human prostate cancer xenografts. The efficacy of selected bispecific antibodies was tested in both immediate treatment and therapeutic treatment dosing models.

Efficacy of Anti-MUC16/Anti-CD3 Bispecific Antibodies in Human Tumor Xenograft Models

[0217] To assess the in vivo efficacy of the anti-MUC16/anti-CD3 bispecifics in human tumor xenograft studies, NOD scid gamma (NSG) mice (Jackson Laboratories, Bar Harbor, Me.) were pre-implanted with human peripheral blood mononuclear cells (PBMCs; ReachBio LLC., Seattle, Wash.) and then given ascites cells from the human ovarian cancer cell line OVCAR-3 (American Type Tissue Culture, Manassas, Va.) transduced with luciferase (OVCAR-3/Luc). OVCAR-3 cells endogenously express MUC-16.

[0218] Briefly, NSG mice were injected intraperitoneally (i.p.) with 5.0.times.10.sup.6 human PBMCs. 8d later, 1.5.times.10.sup.6 ascites cells from the OVCAR-3/Luc cell line, previously passaged in vivo, were administered i.p. to the NSG mice engrafted with PBMCs. In the immediate treatment group, mice were treated i.p. on the day of OVCAR-3/Luc cell implantation with MUC16/CD3 Bispecific antibodies BSMUC16/CD3-001 or BSMUC16/CD3-005, or an isotype control, at a dose of 10 ug/mouse (N=5 mice/treatment group). In the therapeutic dose model, mice were treated i.p. 7d post tumor implantation with the MUC16/CD3 Bispecific or control antibodies described above, at a dose of 10 ug/mouse (N=5/treatment group).

[0219] In all studies, tumor growth was monitored via bioluminescent imaging (BLI). Mice were injected i.p. with the luciferase substrate D-luciferin suspended in PBS (150 mg/kg) and imaged under isoflurane anesthesia after 10 min. BLI was performed using the Xenogen IVIS system (Perkin Elmer, Hopkinton, Mass.) and BLI signals were extracted using Living Image software (Xenogen/Perkin Elmer). Regions of interest were drawn around each cell mass and photon intensities were recorded as photons(p)/sec(s)/cm.sup.2/steradian(sr). For the immediate-treatment group, data is shown as BLI levels 26d post tumor implantation (Table 15). For the therapeutic-treatment group, data is shown as fold-change in BLI between day 6 (1 d before treatment) and at study endpoint (26d post tumor implantation; Table 16).

[0220] As the results show, both BSMUC16/CD3-001 and BSMUC16/CD3-005 showed similar efficacy in suppressing tumor growth compared to the isotype control when BLI was measured at Day 26 in the immediate dosing model. Both anti-MUC16/anti-CD3 bispecific antibodies also suppressed the growth of established tumors when administered 7d post tumor implantation, compared to the control. In summary, the bispecific anti-MUC16/anti-CD3 antibodies of this invention display potent anti-tumor efficacy in several models.

TABLE-US-00017 TABLE 15 Efficacy of anti-MUC16/anti-CD3 Bispecific Antibodies in Immune- Compromised Xenograft Model: Immediate Dosing Avg Bioluminescent Radiance Tumor Model/ (photons/sec/cm.sup.2/ Mouse Strain/ Bispecific steradian) Day 26 Dose Antibody Identifier N (mean .+-. SD) OVCAR-3/Luc/ BSMUC16/CD3-001 5 1.4 .times. 10.sup.3 .+-. 3.5 .times. 10.sup.2 NSG/ BSMUC16/CD3-005 5 1.5 .times. 10.sup.3 .+-. 9.7 .times. 10.sup.2 10 ug/mouse Isotype Control 5 2.0 .times. 10.sup.7 .+-. 1.0 .times. 10.sup.6

TABLE-US-00018 TABLE 16 Efficacy of anti-Muc16/anti-CD3 Bispecific Antibodies in Immune-Compromised Xenograft Model: Therapeutic Treatment Fold change in Avg Bioluminescent Tumor Model/ Radiance [p/s/cm.sup.2/sr] Mouse Strain/ Bispecific Antibody at Day 26 relative to Day 6 Dose Identifier N (mean .+-. SD) OVCAR-3/ BSMUC16/CD3-001 5 2.0 .+-. 5.0 Luc/NSG/ BSMUC16/CD3-005 5 0.01 .+-. 0.02 10 ug/mouse Isotype Control 5 21.0 .+-. 8.0

Example 9: Pharmacokinetic Assessment of Anti-MUC16.times.CD3 Bispecific Antibodies

[0221] Assessment of the pharmacokinetics of anti-MUC16.times.CD3 bispecific antibodies BSMUC16/CD3-001 and BSMUC16/CD3-005 and an isotype control were conducted in humanized MUC16.times. CD3 mice (mice homozygous for human MUC16 and CD3 expression, MUC16.sup.hu/hu.times. CD3.sup.hu/hu), CD3 humanized mice (mice homozygous for human CD3 expression, C.sub.D3 hu/hu)) and strain-matched (75% C57BL, 25%129Sv) wild-type (WT) mice. Cohorts contained 4-5 mice per tested antibody and per mouse strain. All mice received a single intra-peritoneal (i.p.) 0.4 mg/kg dose. Blood samples were collected at 3 and 6 hours, 1, 3, 7, 14 and 28 days post dosing. Blood was processed into serum and frozen at -80.degree. C. until analyzed.

[0222] Circulating antibody concentrations were determined by total human IgG antibody analysis using the GyroLab xPlore.TM. (Gyros, Uppsala, Sweden). Briefly, a biotinylated goat anti-human IgG polyclonal antibody (Jackson ImmunoResearch, West Grove, Pa.) was captured onto streptavidin coated beads on a Gyrolab Bioaffy 200 CD (Gyros) in order to capture the human IgG present in the sera. After affinity column capture, bound human IgG antibody in samples was detected with Alexa-647 labeled goat anti-human IgG (Jackson ImmunoResearch). Fluorescent signal on the column allowed for the detection of bound IgG and response units (RU) were read by the instrument. Sample concentrations were determined by interpolation from a standard curve that was fit using a 5-parameter logistic curve fit using the Gyrolab Evaluator Software.

[0223] PK parameters were determined by non-compartmental analysis (NCA) using Phoenix.RTM.WinNonlin.RTM. software Version 6.3 (Certara, L. P., Princeton, N.J.) and an extravascular dosing model. Using the respective mean concentration values for each antibody, all PK parameters including observed maximum concentration in serum (C.sub.max), estimated half-life observed (t.sub.1/2), and area under the concentration curve versus time up to the last measureable concentration (AUC.sub.last) were determined using a linear trapezoidal rule with linear interpolation and uniform weighting

[0224] Following i.p. administration of antibodies in WT mice, the total IgG concentration-time profiles of BSMUC16/CD3-001, BSMUC16/CD3-005 and the isotype control were all similar, characterized first by a brief drug distribution followed by a single drug elimination phase throughout the remainder of the study. Maximum serum concentrations (C.sub.max) and calculated drug exposure (AUC.sub.last) of the three antibodies were comparable (within 1.3-fold of each other). Following i.p. administration of antibodies in CD3.sup.hu/hu mice, BSMUC16/CD3-001, BSMUC16/CD3-005 and isotype control had comparable C.sub.max concentrations (4.6, 3.6 and 4.1 .mu.g/mL, respectively). BSMUC16/CD3-005 and the isotype control exhibited similar drug elimination curves, while BSMUC16/CD3-001 exhibited steeper drug elimination than both, suggesting that human CD3 target binding drives clearance. Terminal antibody concentration for BSMUC16/CD3-001 was 0.03 .mu.g/mL, which is about 28-fold less than terminal antibody concentrations determined for the isotype control (0.85 .mu.g/mL) and 22-fold less than BSMUC16/CD3-005 (0.66 .mu.g/mL) serum concentrations.

[0225] In MUC16.sup.hu/hu.times.CD3.sup.hu/hu double-humanized mice, the Muc16.times.CD3 bispecific and isotype control antibodies had comparable C.sub.max concentrations (C.sub.max range: 4.5-6.9 .mu.g/mL). Both bispecific antibodies exhibited steeper drug elimination than the isotype control suggesting a target-mediated effect. Terminal antibody concentrations for BSMUC16/CD3-001 and BSMUC16/CD3-005 were about 29-fold and 2.9-fold less, respectively, than terminal antibody concentrations determined for the isotype control (0.86 .mu.g/mL).

[0226] A summary of the data for total anti-MUC16.times.CD3 bispecific antibodies and isotype control antibody concentrations are summarized in Table 17. Mean PK parameters are described in Tables 18A and 18B. Mean total antibody concentrations versus time are shown in FIGS. 2A, 2B and 2C. In conclusion, MUC16.times.CD3 bispecific antibodies exhibited similar C.sub.max and drug elimination curves in WT mice, but BSMUC16/CD3-001 displayed steeper elimination rates than BSMUC16/CD3-005 and the isotype control in CD3 single-humanized mice and MUC16/CD3 double humanized mice. Since the bispecific antibodies administered in this PK study are comprised of the same anti-MUC16 binding arm, the results suggest that the strength of binding of the CD3 targeting arm may play a role in drug exposure levels (AUC.sub.last) and drug elimination rates. Neither BSMUC16/CD3-001 or BSMUC16/CD3-005 bind mouse MUC16 or mouse CD3.

TABLE-US-00019 TABLE 17 Mean Concentrations of Total IgG in Serum Following a Single 0.4 mg/kg Intra- peritoneal Injection of BSMUC16/CD3-001, BSMUC16/CD3-005 and Isotype Control Antibodies in WT Mice, Humanized CD3 Mice and Humanized MUC16 .times. CD3 mice Total mAb Concentration In Mouse Serum MUC16.sup.hu/hu .times. WT CD3.sup.hu/hu CD3.sup.hu/hu Mean Mean Mean Antibody Time (d) (.mu.g/mL) +/-SD (.mu.g/mL) +/-SD (.mu.g/mL) +/-SD BSMUC16/CD3- 0.13 5.39 0.34 4.30 0.29 6.77 1.52 001 0.25 5.80 0.36 4.26 1.07 6.63 1.06 1.00 4.13 0.43 2.87 0.71 4.89 0.53 3.00 3.19 0.53 1.44 0.27 2.50 0.22 7.00 2.61 0.73 0.72 0.13 1.20 0.22 14.00 1.44 0.69 0.18 0.05 0.28 0.08 21.00 0.93 ND 0.07 0.02 0.06 0.05 28.00 0.60 ND 0.04 0.01 0.03 0.02 BSMUC16/CD3- 0.13 4.23 0.62 3.35 1.15 4.35 0.24 005 0.25 4.53 0.55 3.40 0.96 4.45 0.49 1.00 3.47 0.32 2.72 0.42 3.00 0.61 3.00 2.51 0.13 1.95 0.37 1.98 0.41 7.00 2.02 0.24 2.31 0.67 1.58 0.36 14.00 1.19 0.17 1.01 0.23 0.78 0.26 21.00 1.19 0.29 1.19 0.11 0.66 0.29 28.00 0.71 0.20 0.66 0.28 0.30 0.22 Isotype Control 0.13 5.07 1.16 5.43 1.30 6.56 0.70 0.25 5.91 1.10 5.67 1.91 6.48 0.90 1.00 2.64 0.24 2.98 1.14 2.82 0.30 3.00 2.05 0.06 2.29 0.83 1.57 0.37 7.00 1.80 0.25 2.14 0.85 1.96 0.37 14.00 1.22 0.28 1.48 0.66 1.34 0.37 21.00 1.20 0.58 1.43 0.72 1.24 0.44 28.00 0.73 0.24 0.85 0.29 0.86 0.41 Time: (h, when noted) = time in hours post single-dose injection; D = Day of study; SD = Standard deviation; ND = Not determined due to exclusion of mice with drug clearing anti-drug titers

TABLE-US-00020 TABLE 18A Summary of Pharmacokinetic Parameters: CD3.sup.hu/hu humanized mice WT mice CD3.sup.hu/hu mice Isotype BSMUC16/ BSMUC16/CD3- Isotype BSMUC16/ BSMUC16/ Parameter Units Control CD3-001 005 Control CD3-001 CD3-005 C.sub.max .mu.g/mL 5 .+-. 3 6 .+-. 0.4 5 .+-. 0.5 4.1 .+-. 3 4.6 .+-. 0.8 3.5 .+-. 1 T.sub.1/2 d 11 .+-. 4 7 .+-. 3 12 .+-. 2 14 .+-. 0.5 3.9 .+-. 0.6 11 .+-. 5 AUC.sub.last d .mu.g/mL 35 .+-. 18 40 .+-. 11 45 .+-. 5 49 .+-. 20 16 .+-. 3 36 .+-. 13 C.sub.max = Peak concentration; AUC = Area under the concentration-time curve; AUC.sub.last = AUC computed from time zero to the time of the last positive concentration; T.sub.1/2 = Estimated half-life observed; d = day

TABLE-US-00021 TABLE 18B Summary of Pharmacokinetic Parameters: MUC16.sup.hu/hu .times. CD3.sup.hu/hu double- humanized mice WT mice MUC16.sup.hu/hu .times. CD3.sup.hu/hu mice Isotype BSMUC16/ BSMUC16/ Isotype BSMUC16/ BSMUC16/ Parameter Units Control CD3-001 CD3-005 Control CD3-001 CD3-005 C.sub.max .mu.g/mL 5 .+-. 3 6 .+-. 0.4 5 .+-. 0.5 6.7 .+-. 0.7 6.9 .+-. 1 4.5 .+-. 4 T.sub.1/2 d 11 .+-. 4 7 .+-. 3 12 .+-. 2 12.9 .+-. 4 3.3 .+-. 0.8 8.2 .+-. 4 AUC.sub.last d .mu.g/mL 35 .+-. 18 40 .+-. 11 45 .+-. 5 46 .+-. 10 27 .+-. 3 34 .+-. 11 C.sub.max = Peak concentration; AUC = Area under the concentration-time curve; AUC.sub.last = AUC computed from time zero to the time of the last positive concentration; T.sub.1/2 = Estimated half-life observed; d = day

Example 10: Anti-STEAP2/Anti-CD3 Bispecific Antibodies Display Potent Anti-Tumor Efficacy In Vivo

[0227] To determine the in vivo efficacy of exemplary anti-STEAP2/anti-CD3 bispecific antibodies identified as having weak or no detectable binding affinity to human and cynomolgus CD3, studies were performed in immunocompromised mice bearing human prostate cancer xenografts.

[0228] To assess the in vivo efficacy of the anti-STEAP2/anti-CD3 bispecifics in human tumor xenograft studies, NOD scid gamma (NSG) mice (Jackson Laboratories, Bar Harbor, Me.) were co-implanted with human peripheral blood mononuclear cells (PBMCs; ReachBio LLC., Seattle, Wash.) along with human prostate cancer C4-2 cells (MD Anderson Cancer Center, Houston Tex.) which endogenously express STEAP2.

[0229] Briefly, 5.0.times.10.sup.6 C4-2 cells were co-implanted subcutaneously (s.c.) with 1.25.times.10.sup.6 human PBMCs in a 50:50 mix of matrigel matrix (BD Biosciences, San Jose, Calif.) into the right flank of male NSG mice. Mice were treated intraperitoneally (i.p.) on the day of implantation (immediate treatment model) with anti-STEAP2/anti-CD3 bispecifics BSSTEAP2/CD3-001, BSSTEAP2/CD3-002 or BSSTEAP2/CD3-003, or an isotype control (that dos not bind C4-2 tumor cells), at a dose of 0.1 or 0.01 mg/kg (N=5 mice/group).

[0230] Tumor size was measured 2.times./week using calipers and tumor volume calculated as Volume=(length.times.width.sup.2)/2. Data is shown as tumor size (mm.sup.3) at study endpoint, 46d post-tumor implantation (Table 19).

[0231] As the results in Table 19 show, BSSTEAP2/CD3-001, BSSTEAP2/CD3-002 and BSSTEAP2/CD3-003 significantly suppressed tumor growth compared to an isotype control when tumor sizes were measured at study endpoint. Importantly, the anti-STEAP.sup.2/anti-CD3 bispecific antibodies were efficacious in inhibiting C4-2 tumor growth even at the lowest dose of 0.1 mg/kg.

TABLE-US-00022 TABLE 19 Efficacy of anti-STEAP2/anti-CD3 Bispecific Antibodies in Immune- Compromised Xenograft Model: Immediate Dosing Tumor Tumor Size (mm.sup.3) Model/ 46 d post-tumor Mouse Bispecific Antibody Dose implantation Strain Identifier (mg/kg) N (mean .+-. SD) C4-2/ BSSTEAP2/CD3-001 0.1 5 18.0 .+-. 14.0 NSG 0.01 5 23.0 .+-. 220 BSSTEAP2/CD3-002 0.1 5 15.0 .+-. 12.0 0.01 5 17.0 .+-. 8.0 BSSTEAP2/CD3-003 0.1 5 19.0 .+-. 12.0 0.01 5 25.0 .+-. 21.0 Control Bispecific 0.1 5 1020.0 .+-. 922.0 The present invention is not to be limited in scope by the specific embodiments

[0232] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Sequence CWU 1

1

2011372DNAArtificial Sequencesynthetic 1gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300agtggttatg gcaagtttta tcattatgga ctggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 3722124PRTArtificial Sequencesynthetic 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 324DNAArtificial Sequencesynthetic 3gggtttacat tcgacgatta cagc 2448PRTArtificial Sequencesynthetic 4Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 524DNAArtificial Sequencesynthetic 5atatcatgga actcaggaag caag 2468PRTArtificial Sequencesynthetic 6Ile Ser Trp Asn Ser Gly Ser Lys 1 5 751DNAArtificial Sequencesynthetic 7gcaaaatacg gcagtggtta tggcaagttt tatcattatg gactggacgt g 51817PRTArtificial Sequencesynthetic 8Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 1 5 10 15 Val 9372DNAArtificial Sequencesynthetic 9gaagtacagt tggtagaatc tggaggagga ctcgtgcaac caggacgatc attgcggttg 60agttgtgctg ctagtggatt cacattcgac gactatgcta tgcattgggt aagacaggct 120ccaggaaaag gactcgaatg ggtgtcagga ataagttgga actccggaag cattgggtac 180gcagattcag tcaaagggcg attcaccata tcccgagata acgctaagaa ctcactttac 240cttcaaatga actctcttcg agcagaggac actgcacttt attattgcgc taaggacggc 300tccggttatg gatattttta ttattatgga atggacgtat ggggacaagg cactactgtt 360accgttagtt cc 37210124PRTArtificial Sequencesynthetic 10Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Tyr Phe Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 1124DNAArtificial Sequencesynthetic 11ggattcacat tcgacgacta tgct 24128PRTArtificial Sequencesynthetic 12Gly Phe Thr Phe Asp Asp Tyr Ala 1 5 1324DNAArtificial Sequencesynthetic 13ataagttgga actccggaag catt 24148PRTArtificial Sequencesynthetic 14Ile Ser Trp Asn Ser Gly Ser Ile 1 5 1551DNAArtificial Sequencesynthetic 15gctaaggacg gctccggtta tggatatttt tattattatg gaatggacgt a 511617PRTArtificial Sequencesynthetic 16Ala Lys Asp Gly Ser Gly Tyr Gly Tyr Phe Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val 17372DNAArtificial Sequencesynthetic 17gaagtacaac tggtcgaatc tggaggaggt cttgttcaac ctggtcgatc acttcgcctt 60tcttgtgccg cttctggttt cactttcgac gattatagca tgcattgggt acgacaggct 120cccggaaaag ggctggaatg ggtgtcagga attagttgga actcaggaag tattggatac 180gctgattcag tcaaaggacg cttcacaatc tcaagggaca acgctaaaaa ctcactttat 240ttgcaaatga actctctccg cgctgaagat accgctctct attattgcgc caaagatggg 300tctggttacg gttattttta ctactatgga atggacgttt ggggccaagg aacaactgtc 360acagtatcat cc 37218124PRTArtificial Sequencesynthetic 18Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Tyr Phe Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 1924DNAArtificial Sequencesynthetic 19ggtttcactt tcgacgatta tagc 24208PRTArtificial Sequencesynthetic 20Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 2124DNAArtificial Sequencesynthetic 21attagttgga actcaggaag tatt 24228PRTArtificial Sequencesynthetic 22Ile Ser Trp Asn Ser Gly Ser Ile 1 5 2351DNAArtificial Sequencesynthetic 23gccaaagatg ggtctggtta cggttatttt tactactatg gaatggacgt t 512417PRTArtificial Sequencesynthetic 24Ala Lys Asp Gly Ser Gly Tyr Gly Tyr Phe Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val 25372DNAArtificial Sequencesynthetic 25gaagttcaac ttgtggaaag tggcggagga ttggttcaac caggacgttc attgaggctt 60tcatgcgcag cttccggatt tacatttgac gattacgcaa tgcactgggt tagacaggca 120ccaggaaaag gactggagtg ggtgagcggg atttcatgga acagcggcag tatcggttat 180gcagactcag ttaaaggaag attcaccatc agtagagaca acgcaaaaaa ttccctttat 240ctccaaatga actctcttag ggccgaagat acagcattgt actactgcgc aaaagacgga 300tcaggttacg gaaaatttta ctactatggt atggatgtat ggggtcaggg aaccacagta 360actgtatcaa gc 37226124PRTArtificial Sequencesynthetic 26Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 2724DNAArtificial Sequencesynthetic 27ggatttacat ttgacgatta cgca 24288PRTArtificial Sequencesynthetic 28Gly Phe Thr Phe Asp Asp Tyr Ala 1 5 2924DNAArtificial Sequencesynthetic 29atttcatgga acagcggcag tatc 24308PRTArtificial Sequencesynthetic 30Ile Ser Trp Asn Ser Gly Ser Ile 1 5 3151DNAArtificial Sequencesynthetic 31gcaaaagacg gatcaggtta cggaaaattt tactactatg gtatggatgt a 513217PRTArtificial Sequencesynthetic 32Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val 33372DNAArtificial Sequencesynthetic 33gaagtgcaac tcgttgaaag cggaggagga ctggtccagc ccggcagatc tctcagattg 60tcttgcgctg catccggatt tacatttgac gactattcaa tgcactgggt acggcaagcc 120ccaggtaaag gactcgaatg ggtaagcggc atatcttgga actcaggcag tattggctac 180gcagattcag taaaaggaag attcactatt tcaagggata atgctaagaa cagtctctac 240ttgcaaatga atagcttgcg cgcagaagat acagcacttt attattgtgc aaaagatgga 300agcggttatg ggaaatttta ttattatggt atggatgtat ggggtcaagg tacaacagtt 360actgtgtcaa gt 37234124PRTArtificial Sequencesynthetic 34Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 3524DNAArtificial Sequencesynthetic 35ggatttacat ttgacgacta ttca 24368PRTArtificial Sequencesynthetic 36Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 3724DNAArtificial Sequencesynthetic 37atatcttgga actcaggcag tatt 24388PRTArtificial Sequencesynthetic 38Ile Ser Trp Asn Ser Gly Ser Ile 1 5 3951DNAArtificial Sequencesynthetic 39gcaaaagatg gaagcggtta tgggaaattt tattattatg gtatggatgt a 514017PRTArtificial Sequencesynthetic 40Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val 41372DNAArtificial Sequencesynthetic 41gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag catcggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300agtggttatg gcaagtttta tcattatgga ctggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 37242124PRTArtificial Sequencesynthetic 42Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 4324DNAArtificial Sequencesynthetic 43gggtttacat tcgacgatta cagc 24448PRTArtificial Sequencesynthetic 44Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 4524DNAArtificial Sequencesynthetic 45atatcatgga actcaggaag catc 24468PRTArtificial Sequencesynthetic 46Ile Ser Trp Asn Ser Gly Ser Ile 1 5 4751DNAArtificial Sequencesynthetic 47gcaaaatacg gcagtggtta tggcaagttt tatcattatg gactggacgt g 514817PRTArtificial Sequencesynthetic 48Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 1 5 10 15 Val 49372DNAArtificial Sequencesynthetic 49gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaagacggc 300agtggttatg gcaagtttta tcattatgga ctggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 37250124PRTArtificial Sequencesynthetic 50Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 5124DNAArtificial Sequencesynthetic 51gggtttacat tcgacgatta cagc 24528PRTArtificial Sequencesynthetic 52Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 5324DNAArtificial Sequencesynthetic 53atatcatgga actcaggaag caag 24548PRTArtificial Sequencesynthetic 54Ile Ser Trp Asn Ser Gly Ser Lys 1 5 5551DNAArtificial Sequencesynthetic 55gcaaaagacg gcagtggtta tggcaagttt tatcattatg gactggacgt g 515617PRTArtificial Sequencesynthetic 56Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 1 5 10 15 Val 57372DNAArtificial Sequencesynthetic 57gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300agtggttatg gcaagtttta ttattatgga ctggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 37258124PRTArtificial Sequencesynthetic 58Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 5924DNAArtificial Sequencesynthetic 59gggtttacat tcgacgatta cagc 24608PRTArtificial Sequencesynthetic 60Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 6124DNAArtificial Sequencesynthetic 61atatcatgga actcaggaag caag 24628PRTArtificial Sequencesynthetic 62Ile Ser Trp Asn Ser Gly Ser Lys 1 5

6351DNAArtificial Sequencesynthetic 63gcaaaatacg gcagtggtta tggcaagttt tattattatg gactggacgt g 516417PRTArtificial Sequencesynthetic 64Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Leu Asp 1 5 10 15 Val 65372DNAArtificial Sequencesynthetic 65gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300agtggttatg gcaagtttta tcattatgga atggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 37266124PRTArtificial Sequencesynthetic 66Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 6724DNAArtificial Sequencesynthetic 67gggtttacat tcgacgatta cagc 24688PRTArtificial Sequencesynthetic 68Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 6924DNAArtificial Sequencesynthetic 69atatcatgga actcaggaag caag 24708PRTArtificial Sequencesynthetic 70Ile Ser Trp Asn Ser Gly Ser Lys 1 5 7151DNAArtificial Sequencesynthetic 71gcaaaatacg gcagtggtta tggcaagttt tatcattatg gaatggacgt g 517217PRTArtificial Sequencesynthetic 72Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Met Asp 1 5 10 15 Val 73372DNAArtificial Sequencesynthetic 73gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag catcggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaagacggc 300agtggttatg gcaagtttta tcattatgga ctggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 37274124PRTArtificial Sequencesynthetic 74Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 7524DNAArtificial Sequencesynthetic 75gggtttacat tcgacgatta cagc 24768PRTArtificial Sequencesynthetic 76Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 7724DNAArtificial Sequencesynthetic 77atatcatgga actcaggaag catc 24788PRTArtificial Sequencesynthetic 78Ile Ser Trp Asn Ser Gly Ser Ile 1 5 7951DNAArtificial Sequencesynthetic 79gcaaaagacg gcagtggtta tggcaagttt tatcattatg gactggacgt g 518017PRTArtificial Sequencesynthetic 80Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 1 5 10 15 Val 81372DNAArtificial Sequencesynthetic 81gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag catcggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300agtggttatg gcaagtttta ttattatgga ctggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 37282124PRTArtificial Sequencesynthetic 82Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 8324DNAArtificial Sequencesynthetic 83gggtttacat tcgacgatta cagc 24848PRTArtificial Sequencesynthetic 84Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 8524DNAArtificial Sequencesynthetic 85atatcatgga actcaggaag catc 24868PRTArtificial Sequencesynthetic 86Ile Ser Trp Asn Ser Gly Ser Ile 1 5 8751DNAArtificial Sequencesynthetic 87gcaaaatacg gcagtggtta tggcaagttt tattattatg gactggacgt g 518817PRTArtificial Sequencesynthetic 88Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Leu Asp 1 5 10 15 Val 89372DNAArtificial Sequencesynthetic 89gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag catcggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300agtggttatg gcaagtttta tcattatgga atggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 37290124PRTArtificial Sequencesynthetic 90Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 9124DNAArtificial Sequencesynthetic 91gggtttacat tcgacgatta cagc 24928PRTArtificial Sequencesynthetic 92Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 9324DNAArtificial Sequencesynthetic 93atatcatgga actcaggaag catc 24948PRTArtificial Sequencesynthetic 94Ile Ser Trp Asn Ser Gly Ser Ile 1 5 9551DNAArtificial Sequencesynthetic 95gcaaaatacg gcagtggtta tggcaagttt tatcattatg gaatggacgt g 519617PRTArtificial Sequencesynthetic 96Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Met Asp 1 5 10 15 Val 97372DNAArtificial Sequencesynthetic 97gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaagacggc 300agtggttatg gcaagtttta ttattatgga ctggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 37298124PRTArtificial Sequencesynthetic 98Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 9924DNAArtificial Sequencesynthetic 99gggtttacat tcgacgatta cagc 241008PRTArtificial Sequencesynthetic 100Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 10124DNAArtificial Sequencesynthetic 101atatcatgga actcaggaag caag 241028PRTArtificial Sequencesynthetic 102Ile Ser Trp Asn Ser Gly Ser Lys 1 5 10351DNAArtificial Sequencesynthetic 103gcaaaagacg gcagtggtta tggcaagttt tattattatg gactggacgt g 5110417PRTArtificial Sequencesynthetic 104Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Leu Asp 1 5 10 15 Val 105372DNAArtificial Sequencesynthetic 105gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaagacggc 300agtggttatg gcaagtttta tcattatgga atggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 372106124PRTArtificial Sequencesynthetic 106Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 10724DNAArtificial Sequencesynthetic 107gggtttacat tcgacgatta cagc 241088PRTArtificial Sequencesynthetic 108Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 10924DNAArtificial Sequencesynthetic 109atatcatgga actcaggaag caag 241108PRTArtificial Sequencesynthetic 110Ile Ser Trp Asn Ser Gly Ser Lys 1 5 11151DNAArtificial Sequencesynthetic 111gcaaaagacg gcagtggtta tggcaagttt tatcattatg gaatggacgt g 5111217PRTArtificial Sequencesynthetic 112Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Met Asp 1 5 10 15 Val 113372DNAArtificial Sequencesynthetic 113gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300agtggttatg gcaagtttta ttattatgga atggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 372114124PRTArtificial Sequencesynthetic 114Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 11524DNAArtificial Sequencesynthetic 115gggtttacat tcgacgatta cagc 241168PRTArtificial Sequencesynthetic 116Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 11724DNAArtificial Sequencesynthetic 117atatcatgga actcaggaag caag 241188PRTArtificial Sequencesynthetic 118Ile Ser Trp Asn Ser Gly Ser Lys 1 5 11951DNAArtificial Sequencesynthetic 119gcaaaatacg gcagtggtta tggcaagttt tattattatg gaatggacgt g 5112017PRTArtificial Sequencesynthetic 120Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val 121372DNAArtificial Sequencesynthetic 121gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag catcggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaagacggc 300agtggttatg gcaagtttta ttattatgga ctggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 372122124PRTArtificial Sequencesynthetic 122Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 12324DNAArtificial Sequencesynthetic 123gggtttacat tcgacgatta cagc

241248PRTArtificial Sequencesynthetic 124Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 12524DNAArtificial Sequencesynthetic 125atatcatgga actcaggaag catc 241268PRTArtificial Sequencesynthetic 126Ile Ser Trp Asn Ser Gly Ser Ile 1 5 12751DNAArtificial Sequencesynthetic 127gcaaaagacg gcagtggtta tggcaagttt tattattatg gactggacgt g 5112817PRTArtificial Sequencesynthetic 128Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Leu Asp 1 5 10 15 Val 129372DNAArtificial Sequencesynthetic 129gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag catcggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaagacggc 300agtggttatg gcaagtttta tcattatgga atggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 372130124PRTArtificial Sequencesynthetic 130Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 13124DNAArtificial Sequencesynthetic 131gggtttacat tcgacgatta cagc 241328PRTArtificial Sequencesynthetic 132Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 13324DNAArtificial Sequencesynthetic 133atatcatgga actcaggaag catc 241348PRTArtificial Sequencesynthetic 134Ile Ser Trp Asn Ser Gly Ser Ile 1 5 13551DNAArtificial Sequencesynthetic 135gcaaaagacg gcagtggtta tggcaagttt tatcattatg gaatggacgt g 5113617PRTArtificial Sequencesynthetic 136Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Met Asp 1 5 10 15 Val 137372DNAArtificial Sequencesynthetic 137gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag catcggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaatacggc 300agtggttatg gcaagtttta ttattatgga atggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 372138124PRTArtificial Sequencesynthetic 138Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 13924DNAArtificial Sequencesynthetic 139gggtttacat tcgacgatta cagc 241408PRTArtificial Sequencesynthetic 140Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 14124DNAArtificial Sequencesynthetic 141atatcatgga actcaggaag catc 241428PRTArtificial Sequencesynthetic 142Ile Ser Trp Asn Ser Gly Ser Ile 1 5 14351DNAArtificial Sequencesynthetic 143gcaaaatacg gcagtggtta tggcaagttt tattattatg gaatggacgt g 5114417PRTArtificial Sequencesynthetic 144Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val 145372DNAArtificial Sequencesynthetic 145gaagtacagc ttgtagaatc cggcggagga ctggtacaac ctggaagaag tcttagactg 60agttgcgcag ctagtgggtt tacattcgac gattacagca tgcattgggt gaggcaagct 120cctggtaaag gattggaatg ggttagcggg atatcatgga actcaggaag caagggatac 180gccgacagcg tgaaaggccg atttacaata tctagggaca acgcaaaaaa ctctctctac 240cttcaaatga actctcttag ggcagaagac acagcattgt attattgcgc aaaagacggc 300agtggttatg gcaagtttta ttattatgga atggacgtgt ggggacaagg gacaacagtg 360acagtgagta gc 372146124PRTArtificial Sequencesynthetic 146Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 14724DNAArtificial Sequencesynthetic 147gggtttacat tcgacgatta cagc 241488PRTArtificial Sequencesynthetic 148Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 14924DNAArtificial Sequencesynthetic 149atatcatgga actcaggaag caag 241508PRTArtificial Sequencesynthetic 150Ile Ser Trp Asn Ser Gly Ser Lys 1 5 15151DNAArtificial Sequencesynthetic 151gcaaaagacg gcagtggtta tggcaagttt tattattatg gaatggacgt g 5115217PRTArtificial Sequencesynthetic 152Ala Lys Asp Gly Ser Gly Tyr Gly Lys Phe Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val 153372DNAArtificial Sequencesynthetic 153gaagtgcagc tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60tcctgtgtag cctctggatt cacctttgat gattattcca tgcactgggt ccggcaagct 120ccagggaagg gcctggagtg ggtctcaggt attagttgga atagtggtag caaagactat 180gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag agctgaagac acggccttgt attactgtgc aaaatatgga 300agtggctacg ggaagttcta ccactacggt ttggacgtct ggggccaagg gaccacggtc 360accgtctcct ca 372154124PRTArtificial Sequencesynthetic 154Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 15524DNAArtificial Sequencesynthetic 155ggattcacct ttgatgatta ttcc 241568PRTArtificial Sequencesynthetic 156Gly Phe Thr Phe Asp Asp Tyr Ser 1 5 15724DNAArtificial Sequencesynthetic 157attagttgga atagtggtag caaa 241588PRTArtificial Sequencesynthetic 158Ile Ser Trp Asn Ser Gly Ser Lys 1 5 15951DNAArtificial Sequencesynthetic 159gcaaaatatg gaagtggcta cgggaagttc taccactacg gtttggacgt c 5116017PRTArtificial Sequencesynthetic 160Ala Lys Tyr Gly Ser Gly Tyr Gly Lys Phe Tyr His Tyr Gly Leu Asp 1 5 10 15 Val 161324DNAArtificial Sequencesynthetic 161gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300caagggacac gactggagat taaa 324162108PRTArtificial Sequencesynthetic 162Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 16318DNAArtificial Sequencesynthetic 163cagagcatta gcagctat 181646PRTArtificial Sequencesynthetic 164Gln Ser Ile Ser Ser Tyr 1 5 1659DNAArtificial Sequencesynthetic 165gctgcatcc 9 1663PRTArtificial Sequencesynthetic 166Ala Ala Ser 1 16730DNAArtificial Sequencesynthetic 167caacagagtt acagtacccc tccgatcacc 3016810PRTArtificial Sequencesynthetic 168Gln Gln Ser Tyr Ser Thr Pro Pro Ile Thr 1 5 10 169207PRTArtificial Sequencesynthetic 169Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser 1 5 10 15 Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60 Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65 70 75 80 His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110 Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met 115 120 125 Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu 130 135 140 Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys 145 150 155 160 Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn 165 170 175 Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg 180 185 190 Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 195 200 205 170171PRTArtificial Sequencesynthetic 170Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu 1 5 10 15 Ser Gln Val Ser Pro Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg 20 25 30 Val Phe Val Asn Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val 35 40 45 Gly Thr Leu Leu Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile 50 55 60 Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys 65 70 75 80 Asp Lys Glu Ser Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys 85 90 95 Val Glu Leu Asp Pro Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val 100 105 110 Ile Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His 115 120 125 Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg 130 135 140 Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr 145 150 155 160 Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys 165 170 171750PRTArtificial Sequencesynthetic 171Met Trp Asn Leu Leu His Glu Thr Asp Ser Ala Val Ala Thr Ala Arg 1 5 10 15 Arg Pro Arg Trp Leu Cys Ala Gly Ala Leu Val Leu Ala Gly Gly Phe 20 25 30 Phe Leu Leu Gly Phe Leu Phe Gly Trp Phe Ile Lys Ser Ser Asn Glu 35 40 45 Ala Thr Asn Ile Thr Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu 50 55 60 Leu Lys Ala Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln Ile 65 70 75 80 Pro His Leu Ala Gly Thr Glu Gln Asn Phe Gln Leu Ala Lys Gln Ile 85 90 95 Gln Ser Gln Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala His 100 105 110 Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro Asn Tyr Ile 115 120 125 Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe Asn Thr Ser Leu Phe 130 135 140 Glu Pro Pro Pro Pro Gly Tyr Glu Asn Val Ser Asp Ile Val Pro Pro 145 150 155 160 Phe Ser Ala Phe Ser Pro Gln Gly Met Pro Glu Gly Asp Leu Val Tyr 165 170 175 Val Asn Tyr Ala Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met 180 185 190 Lys Ile Asn Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys Val 195 200 205 Phe Arg Gly Asn Lys Val Lys Asn Ala Gln Leu Ala Gly Ala Lys Gly 210 215 220 Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val Lys 225 230 235 240 Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly Val Gln Arg Gly 245 250 255 Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro Leu Thr Pro Gly Tyr 260 265 270 Pro Ala Asn Glu Tyr Ala Tyr Arg Arg Gly Ile Ala Glu Ala Val Gly 275 280 285 Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr Asp Ala Gln Lys 290 295 300 Leu Leu Glu Lys Met Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp Arg 305 310 315 320 Gly Ser Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly Asn 325 330 335 Phe Ser Thr Gln Lys Val Lys Met His Ile His Ser Thr Asn Glu Val 340 345 350 Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val Glu Pro 355 360 365 Asp Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser Trp Val Phe Gly 370 375 380 Gly Ile Asp Pro Gln Ser Gly Ala Ala Val Val His Glu Ile Val Arg 385 390

395 400 Ser Phe Gly Thr Leu Lys Lys Glu Gly Trp Arg Pro Arg Arg Thr Ile 405 410 415 Leu Phe Ala Ser Trp Asp Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr 420 425 430 Glu Trp Ala Glu Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val Ala 435 440 445 Tyr Ile Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val 450 455 460 Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu 465 470 475 480 Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr Glu Ser 485 490 495 Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly Met Pro Arg Ile 500 505 510 Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu Val Phe Phe Gln Arg Leu 515 520 525 Gly Ile Ala Ser Gly Arg Ala Arg Tyr Thr Lys Asn Trp Glu Thr Asn 530 535 540 Lys Phe Ser Gly Tyr Pro Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu 545 550 555 560 Leu Val Glu Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val 565 570 575 Ala Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val 580 585 590 Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala 595 600 605 Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu Met Lys Thr 610 615 620 Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val Lys Asn Phe Thr 625 630 635 640 Glu Ile Ala Ser Lys Phe Ser Glu Arg Leu Gln Asp Phe Asp Lys Ser 645 650 655 Asn Pro Ile Val Leu Arg Met Met Asn Asp Gln Leu Met Phe Leu Glu 660 665 670 Arg Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg 675 680 685 His Val Ile Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu Ser 690 695 700 Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp 705 710 715 720 Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val Ala Ala 725 730 735 Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu Val Ala 740 745 750 172943PRTArtificial Sequencesynthetic 172Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala 1 5 10 15 Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Gly Asn Tyr 20 25 30 Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser 35 40 45 Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly 50 55 60 Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp 65 70 75 80 Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr 85 90 95 Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp 100 105 110 Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu 115 120 125 Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro 130 135 140 Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg 145 150 155 160 Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu 165 170 175 Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly 180 185 190 Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile 195 200 205 Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile 210 215 220 Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His 225 230 235 240 Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys 245 250 255 Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys 260 265 270 Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys 275 280 285 Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys 290 295 300 Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp 305 310 315 320 Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn 325 330 335 Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu 340 345 350 Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly 355 360 365 Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val 370 375 380 Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe 385 390 395 400 Met Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu 405 410 415 Gln Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro 420 425 430 Asn Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile 435 440 445 Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp 450 455 460 Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu 465 470 475 480 Arg Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala 485 490 495 Tyr Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg Leu Leu Gly 500 505 510 Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe 515 520 525 Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser 530 535 540 Gln Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr 545 550 555 560 Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val 565 570 575 Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala 580 585 590 Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys 595 600 605 Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr 610 615 620 Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu 625 630 635 640 Met Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile 645 650 655 Ser Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys 660 665 670 Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ala 675 680 685 Asp Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met 690 695 700 Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met 705 710 715 720 His Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp 725 730 735 Glu Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro 740 745 750 Gln Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu 755 760 765 Ser Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp 770 775 780 Arg Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln 785 790 795 800 Arg Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp 805 810 815 Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys 820 825 830 Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu 835 840 845 Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr 850 855 860 Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val 865 870 875 880 Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala Gln Lys Gly Ser His 885 890 895 Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys 900 905 910 Glu Ala Lys Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala 915 920 925 Glu Tyr Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala 930 935 940 17314507PRTArtificial Sequencesynthetic 173Met Leu Lys Pro Ser Gly Leu Pro Gly Ser Ser Ser Pro Thr Arg Ser 1 5 10 15 Leu Met Thr Gly Ser Arg Ser Thr Lys Ala Thr Pro Glu Met Asp Ser 20 25 30 Gly Leu Thr Gly Ala Thr Leu Ser Pro Lys Thr Ser Thr Gly Ala Ile 35 40 45 Val Val Thr Glu His Thr Leu Pro Phe Thr Ser Pro Asp Lys Thr Leu 50 55 60 Ala Ser Pro Thr Ser Ser Val Val Gly Arg Thr Thr Gln Ser Leu Gly 65 70 75 80 Val Met Ser Ser Ala Leu Pro Glu Ser Thr Ser Arg Gly Met Thr His 85 90 95 Ser Glu Gln Arg Thr Ser Pro Ser Leu Ser Pro Gln Val Asn Gly Thr 100 105 110 Pro Ser Arg Asn Tyr Pro Ala Thr Ser Met Val Ser Gly Leu Ser Ser 115 120 125 Pro Arg Thr Arg Thr Ser Ser Thr Glu Gly Asn Phe Thr Lys Glu Ala 130 135 140 Ser Thr Tyr Thr Leu Thr Val Glu Thr Thr Ser Gly Pro Val Thr Glu 145 150 155 160 Lys Tyr Thr Val Pro Thr Glu Thr Ser Thr Thr Glu Gly Asp Ser Thr 165 170 175 Glu Thr Pro Trp Asp Thr Arg Tyr Ile Pro Val Lys Ile Thr Ser Pro 180 185 190 Met Lys Thr Phe Ala Asp Ser Thr Ala Ser Lys Glu Asn Ala Pro Val 195 200 205 Ser Met Thr Pro Ala Glu Thr Thr Val Thr Asp Ser His Thr Pro Gly 210 215 220 Arg Thr Asn Pro Ser Phe Gly Thr Leu Tyr Ser Ser Phe Leu Asp Leu 225 230 235 240 Ser Pro Lys Gly Thr Pro Asn Ser Arg Gly Glu Thr Ser Leu Glu Leu 245 250 255 Ile Leu Ser Thr Thr Gly Tyr Pro Phe Ser Ser Pro Glu Pro Gly Ser 260 265 270 Ala Gly His Ser Arg Ile Ser Thr Ser Ala Pro Leu Ser Ser Ser Ala 275 280 285 Ser Val Leu Asp Asn Lys Ile Ser Glu Thr Ser Ile Phe Ser Gly Gln 290 295 300 Ser Leu Thr Ser Pro Leu Ser Pro Gly Val Pro Glu Ala Arg Ala Ser 305 310 315 320 Thr Met Pro Asn Ser Ala Ile Pro Phe Ser Met Thr Leu Ser Asn Ala 325 330 335 Glu Thr Ser Ala Glu Arg Val Arg Ser Thr Ile Ser Ser Leu Gly Thr 340 345 350 Pro Ser Ile Ser Thr Lys Gln Thr Ala Glu Thr Ile Leu Thr Phe His 355 360 365 Ala Phe Ala Glu Thr Met Asp Ile Pro Ser Thr His Ile Ala Lys Thr 370 375 380 Leu Ala Ser Glu Trp Leu Gly Ser Pro Gly Thr Leu Gly Gly Thr Ser 385 390 395 400 Thr Ser Ala Leu Thr Thr Thr Ser Pro Ser Thr Thr Leu Val Ser Glu 405 410 415 Glu Thr Asn Thr His His Ser Thr Ser Gly Lys Glu Thr Glu Gly Thr 420 425 430 Leu Asn Thr Ser Met Thr Pro Leu Glu Thr Ser Ala Pro Gly Glu Glu 435 440 445 Ser Glu Met Thr Ala Thr Leu Val Pro Thr Leu Gly Phe Thr Thr Leu 450 455 460 Asp Ser Lys Ile Arg Ser Pro Ser Gln Val Ser Ser Ser His Pro Thr 465 470 475 480 Arg Glu Leu Arg Thr Thr Gly Ser Thr Ser Gly Arg Gln Ser Ser Ser 485 490 495 Thr Ala Ala His Gly Ser Ser Asp Ile Leu Arg Ala Thr Thr Ser Ser 500 505 510 Thr Ser Lys Ala Ser Ser Trp Thr Ser Glu Ser Thr Ala Gln Gln Phe 515 520 525 Ser Glu Pro Gln His Thr Gln Trp Val Glu Thr Ser Pro Ser Met Lys 530 535 540 Thr Glu Arg Pro Pro Ala Ser Thr Ser Val Ala Ala Pro Ile Thr Thr 545 550 555 560 Ser Val Pro Ser Val Val Ser Gly Phe Thr Thr Leu Lys Thr Ser Ser 565 570 575 Thr Lys Gly Ile Trp Leu Glu Glu Thr Ser Ala Asp Thr Leu Ile Gly 580 585 590 Glu Ser Thr Ala Gly Pro Thr Thr His Gln Phe Ala Val Pro Thr Gly 595 600 605 Ile Ser Met Thr Gly Gly Ser Ser Thr Arg Gly Ser Gln Gly Thr Thr 610 615 620 His Leu Leu Thr Arg Ala Thr Ala Ser Ser Glu Thr Ser Ala Asp Leu 625 630 635 640 Thr Leu Ala Thr Asn Gly Val Pro Val Ser Val Ser Pro Ala Val Ser 645 650 655 Lys Thr Ala Ala Gly Ser Ser Pro Pro Gly Gly Thr Lys Pro Ser Tyr 660 665 670 Thr Met Val Ser Ser Val Ile Pro Glu Thr Ser Ser Leu Gln Ser Ser 675 680 685 Ala Phe Arg Glu Gly Thr Ser Leu Gly Leu Thr Pro Leu Asn Thr Arg 690 695 700 His Pro Phe Ser Ser Pro Glu Pro Asp Ser Ala Gly His Thr Lys Ile 705 710 715 720 Ser Thr Ser Ile Pro Leu Leu Ser Ser Ala Ser Val Leu Glu Asp Lys 725 730 735 Val Ser Ala Thr Ser Thr Phe Ser His His Lys Ala Thr Ser Ser Ile 740 745 750 Thr Thr Gly Thr Pro Glu Ile Ser Thr Lys Thr Lys Pro Ser Ser Ala 755 760 765 Val Leu Ser Ser Met Thr Leu Ser Asn Ala Ala Thr Ser Pro Glu Arg 770 775 780 Val Arg Asn Ala Thr Ser Pro Leu Thr His Pro Ser Pro Ser Gly Glu 785 790 795 800 Glu Thr Ala Gly Ser Val Leu Thr Leu Ser Thr Ser Ala Glu Thr Thr 805 810 815 Asp Ser Pro Asn Ile His Pro Thr Gly Thr Leu Thr Ser Glu Ser Ser 820 825 830 Glu Ser Pro Ser Thr Leu Ser Leu Pro Ser Val Ser Gly Val Lys Thr 835 840 845 Thr Phe Ser Ser Ser Thr Pro Ser Thr His Leu Phe Thr Ser Gly Glu 850 855 860 Glu Thr Glu Glu Thr Ser Asn Pro Ser Val Ser Gln Pro Glu Thr Ser 865 870 875 880 Val Ser Arg Val Arg Thr Thr Leu Ala Ser Thr Ser Val Pro Thr Pro 885 890 895 Val Phe Pro Thr Met Asp Thr Trp Pro Thr Arg Ser Ala Gln Phe Ser 900 905 910 Ser Ser His Leu Val Ser Glu Leu Arg Ala Thr Ser Ser Thr Ser Val 915 920 925 Thr Asn Ser Thr Gly Ser Ala Leu Pro Lys Ile Ser His Leu Thr Gly 930 935 940 Thr Ala Thr Met Ser Gln Thr Asn Arg Asp Thr Phe Asn Asp Ser Ala 945 950 955 960 Ala Pro Gln Ser Thr Thr Trp Pro Glu Thr Ser Pro Arg Phe Lys Thr 965 970 975 Gly Leu Pro Ser Ala Thr Thr Thr Val Ser Thr Ser Ala Thr Ser Leu 980 985 990 Ser Ala Thr Val Met Val Ser Lys Phe Thr Ser Pro Ala Thr

Ser Ser 995 1000 1005 Met Glu Ala Thr Ser Ile Arg Glu Pro Ser Thr Thr Ile Leu Thr 1010 1015 1020 Thr Glu Thr Thr Asn Gly Pro Gly Ser Met Ala Val Ala Ser Thr 1025 1030 1035 Asn Ile Pro Ile Gly Lys Gly Tyr Ile Thr Glu Gly Arg Leu Asp 1040 1045 1050 Thr Ser His Leu Pro Ile Gly Thr Thr Ala Ser Ser Glu Thr Ser 1055 1060 1065 Met Asp Phe Thr Met Ala Lys Glu Ser Val Ser Met Ser Val Ser 1070 1075 1080 Pro Ser Gln Ser Met Asp Ala Ala Gly Ser Ser Thr Pro Gly Arg 1085 1090 1095 Thr Ser Gln Phe Val Asp Thr Phe Ser Asp Asp Val Tyr His Leu 1100 1105 1110 Thr Ser Arg Glu Ile Thr Ile Pro Arg Asp Gly Thr Ser Ser Ala 1115 1120 1125 Leu Thr Pro Gln Met Thr Ala Thr His Pro Pro Ser Pro Asp Pro 1130 1135 1140 Gly Ser Ala Arg Ser Thr Trp Leu Gly Ile Leu Ser Ser Ser Pro 1145 1150 1155 Ser Ser Pro Thr Pro Lys Val Thr Met Ser Ser Thr Phe Ser Thr 1160 1165 1170 Gln Arg Val Thr Thr Ser Met Ile Met Asp Thr Val Glu Thr Ser 1175 1180 1185 Arg Trp Asn Met Pro Asn Leu Pro Ser Thr Thr Ser Leu Thr Pro 1190 1195 1200 Ser Asn Ile Pro Thr Ser Gly Ala Ile Gly Lys Ser Thr Leu Val 1205 1210 1215 Pro Leu Asp Thr Pro Ser Pro Ala Thr Ser Leu Glu Ala Ser Glu 1220 1225 1230 Gly Gly Leu Pro Thr Leu Ser Thr Tyr Pro Glu Ser Thr Asn Thr 1235 1240 1245 Pro Ser Ile His Leu Gly Ala His Ala Ser Ser Glu Ser Pro Ser 1250 1255 1260 Thr Ile Lys Leu Thr Met Ala Ser Val Val Lys Pro Gly Ser Tyr 1265 1270 1275 Thr Pro Leu Thr Phe Pro Ser Ile Glu Thr His Ile His Val Ser 1280 1285 1290 Thr Ala Arg Met Ala Tyr Ser Ser Gly Ser Ser Pro Glu Met Thr 1295 1300 1305 Ala Pro Gly Glu Thr Asn Thr Gly Ser Thr Trp Asp Pro Thr Thr 1310 1315 1320 Tyr Ile Thr Thr Thr Asp Pro Lys Asp Thr Ser Ser Ala Gln Val 1325 1330 1335 Ser Thr Pro His Ser Val Arg Thr Leu Arg Thr Thr Glu Asn His 1340 1345 1350 Pro Lys Thr Glu Ser Ala Thr Pro Ala Ala Tyr Ser Gly Ser Pro 1355 1360 1365 Lys Ile Ser Ser Ser Pro Asn Leu Thr Ser Pro Ala Thr Lys Ala 1370 1375 1380 Trp Thr Ile Thr Asp Thr Thr Glu His Ser Thr Gln Leu His Tyr 1385 1390 1395 Thr Lys Leu Ala Glu Lys Ser Ser Gly Phe Glu Thr Gln Ser Ala 1400 1405 1410 Pro Gly Pro Val Ser Val Val Ile Pro Thr Ser Pro Thr Ile Gly 1415 1420 1425 Ser Ser Thr Leu Glu Leu Thr Ser Asp Val Pro Gly Glu Pro Leu 1430 1435 1440 Val Leu Ala Pro Ser Glu Gln Thr Thr Ile Thr Leu Pro Met Ala 1445 1450 1455 Thr Trp Leu Ser Thr Ser Leu Thr Glu Glu Met Ala Ser Thr Asp 1460 1465 1470 Leu Asp Ile Ser Ser Pro Ser Ser Pro Met Ser Thr Phe Ala Ile 1475 1480 1485 Phe Pro Pro Met Ser Thr Pro Ser His Glu Leu Ser Lys Ser Glu 1490 1495 1500 Ala Asp Thr Ser Ala Ile Arg Asn Thr Asp Ser Thr Thr Leu Asp 1505 1510 1515 Gln His Leu Gly Ile Arg Ser Leu Gly Arg Thr Gly Asp Leu Thr 1520 1525 1530 Thr Val Pro Ile Thr Pro Leu Thr Thr Thr Trp Thr Ser Val Ile 1535 1540 1545 Glu His Ser Thr Gln Ala Gln Asp Thr Leu Ser Ala Thr Met Ser 1550 1555 1560 Pro Thr His Val Thr Gln Ser Leu Lys Asp Gln Thr Ser Ile Pro 1565 1570 1575 Ala Ser Ala Ser Pro Ser His Leu Thr Glu Val Tyr Pro Glu Leu 1580 1585 1590 Gly Thr Gln Gly Arg Ser Ser Ser Glu Ala Thr Thr Phe Trp Lys 1595 1600 1605 Pro Ser Thr Asp Thr Leu Ser Arg Glu Ile Glu Thr Gly Pro Thr 1610 1615 1620 Asn Ile Gln Ser Thr Pro Pro Met Asp Asn Thr Thr Thr Gly Ser 1625 1630 1635 Ser Ser Ser Gly Val Thr Leu Gly Ile Ala His Leu Pro Ile Gly 1640 1645 1650 Thr Ser Ser Pro Ala Glu Thr Ser Thr Asn Met Ala Leu Glu Arg 1655 1660 1665 Arg Ser Ser Thr Ala Thr Val Ser Met Ala Gly Thr Met Gly Leu 1670 1675 1680 Leu Val Thr Ser Ala Pro Gly Arg Ser Ile Ser Gln Ser Leu Gly 1685 1690 1695 Arg Val Ser Ser Val Leu Ser Glu Ser Thr Thr Glu Gly Val Thr 1700 1705 1710 Asp Ser Ser Lys Gly Ser Ser Pro Arg Leu Asn Thr Gln Gly Asn 1715 1720 1725 Thr Ala Leu Ser Ser Ser Leu Glu Pro Ser Tyr Ala Glu Gly Ser 1730 1735 1740 Gln Met Ser Thr Ser Ile Pro Leu Thr Ser Ser Pro Thr Thr Pro 1745 1750 1755 Asp Val Glu Phe Ile Gly Gly Ser Thr Phe Trp Thr Lys Glu Val 1760 1765 1770 Thr Thr Val Met Thr Ser Asp Ile Ser Lys Ser Ser Ala Arg Thr 1775 1780 1785 Glu Ser Ser Ser Ala Thr Leu Met Ser Thr Ala Leu Gly Ser Thr 1790 1795 1800 Glu Asn Thr Gly Lys Glu Lys Leu Arg Thr Ala Ser Met Asp Leu 1805 1810 1815 Pro Ser Pro Thr Pro Ser Met Glu Val Thr Pro Trp Ile Ser Leu 1820 1825 1830 Thr Leu Ser Asn Ala Pro Asn Thr Thr Asp Ser Leu Asp Leu Ser 1835 1840 1845 His Gly Val His Thr Ser Ser Ala Gly Thr Leu Ala Thr Asp Arg 1850 1855 1860 Ser Leu Asn Thr Gly Val Thr Arg Ala Ser Arg Leu Glu Asn Gly 1865 1870 1875 Ser Asp Thr Ser Ser Lys Ser Leu Ser Met Gly Asn Ser Thr His 1880 1885 1890 Thr Ser Met Thr Tyr Thr Glu Lys Ser Glu Val Ser Ser Ser Ile 1895 1900 1905 His Pro Arg Pro Glu Thr Ser Ala Pro Gly Ala Glu Thr Thr Leu 1910 1915 1920 Thr Ser Thr Pro Gly Asn Arg Ala Ile Ser Leu Thr Leu Pro Phe 1925 1930 1935 Ser Ser Ile Pro Val Glu Glu Val Ile Ser Thr Gly Ile Thr Ser 1940 1945 1950 Gly Pro Asp Ile Asn Ser Ala Pro Met Thr His Ser Pro Ile Thr 1955 1960 1965 Pro Pro Thr Ile Val Trp Thr Ser Thr Gly Thr Ile Glu Gln Ser 1970 1975 1980 Thr Gln Pro Leu His Ala Val Ser Ser Glu Lys Val Ser Val Gln 1985 1990 1995 Thr Gln Ser Thr Pro Tyr Val Asn Ser Val Ala Val Ser Ala Ser 2000 2005 2010 Pro Thr His Glu Asn Ser Val Ser Ser Gly Ser Ser Thr Ser Ser 2015 2020 2025 Pro Tyr Ser Ser Ala Ser Leu Glu Ser Leu Asp Ser Thr Ile Ser 2030 2035 2040 Arg Arg Asn Ala Ile Thr Ser Trp Leu Trp Asp Leu Thr Thr Ser 2045 2050 2055 Leu Pro Thr Thr Thr Trp Pro Ser Thr Ser Leu Ser Glu Ala Leu 2060 2065 2070 Ser Ser Gly His Ser Gly Val Ser Asn Pro Ser Ser Thr Thr Thr 2075 2080 2085 Glu Phe Pro Leu Phe Ser Ala Ala Ser Thr Ser Ala Ala Lys Gln 2090 2095 2100 Arg Asn Pro Glu Thr Glu Thr His Gly Pro Gln Asn Thr Ala Ala 2105 2110 2115 Ser Thr Leu Asn Thr Asp Ala Ser Ser Val Thr Gly Leu Ser Glu 2120 2125 2130 Thr Pro Val Gly Ala Ser Ile Ser Ser Glu Val Pro Leu Pro Met 2135 2140 2145 Ala Ile Thr Ser Arg Ser Asp Val Ser Gly Leu Thr Ser Glu Ser 2150 2155 2160 Thr Ala Asn Pro Ser Leu Gly Thr Ala Ser Ser Ala Gly Thr Lys 2165 2170 2175 Leu Thr Arg Thr Ile Ser Leu Pro Thr Ser Glu Ser Leu Val Ser 2180 2185 2190 Phe Arg Met Asn Lys Asp Pro Trp Thr Val Ser Ile Pro Leu Gly 2195 2200 2205 Ser His Pro Thr Thr Asn Thr Glu Thr Ser Ile Pro Val Asn Ser 2210 2215 2220 Ala Gly Pro Pro Gly Leu Ser Thr Val Ala Ser Asp Val Ile Asp 2225 2230 2235 Thr Pro Ser Asp Gly Ala Glu Ser Ile Pro Thr Val Ser Phe Ser 2240 2245 2250 Pro Ser Pro Asp Thr Glu Val Thr Thr Ile Ser His Phe Pro Glu 2255 2260 2265 Lys Thr Thr His Ser Phe Arg Thr Ile Ser Ser Leu Thr His Glu 2270 2275 2280 Leu Thr Ser Arg Val Thr Pro Ile Pro Gly Asp Trp Met Ser Ser 2285 2290 2295 Ala Met Ser Thr Lys Pro Thr Gly Ala Ser Pro Ser Ile Thr Leu 2300 2305 2310 Gly Glu Arg Arg Thr Ile Thr Ser Ala Ala Pro Thr Thr Ser Pro 2315 2320 2325 Ile Val Leu Thr Ala Ser Phe Thr Glu Thr Ser Thr Val Ser Leu 2330 2335 2340 Asp Asn Glu Thr Thr Val Lys Thr Ser Asp Ile Leu Asp Ala Arg 2345 2350 2355 Lys Thr Asn Glu Leu Pro Ser Asp Ser Ser Ser Ser Ser Asp Leu 2360 2365 2370 Ile Asn Thr Ser Ile Ala Ser Ser Thr Met Asp Val Thr Lys Thr 2375 2380 2385 Ala Ser Ile Ser Pro Thr Ser Ile Ser Gly Met Thr Ala Ser Ser 2390 2395 2400 Ser Pro Ser Leu Phe Ser Ser Asp Arg Pro Gln Val Pro Thr Ser 2405 2410 2415 Thr Thr Glu Thr Asn Thr Ala Thr Ser Pro Ser Val Ser Ser Asn 2420 2425 2430 Thr Tyr Ser Leu Asp Gly Gly Ser Asn Val Gly Gly Thr Pro Ser 2435 2440 2445 Thr Leu Pro Pro Phe Thr Ile Thr His Pro Val Glu Thr Ser Ser 2450 2455 2460 Ala Leu Leu Ala Trp Ser Arg Pro Val Arg Thr Phe Ser Thr Met 2465 2470 2475 Val Ser Thr Asp Thr Ala Ser Gly Glu Asn Pro Thr Ser Ser Asn 2480 2485 2490 Ser Val Val Thr Ser Val Pro Ala Pro Gly Thr Trp Thr Ser Val 2495 2500 2505 Gly Ser Thr Thr Asp Leu Pro Ala Met Gly Phe Leu Lys Thr Ser 2510 2515 2520 Pro Ala Gly Glu Ala His Ser Leu Leu Ala Ser Thr Ile Glu Pro 2525 2530 2535 Ala Thr Ala Phe Thr Pro His Leu Ser Ala Ala Val Val Thr Gly 2540 2545 2550 Ser Ser Ala Thr Ser Glu Ala Ser Leu Leu Thr Thr Ser Glu Ser 2555 2560 2565 Lys Ala Ile His Ser Ser Pro Gln Thr Pro Thr Thr Pro Thr Ser 2570 2575 2580 Gly Ala Asn Trp Glu Thr Ser Ala Thr Pro Glu Ser Leu Leu Val 2585 2590 2595 Val Thr Glu Thr Ser Asp Thr Thr Leu Thr Ser Lys Ile Leu Val 2600 2605 2610 Thr Asp Thr Ile Leu Phe Ser Thr Val Ser Thr Pro Pro Ser Lys 2615 2620 2625 Phe Pro Ser Thr Gly Thr Leu Ser Gly Ala Ser Phe Pro Thr Leu 2630 2635 2640 Leu Pro Asp Thr Pro Ala Ile Pro Leu Thr Ala Thr Glu Pro Thr 2645 2650 2655 Ser Ser Leu Ala Thr Ser Phe Asp Ser Thr Pro Leu Val Thr Ile 2660 2665 2670 Ala Ser Asp Ser Leu Gly Thr Val Pro Glu Thr Thr Leu Thr Met 2675 2680 2685 Ser Glu Thr Ser Asn Gly Asp Ala Leu Val Leu Lys Thr Val Ser 2690 2695 2700 Asn Pro Asp Arg Ser Ile Pro Gly Ile Thr Ile Gln Gly Val Thr 2705 2710 2715 Glu Ser Pro Leu His Pro Ser Ser Thr Ser Pro Ser Lys Ile Val 2720 2725 2730 Ala Pro Arg Asn Thr Thr Tyr Glu Gly Ser Ile Thr Val Ala Leu 2735 2740 2745 Ser Thr Leu Pro Ala Gly Thr Thr Gly Ser Leu Val Phe Ser Gln 2750 2755 2760 Ser Ser Glu Asn Ser Glu Thr Thr Ala Leu Val Asp Ser Ser Ala 2765 2770 2775 Gly Leu Glu Arg Ala Ser Val Met Pro Leu Thr Thr Gly Ser Gln 2780 2785 2790 Gly Met Ala Ser Ser Gly Gly Ile Arg Ser Gly Ser Thr His Ser 2795 2800 2805 Thr Gly Thr Lys Thr Phe Ser Ser Leu Pro Leu Thr Met Asn Pro 2810 2815 2820 Gly Glu Val Thr Ala Met Ser Glu Ile Thr Thr Asn Arg Leu Thr 2825 2830 2835 Ala Thr Gln Ser Thr Ala Pro Lys Gly Ile Pro Val Lys Pro Thr 2840 2845 2850 Ser Ala Glu Ser Gly Leu Leu Thr Pro Val Ser Ala Ser Ser Ser 2855 2860 2865 Pro Ser Lys Ala Phe Ala Ser Leu Thr Thr Ala Pro Pro Thr Trp 2870 2875 2880 Gly Ile Pro Gln Ser Thr Leu Thr Phe Glu Phe Ser Glu Val Pro 2885 2890 2895 Ser Leu Asp Thr Lys Ser Ala Ser Leu Pro Thr Pro Gly Gln Ser 2900 2905 2910 Leu Asn Thr Ile Pro Asp Ser Asp Ala Ser Thr Ala Ser Ser Ser 2915 2920 2925 Leu Ser Lys Ser Pro Glu Lys Asn Pro Arg Ala Arg Met Met Thr 2930 2935 2940 Ser Thr Lys Ala Ile Ser Ala Ser Ser Phe Gln Ser Thr Gly Phe 2945 2950 2955 Thr Glu Thr Pro Glu Gly Ser Ala Ser Pro Ser Met Ala Gly His 2960 2965 2970 Glu Pro Arg Val Pro Thr Ser Gly Thr Gly Asp Pro Arg Tyr Ala 2975 2980 2985 Ser Glu Ser Met Ser Tyr Pro Asp Pro Ser Lys Ala Ser Ser Ala 2990 2995 3000 Met Thr Ser Thr Ser Leu Ala Ser Lys Leu Thr Thr Leu Phe Ser 3005 3010 3015 Thr Gly Gln Ala Ala Arg Ser Gly Ser Ser Ser Ser Pro Ile Ser 3020 3025 3030 Leu Ser Thr Glu Lys Glu Thr Ser Phe Leu Ser Pro Thr Ala Ser 3035 3040 3045 Thr Ser Arg Lys Thr Ser Leu Phe Leu Gly Pro Ser Met Ala Arg 3050 3055 3060 Gln Pro Asn Ile Leu Val His Leu Gln Thr Ser Ala Leu Thr Leu 3065 3070 3075 Ser Pro Thr Ser Thr Leu Asn Met Ser Gln Glu Glu Pro Pro Glu 3080 3085 3090 Leu Thr Ser Ser Gln Thr Ile Ala Glu Glu Glu Gly Thr Thr Ala 3095 3100 3105 Glu Thr Gln Thr Leu Thr Phe Thr Pro Ser Glu Thr Pro Thr Ser 3110 3115 3120 Leu Leu Pro Val Ser Ser Pro Thr Glu Pro Thr Ala Arg Arg Lys 3125 3130 3135 Ser Ser Pro Glu Thr Trp Ala Ser Ser Ile Ser Val Pro Ala Lys 3140 3145 3150 Thr Ser Leu Val Glu Thr Thr Asp Gly Thr Leu Val Thr Thr Ile 3155 3160 3165 Lys Met Ser Ser Gln Ala Ala Gln Gly Asn Ser Thr Trp Pro Ala 3170 3175 3180 Pro Ala Glu Glu Thr Gly Ser Ser Pro Ala Gly Thr Ser Pro Gly 3185 3190

3195 Ser Pro Glu Met Ser Thr Thr Leu Lys Ile Met Ser Ser Lys Glu 3200 3205 3210 Pro Ser Ile Ser Pro Glu Ile Arg Ser Thr Val Arg Asn Ser Pro 3215 3220 3225 Trp Lys Thr Pro Glu Thr Thr Val Pro Met Glu Thr Thr Val Glu 3230 3235 3240 Pro Val Thr Leu Gln Ser Thr Ala Leu Gly Ser Gly Ser Thr Ser 3245 3250 3255 Ile Ser His Leu Pro Thr Gly Thr Thr Ser Pro Thr Lys Ser Pro 3260 3265 3270 Thr Glu Asn Met Leu Ala Thr Glu Arg Val Ser Leu Ser Pro Ser 3275 3280 3285 Pro Pro Glu Ala Trp Thr Asn Leu Tyr Ser Gly Thr Pro Gly Gly 3290 3295 3300 Thr Arg Gln Ser Leu Ala Thr Met Ser Ser Val Ser Leu Glu Ser 3305 3310 3315 Pro Thr Ala Arg Ser Ile Thr Gly Thr Gly Gln Gln Ser Ser Pro 3320 3325 3330 Glu Leu Val Ser Lys Thr Thr Gly Met Glu Phe Ser Met Trp His 3335 3340 3345 Gly Ser Thr Gly Gly Thr Thr Gly Asp Thr His Val Ser Leu Ser 3350 3355 3360 Thr Ser Ser Asn Ile Leu Glu Asp Pro Val Thr Ser Pro Asn Ser 3365 3370 3375 Val Ser Ser Leu Thr Asp Lys Ser Lys His Lys Thr Glu Thr Trp 3380 3385 3390 Val Ser Thr Thr Ala Ile Pro Ser Thr Val Leu Asn Asn Lys Ile 3395 3400 3405 Met Ala Ala Glu Gln Gln Thr Ser Arg Ser Val Asp Glu Ala Tyr 3410 3415 3420 Ser Ser Thr Ser Ser Trp Ser Asp Gln Thr Ser Gly Ser Asp Ile 3425 3430 3435 Thr Leu Gly Ala Ser Pro Asp Val Thr Asn Thr Leu Tyr Ile Thr 3440 3445 3450 Ser Thr Ala Gln Thr Thr Ser Leu Val Ser Leu Pro Ser Gly Asp 3455 3460 3465 Gln Gly Ile Thr Ser Leu Thr Asn Pro Ser Gly Gly Lys Thr Ser 3470 3475 3480 Ser Ala Ser Ser Val Thr Ser Pro Ser Ile Gly Leu Glu Thr Leu 3485 3490 3495 Arg Ala Asn Val Ser Ala Val Lys Ser Asp Ile Ala Pro Thr Ala 3500 3505 3510 Gly His Leu Ser Gln Thr Ser Ser Pro Ala Glu Val Ser Ile Leu 3515 3520 3525 Asp Val Thr Thr Ala Pro Thr Pro Gly Ile Ser Thr Thr Ile Thr 3530 3535 3540 Thr Met Gly Thr Asn Ser Ile Ser Thr Thr Thr Pro Asn Pro Glu 3545 3550 3555 Val Gly Met Ser Thr Met Asp Ser Thr Pro Ala Thr Glu Arg Arg 3560 3565 3570 Thr Thr Ser Thr Glu His Pro Ser Thr Trp Ser Ser Thr Ala Ala 3575 3580 3585 Ser Asp Ser Trp Thr Val Thr Asp Met Thr Ser Asn Leu Lys Val 3590 3595 3600 Ala Arg Ser Pro Gly Thr Ile Ser Thr Met His Thr Thr Ser Phe 3605 3610 3615 Leu Ala Ser Ser Thr Glu Leu Asp Ser Met Ser Thr Pro His Gly 3620 3625 3630 Arg Ile Thr Val Ile Gly Thr Ser Leu Val Thr Pro Ser Ser Asp 3635 3640 3645 Ala Ser Ala Val Lys Thr Glu Thr Ser Thr Ser Glu Arg Thr Leu 3650 3655 3660 Ser Pro Ser Asp Thr Thr Ala Ser Thr Pro Ile Ser Thr Phe Ser 3665 3670 3675 Arg Val Gln Arg Met Ser Ile Ser Val Pro Asp Ile Leu Ser Thr 3680 3685 3690 Ser Trp Thr Pro Ser Ser Thr Glu Ala Glu Asp Val Pro Val Ser 3695 3700 3705 Met Val Ser Thr Asp His Ala Ser Thr Lys Thr Asp Pro Asn Thr 3710 3715 3720 Pro Leu Ser Thr Phe Leu Phe Asp Ser Leu Ser Thr Leu Asp Trp 3725 3730 3735 Asp Thr Gly Arg Ser Leu Ser Ser Ala Thr Ala Thr Thr Ser Ala 3740 3745 3750 Pro Gln Gly Ala Thr Thr Pro Gln Glu Leu Thr Leu Glu Thr Met 3755 3760 3765 Ile Ser Pro Ala Thr Ser Gln Leu Pro Phe Ser Ile Gly His Ile 3770 3775 3780 Thr Ser Ala Val Thr Pro Ala Ala Met Ala Arg Ser Ser Gly Val 3785 3790 3795 Thr Phe Ser Arg Pro Asp Pro Thr Ser Lys Lys Ala Glu Gln Thr 3800 3805 3810 Ser Thr Gln Leu Pro Thr Thr Thr Ser Ala His Pro Gly Gln Val 3815 3820 3825 Pro Arg Ser Ala Ala Thr Thr Leu Asp Val Ile Pro His Thr Ala 3830 3835 3840 Lys Thr Pro Asp Ala Thr Phe Gln Arg Gln Gly Gln Thr Ala Leu 3845 3850 3855 Thr Thr Glu Ala Arg Ala Thr Ser Asp Ser Trp Asn Glu Lys Glu 3860 3865 3870 Lys Ser Thr Pro Ser Ala Pro Trp Ile Thr Glu Met Met Asn Ser 3875 3880 3885 Val Ser Glu Asp Thr Ile Lys Glu Val Thr Ser Ser Ser Ser Val 3890 3895 3900 Leu Arg Thr Leu Asn Thr Leu Asp Ile Asn Leu Glu Ser Gly Thr 3905 3910 3915 Thr Ser Ser Pro Ser Trp Lys Ser Ser Pro Tyr Glu Arg Ile Ala 3920 3925 3930 Pro Ser Glu Ser Thr Thr Asp Lys Glu Ala Ile His Pro Ser Thr 3935 3940 3945 Asn Thr Val Glu Thr Thr Gly Trp Val Thr Ser Ser Glu His Ala 3950 3955 3960 Ser His Ser Thr Ile Pro Ala His Ser Ala Ser Ser Lys Leu Thr 3965 3970 3975 Ser Pro Val Val Thr Thr Ser Thr Arg Glu Gln Ala Ile Val Ser 3980 3985 3990 Met Ser Thr Thr Thr Trp Pro Glu Ser Thr Arg Ala Arg Thr Glu 3995 4000 4005 Pro Asn Ser Phe Leu Thr Ile Glu Leu Arg Asp Val Ser Pro Tyr 4010 4015 4020 Met Asp Thr Ser Ser Thr Thr Gln Thr Ser Ile Ile Ser Ser Pro 4025 4030 4035 Gly Ser Thr Ala Ile Thr Lys Gly Pro Arg Thr Glu Ile Thr Ser 4040 4045 4050 Ser Lys Arg Ile Ser Ser Ser Phe Leu Ala Gln Ser Met Arg Ser 4055 4060 4065 Ser Asp Ser Pro Ser Glu Ala Ile Thr Arg Leu Ser Asn Phe Pro 4070 4075 4080 Ala Met Thr Glu Ser Gly Gly Met Ile Leu Ala Met Gln Thr Ser 4085 4090 4095 Pro Pro Gly Ala Thr Ser Leu Ser Ala Pro Thr Leu Asp Thr Ser 4100 4105 4110 Ala Thr Ala Ser Trp Thr Gly Thr Pro Leu Ala Thr Thr Gln Arg 4115 4120 4125 Phe Thr Tyr Ser Glu Lys Thr Thr Leu Phe Ser Lys Gly Pro Glu 4130 4135 4140 Asp Thr Ser Gln Pro Ser Pro Pro Ser Val Glu Glu Thr Ser Ser 4145 4150 4155 Ser Ser Ser Leu Val Pro Ile His Ala Thr Thr Ser Pro Ser Asn 4160 4165 4170 Ile Leu Leu Thr Ser Gln Gly His Ser Pro Ser Ser Thr Pro Pro 4175 4180 4185 Val Thr Ser Val Phe Leu Ser Glu Thr Ser Gly Leu Gly Lys Thr 4190 4195 4200 Thr Asp Met Ser Arg Ile Ser Leu Glu Pro Gly Thr Ser Leu Pro 4205 4210 4215 Pro Asn Leu Ser Ser Thr Ala Gly Glu Ala Leu Ser Thr Tyr Glu 4220 4225 4230 Ala Ser Arg Asp Thr Lys Ala Ile His His Ser Ala Asp Thr Ala 4235 4240 4245 Val Thr Asn Met Glu Ala Thr Ser Ser Glu Tyr Ser Pro Ile Pro 4250 4255 4260 Gly His Thr Lys Pro Ser Lys Ala Thr Ser Pro Leu Val Thr Ser 4265 4270 4275 His Ile Met Gly Asp Ile Thr Ser Ser Thr Ser Val Phe Gly Ser 4280 4285 4290 Ser Glu Thr Thr Glu Ile Glu Thr Val Ser Ser Val Asn Gln Gly 4295 4300 4305 Leu Gln Glu Arg Ser Thr Ser Gln Val Ala Ser Ser Ala Thr Glu 4310 4315 4320 Thr Ser Thr Val Ile Thr His Val Ser Ser Gly Asp Ala Thr Thr 4325 4330 4335 His Val Thr Lys Thr Gln Ala Thr Phe Ser Ser Gly Thr Ser Ile 4340 4345 4350 Ser Ser Pro His Gln Phe Ile Thr Ser Thr Asn Thr Phe Thr Asp 4355 4360 4365 Val Ser Thr Asn Pro Ser Thr Ser Leu Ile Met Thr Glu Ser Ser 4370 4375 4380 Gly Val Thr Ile Thr Thr Gln Thr Gly Pro Thr Gly Ala Ala Thr 4385 4390 4395 Gln Gly Pro Tyr Leu Leu Asp Thr Ser Thr Met Pro Tyr Leu Thr 4400 4405 4410 Glu Thr Pro Leu Ala Val Thr Pro Asp Phe Met Gln Ser Glu Lys 4415 4420 4425 Thr Thr Leu Ile Ser Lys Gly Pro Lys Asp Val Ser Trp Thr Ser 4430 4435 4440 Pro Pro Ser Val Ala Glu Thr Ser Tyr Pro Ser Ser Leu Thr Pro 4445 4450 4455 Phe Leu Val Thr Thr Ile Pro Pro Ala Thr Ser Thr Leu Gln Gly 4460 4465 4470 Gln His Thr Ser Ser Pro Val Ser Ala Thr Ser Val Leu Thr Ser 4475 4480 4485 Gly Leu Val Lys Thr Thr Asp Met Leu Asn Thr Ser Met Glu Pro 4490 4495 4500 Val Thr Asn Ser Pro Gln Asn Leu Asn Asn Pro Ser Asn Glu Ile 4505 4510 4515 Leu Ala Thr Leu Ala Ala Thr Thr Asp Ile Glu Thr Ile His Pro 4520 4525 4530 Ser Ile Asn Lys Ala Val Thr Asn Met Gly Thr Ala Ser Ser Ala 4535 4540 4545 His Val Leu His Ser Thr Leu Pro Val Ser Ser Glu Pro Ser Thr 4550 4555 4560 Ala Thr Ser Pro Met Val Pro Ala Ser Ser Met Gly Asp Ala Leu 4565 4570 4575 Ala Ser Ile Ser Ile Pro Gly Ser Glu Thr Thr Asp Ile Glu Gly 4580 4585 4590 Glu Pro Thr Ser Ser Leu Thr Ala Gly Arg Lys Glu Asn Ser Thr 4595 4600 4605 Leu Gln Glu Met Asn Ser Thr Thr Glu Ser Asn Ile Ile Leu Ser 4610 4615 4620 Asn Val Ser Val Gly Ala Ile Thr Glu Ala Thr Lys Met Glu Val 4625 4630 4635 Pro Ser Phe Asp Ala Thr Phe Ile Pro Thr Pro Ala Gln Ser Thr 4640 4645 4650 Lys Phe Pro Asp Ile Phe Ser Val Ala Ser Ser Arg Leu Ser Asn 4655 4660 4665 Ser Pro Pro Met Thr Ile Ser Thr His Met Thr Thr Thr Gln Thr 4670 4675 4680 Gly Ser Ser Gly Ala Thr Ser Lys Ile Pro Leu Ala Leu Asp Thr 4685 4690 4695 Ser Thr Leu Glu Thr Ser Ala Gly Thr Pro Ser Val Val Thr Glu 4700 4705 4710 Gly Phe Ala His Ser Lys Ile Thr Thr Ala Met Asn Asn Asp Val 4715 4720 4725 Lys Asp Val Ser Gln Thr Asn Pro Pro Phe Gln Asp Glu Ala Ser 4730 4735 4740 Ser Pro Ser Ser Gln Ala Pro Val Leu Val Thr Thr Leu Pro Ser 4745 4750 4755 Ser Val Ala Phe Thr Pro Gln Trp His Ser Thr Ser Ser Pro Val 4760 4765 4770 Ser Met Ser Ser Val Leu Thr Ser Ser Leu Val Lys Thr Ala Gly 4775 4780 4785 Lys Val Asp Thr Ser Leu Glu Thr Val Thr Ser Ser Pro Gln Ser 4790 4795 4800 Met Ser Asn Thr Leu Asp Asp Ile Ser Val Thr Ser Ala Ala Thr 4805 4810 4815 Thr Asp Ile Glu Thr Thr His Pro Ser Ile Asn Thr Val Val Thr 4820 4825 4830 Asn Val Gly Thr Thr Gly Ser Ala Phe Glu Ser His Ser Thr Val 4835 4840 4845 Ser Ala Tyr Pro Glu Pro Ser Lys Val Thr Ser Pro Asn Val Thr 4850 4855 4860 Thr Ser Thr Met Glu Asp Thr Thr Ile Ser Arg Ser Ile Pro Lys 4865 4870 4875 Ser Ser Lys Thr Thr Arg Thr Glu Thr Glu Thr Thr Ser Ser Leu 4880 4885 4890 Thr Pro Lys Leu Arg Glu Thr Ser Ile Ser Gln Glu Ile Thr Ser 4895 4900 4905 Ser Thr Glu Thr Ser Thr Val Pro Tyr Lys Glu Leu Thr Gly Ala 4910 4915 4920 Thr Thr Glu Val Ser Arg Thr Asp Val Thr Ser Ser Ser Ser Thr 4925 4930 4935 Ser Phe Pro Gly Pro Asp Gln Ser Thr Val Ser Leu Asp Ile Ser 4940 4945 4950 Thr Glu Thr Asn Thr Arg Leu Ser Thr Ser Pro Ile Met Thr Glu 4955 4960 4965 Ser Ala Glu Ile Thr Ile Thr Thr Gln Thr Gly Pro His Gly Ala 4970 4975 4980 Thr Ser Gln Asp Thr Phe Thr Met Asp Pro Ser Asn Thr Thr Pro 4985 4990 4995 Gln Ala Gly Ile His Ser Ala Met Thr His Gly Phe Ser Gln Leu 5000 5005 5010 Asp Val Thr Thr Leu Met Ser Arg Ile Pro Gln Asp Val Ser Trp 5015 5020 5025 Thr Ser Pro Pro Ser Val Asp Lys Thr Ser Ser Pro Ser Ser Phe 5030 5035 5040 Leu Ser Ser Pro Ala Met Thr Thr Pro Ser Leu Ile Ser Ser Thr 5045 5050 5055 Leu Pro Glu Asp Lys Leu Ser Ser Pro Met Thr Ser Leu Leu Thr 5060 5065 5070 Ser Gly Leu Val Lys Ile Thr Asp Ile Leu Arg Thr Arg Leu Glu 5075 5080 5085 Pro Val Thr Ser Ser Leu Pro Asn Phe Ser Ser Thr Ser Asp Lys 5090 5095 5100 Ile Leu Ala Thr Ser Lys Asp Ser Lys Asp Thr Lys Glu Ile Phe 5105 5110 5115 Pro Ser Ile Asn Thr Glu Glu Thr Asn Val Lys Ala Asn Asn Ser 5120 5125 5130 Gly His Glu Ser His Ser Pro Ala Leu Ala Asp Ser Glu Thr Pro 5135 5140 5145 Lys Ala Thr Thr Gln Met Val Ile Thr Thr Thr Val Gly Asp Pro 5150 5155 5160 Ala Pro Ser Thr Ser Met Pro Val His Gly Ser Ser Glu Thr Thr 5165 5170 5175 Asn Ile Lys Arg Glu Pro Thr Tyr Phe Leu Thr Pro Arg Leu Arg 5180 5185 5190 Glu Thr Ser Thr Ser Gln Glu Ser Ser Phe Pro Thr Asp Thr Ser 5195 5200 5205 Phe Leu Leu Ser Lys Val Pro Thr Gly Thr Ile Thr Glu Val Ser 5210 5215 5220 Ser Thr Gly Val Asn Ser Ser Ser Lys Ile Ser Thr Pro Asp His 5225 5230 5235 Asp Lys Ser Thr Val Pro Pro Asp Thr Phe Thr Gly Glu Ile Pro 5240 5245 5250 Arg Val Phe Thr Ser Ser Ile Lys Thr Lys Ser Ala Glu Met Thr 5255 5260 5265 Ile Thr Thr Gln Ala Ser Pro Pro Glu Ser Ala Ser His Ser Thr 5270 5275 5280 Leu Pro Leu Asp Thr Ser Thr Thr Leu Ser Gln Gly Gly Thr His 5285 5290 5295 Ser Thr Val Thr Gln Gly Phe Pro Tyr Ser Glu Val Thr Thr Leu 5300 5305 5310 Met Gly Met Gly Pro Gly Asn Val Ser Trp Met Thr Thr Pro Pro 5315 5320 5325 Val Glu Glu Thr Ser Ser Val Ser Ser Leu Met Ser Ser Pro Ala 5330 5335 5340 Met Thr Ser Pro Ser Pro Val Ser Ser Thr Ser Pro Gln Ser Ile 5345 5350 5355 Pro Ser Ser Pro Leu Pro Val Thr Ala Leu Pro Thr Ser Val Leu 5360 5365 5370 Val Thr Thr Thr Asp Val Leu Gly Thr Thr Ser Pro Glu Ser Val 5375 5380 5385 Thr Ser Ser Pro Pro Asn Leu

Ser Ser Ile Thr His Glu Arg Pro 5390 5395 5400 Ala Thr Tyr Lys Asp Thr Ala His Thr Glu Ala Ala Met His His 5405 5410 5415 Ser Thr Asn Thr Ala Val Thr Asn Val Gly Thr Ser Gly Ser Gly 5420 5425 5430 His Lys Ser Gln Ser Ser Val Leu Ala Asp Ser Glu Thr Ser Lys 5435 5440 5445 Ala Thr Pro Leu Met Ser Thr Thr Ser Thr Leu Gly Asp Thr Ser 5450 5455 5460 Val Ser Thr Ser Thr Pro Asn Ile Ser Gln Thr Asn Gln Ile Gln 5465 5470 5475 Thr Glu Pro Thr Ala Ser Leu Ser Pro Arg Leu Arg Glu Ser Ser 5480 5485 5490 Thr Ser Glu Lys Thr Ser Ser Thr Thr Glu Thr Asn Thr Ala Phe 5495 5500 5505 Ser Tyr Val Pro Thr Gly Ala Ile Thr Gln Ala Ser Arg Thr Glu 5510 5515 5520 Ile Ser Ser Ser Arg Thr Ser Ile Ser Asp Leu Asp Arg Pro Thr 5525 5530 5535 Ile Ala Pro Asp Ile Ser Thr Gly Met Ile Thr Arg Leu Phe Thr 5540 5545 5550 Ser Pro Ile Met Thr Lys Ser Ala Glu Met Thr Val Thr Thr Gln 5555 5560 5565 Thr Thr Thr Pro Gly Ala Thr Ser Gln Gly Ile Leu Pro Trp Asp 5570 5575 5580 Thr Ser Thr Thr Leu Phe Gln Gly Gly Thr His Ser Thr Val Ser 5585 5590 5595 Gln Gly Phe Pro His Ser Glu Ile Thr Thr Leu Arg Ser Arg Thr 5600 5605 5610 Pro Gly Asp Val Ser Trp Met Thr Thr Pro Pro Val Glu Glu Thr 5615 5620 5625 Ser Ser Gly Phe Ser Leu Met Ser Pro Ser Met Thr Ser Pro Ser 5630 5635 5640 Pro Val Ser Ser Thr Ser Pro Glu Ser Ile Pro Ser Ser Pro Leu 5645 5650 5655 Pro Val Thr Ala Leu Leu Thr Ser Val Leu Val Thr Thr Thr Asn 5660 5665 5670 Val Leu Gly Thr Thr Ser Pro Glu Pro Val Thr Ser Ser Pro Pro 5675 5680 5685 Asn Leu Ser Ser Pro Thr Gln Glu Arg Leu Thr Thr Tyr Lys Asp 5690 5695 5700 Thr Ala His Thr Glu Ala Met His Ala Ser Met His Thr Asn Thr 5705 5710 5715 Ala Val Ala Asn Val Gly Thr Ser Ile Ser Gly His Glu Ser Gln 5720 5725 5730 Ser Ser Val Pro Ala Asp Ser His Thr Ser Lys Ala Thr Ser Pro 5735 5740 5745 Met Gly Ile Thr Phe Ala Met Gly Asp Thr Ser Val Ser Thr Ser 5750 5755 5760 Thr Pro Ala Phe Phe Glu Thr Arg Ile Gln Thr Glu Ser Thr Ser 5765 5770 5775 Ser Leu Ile Pro Gly Leu Arg Asp Thr Arg Thr Ser Glu Glu Ile 5780 5785 5790 Asn Thr Val Thr Glu Thr Ser Thr Val Leu Ser Glu Val Pro Thr 5795 5800 5805 Thr Thr Thr Thr Glu Val Ser Arg Thr Glu Val Ile Thr Ser Ser 5810 5815 5820 Arg Thr Thr Ile Ser Gly Pro Asp His Ser Lys Met Ser Pro Tyr 5825 5830 5835 Ile Ser Thr Glu Thr Ile Thr Arg Leu Ser Thr Phe Pro Phe Val 5840 5845 5850 Thr Gly Ser Thr Glu Met Ala Ile Thr Asn Gln Thr Gly Pro Ile 5855 5860 5865 Gly Thr Ile Ser Gln Ala Thr Leu Thr Leu Asp Thr Ser Ser Thr 5870 5875 5880 Ala Ser Trp Glu Gly Thr His Ser Pro Val Thr Gln Arg Phe Pro 5885 5890 5895 His Ser Glu Glu Thr Thr Thr Met Ser Arg Ser Thr Lys Gly Val 5900 5905 5910 Ser Trp Gln Ser Pro Pro Ser Val Glu Glu Thr Ser Ser Pro Ser 5915 5920 5925 Ser Pro Val Pro Leu Pro Ala Ile Thr Ser His Ser Ser Leu Tyr 5930 5935 5940 Ser Ala Val Ser Gly Ser Ser Pro Thr Ser Ala Leu Pro Val Thr 5945 5950 5955 Ser Leu Leu Thr Ser Gly Arg Arg Lys Thr Ile Asp Met Leu Asp 5960 5965 5970 Thr His Ser Glu Leu Val Thr Ser Ser Leu Pro Ser Ala Ser Ser 5975 5980 5985 Phe Ser Gly Glu Ile Leu Thr Ser Glu Ala Ser Thr Asn Thr Glu 5990 5995 6000 Thr Ile His Phe Ser Glu Asn Thr Ala Glu Thr Asn Met Gly Thr 6005 6010 6015 Thr Asn Ser Met His Lys Leu His Ser Ser Val Ser Ile His Ser 6020 6025 6030 Gln Pro Ser Gly His Thr Pro Pro Lys Val Thr Gly Ser Met Met 6035 6040 6045 Glu Asp Ala Ile Val Ser Thr Ser Thr Pro Gly Ser Pro Glu Thr 6050 6055 6060 Lys Asn Val Asp Arg Asp Ser Thr Ser Pro Leu Thr Pro Glu Leu 6065 6070 6075 Lys Glu Asp Ser Thr Ala Leu Val Met Asn Ser Thr Thr Glu Ser 6080 6085 6090 Asn Thr Val Phe Ser Ser Val Ser Leu Asp Ala Ala Thr Glu Val 6095 6100 6105 Ser Arg Ala Glu Val Thr Tyr Tyr Asp Pro Thr Phe Met Pro Ala 6110 6115 6120 Ser Ala Gln Ser Thr Lys Ser Pro Asp Ile Ser Pro Glu Ala Ser 6125 6130 6135 Ser Ser His Ser Asn Ser Pro Pro Leu Thr Ile Ser Thr His Lys 6140 6145 6150 Thr Ile Ala Thr Gln Thr Gly Pro Ser Gly Val Thr Ser Leu Gly 6155 6160 6165 Gln Leu Thr Leu Asp Thr Ser Thr Ile Ala Thr Ser Ala Gly Thr 6170 6175 6180 Pro Ser Ala Arg Thr Gln Asp Phe Val Asp Ser Glu Thr Thr Ser 6185 6190 6195 Val Met Asn Asn Asp Leu Asn Asp Val Leu Lys Thr Ser Pro Phe 6200 6205 6210 Ser Ala Glu Glu Ala Asn Ser Leu Ser Ser Gln Ala Pro Leu Leu 6215 6220 6225 Val Thr Thr Ser Pro Ser Pro Val Thr Ser Thr Leu Gln Glu His 6230 6235 6240 Ser Thr Ser Ser Leu Val Ser Val Thr Ser Val Pro Thr Pro Thr 6245 6250 6255 Leu Ala Lys Ile Thr Asp Met Asp Thr Asn Leu Glu Pro Val Thr 6260 6265 6270 Arg Ser Pro Gln Asn Leu Arg Asn Thr Leu Ala Thr Ser Glu Ala 6275 6280 6285 Thr Thr Asp Thr His Thr Met His Pro Ser Ile Asn Thr Ala Val 6290 6295 6300 Ala Asn Val Gly Thr Thr Ser Ser Pro Asn Glu Phe Tyr Phe Thr 6305 6310 6315 Val Ser Pro Asp Ser Asp Pro Tyr Lys Ala Thr Ser Ala Val Val 6320 6325 6330 Ile Thr Ser Thr Ser Gly Asp Ser Ile Val Ser Thr Ser Met Pro 6335 6340 6345 Arg Ser Ser Ala Met Lys Lys Ile Glu Ser Glu Thr Thr Phe Ser 6350 6355 6360 Leu Ile Phe Arg Leu Arg Glu Thr Ser Thr Ser Gln Lys Ile Gly 6365 6370 6375 Ser Ser Ser Asp Thr Ser Thr Val Phe Asp Lys Ala Phe Thr Ala 6380 6385 6390 Ala Thr Thr Glu Val Ser Arg Thr Glu Leu Thr Ser Ser Ser Arg 6395 6400 6405 Thr Ser Ile Gln Gly Thr Glu Lys Pro Thr Met Ser Pro Asp Thr 6410 6415 6420 Ser Thr Arg Ser Val Thr Met Leu Ser Thr Phe Ala Gly Leu Thr 6425 6430 6435 Lys Ser Glu Glu Arg Thr Ile Ala Thr Gln Thr Gly Pro His Arg 6440 6445 6450 Ala Thr Ser Gln Gly Thr Leu Thr Trp Asp Thr Ser Ile Thr Thr 6455 6460 6465 Ser Gln Ala Gly Thr His Ser Ala Met Thr His Gly Phe Ser Gln 6470 6475 6480 Leu Asp Leu Ser Thr Leu Thr Ser Arg Val Pro Glu Tyr Ile Ser 6485 6490 6495 Gly Thr Ser Pro Pro Ser Val Glu Lys Thr Ser Ser Ser Ser Ser 6500 6505 6510 Leu Leu Ser Leu Pro Ala Ile Thr Ser Pro Ser Pro Val Pro Thr 6515 6520 6525 Thr Leu Pro Glu Ser Arg Pro Ser Ser Pro Val His Leu Thr Ser 6530 6535 6540 Leu Pro Thr Ser Gly Leu Val Lys Thr Thr Asp Met Leu Ala Ser 6545 6550 6555 Val Ala Ser Leu Pro Pro Asn Leu Gly Ser Thr Ser His Lys Ile 6560 6565 6570 Pro Thr Thr Ser Glu Asp Ile Lys Asp Thr Glu Lys Met Tyr Pro 6575 6580 6585 Ser Thr Asn Ile Ala Val Thr Asn Val Gly Thr Thr Thr Ser Glu 6590 6595 6600 Lys Glu Ser Tyr Ser Ser Val Pro Ala Tyr Ser Glu Pro Pro Lys 6605 6610 6615 Val Thr Ser Pro Met Val Thr Ser Phe Asn Ile Arg Asp Thr Ile 6620 6625 6630 Val Ser Thr Ser Met Pro Gly Ser Ser Glu Ile Thr Arg Ile Glu 6635 6640 6645 Met Glu Ser Thr Phe Ser Leu Ala His Gly Leu Lys Gly Thr Ser 6650 6655 6660 Thr Ser Gln Asp Pro Ile Val Ser Thr Glu Lys Ser Ala Val Leu 6665 6670 6675 His Lys Leu Thr Thr Gly Ala Thr Glu Thr Ser Arg Thr Glu Val 6680 6685 6690 Ala Ser Ser Arg Arg Thr Ser Ile Pro Gly Pro Asp His Ser Thr 6695 6700 6705 Glu Ser Pro Asp Ile Ser Thr Glu Val Ile Pro Ser Leu Pro Ile 6710 6715 6720 Ser Leu Gly Ile Thr Glu Ser Ser Asn Met Thr Ile Ile Thr Arg 6725 6730 6735 Thr Gly Pro Pro Leu Gly Ser Thr Ser Gln Gly Thr Phe Thr Leu 6740 6745 6750 Asp Thr Pro Thr Thr Ser Ser Arg Ala Gly Thr His Ser Met Ala 6755 6760 6765 Thr Gln Glu Phe Pro His Ser Glu Met Thr Thr Val Met Asn Lys 6770 6775 6780 Asp Pro Glu Ile Leu Ser Trp Thr Ile Pro Pro Ser Ile Glu Lys 6785 6790 6795 Thr Ser Phe Ser Ser Ser Leu Met Pro Ser Pro Ala Met Thr Ser 6800 6805 6810 Pro Pro Val Ser Ser Thr Leu Pro Lys Thr Ile His Thr Thr Pro 6815 6820 6825 Ser Pro Met Thr Ser Leu Leu Thr Pro Ser Leu Val Met Thr Thr 6830 6835 6840 Asp Thr Leu Gly Thr Ser Pro Glu Pro Thr Thr Ser Ser Pro Pro 6845 6850 6855 Asn Leu Ser Ser Thr Ser His Glu Ile Leu Thr Thr Asp Glu Asp 6860 6865 6870 Thr Thr Ala Ile Glu Ala Met His Pro Ser Thr Ser Thr Ala Ala 6875 6880 6885 Thr Asn Val Glu Thr Thr Ser Ser Gly His Gly Ser Gln Ser Ser 6890 6895 6900 Val Leu Ala Asp Ser Glu Lys Thr Lys Ala Thr Ala Pro Met Asp 6905 6910 6915 Thr Thr Ser Thr Met Gly His Thr Thr Val Ser Thr Ser Met Ser 6920 6925 6930 Val Ser Ser Glu Thr Thr Lys Ile Lys Arg Glu Ser Thr Tyr Ser 6935 6940 6945 Leu Thr Pro Gly Leu Arg Glu Thr Ser Ile Ser Gln Asn Ala Ser 6950 6955 6960 Phe Ser Thr Asp Thr Ser Ile Val Leu Ser Glu Val Pro Thr Gly 6965 6970 6975 Thr Thr Ala Glu Val Ser Arg Thr Glu Val Thr Ser Ser Gly Arg 6980 6985 6990 Thr Ser Ile Pro Gly Pro Ser Gln Ser Thr Val Leu Pro Glu Ile 6995 7000 7005 Ser Thr Arg Thr Met Thr Arg Leu Phe Ala Ser Pro Thr Met Thr 7010 7015 7020 Glu Ser Ala Glu Met Thr Ile Pro Thr Gln Thr Gly Pro Ser Gly 7025 7030 7035 Ser Thr Ser Gln Asp Thr Leu Thr Leu Asp Thr Ser Thr Thr Lys 7040 7045 7050 Ser Gln Ala Lys Thr His Ser Thr Leu Thr Gln Arg Phe Pro His 7055 7060 7065 Ser Glu Met Thr Thr Leu Met Ser Arg Gly Pro Gly Asp Met Ser 7070 7075 7080 Trp Gln Ser Ser Pro Ser Leu Glu Asn Pro Ser Ser Leu Pro Ser 7085 7090 7095 Leu Leu Ser Leu Pro Ala Thr Thr Ser Pro Pro Pro Ile Ser Ser 7100 7105 7110 Thr Leu Pro Val Thr Ile Ser Ser Ser Pro Leu Pro Val Thr Ser 7115 7120 7125 Leu Leu Thr Ser Ser Pro Val Thr Thr Thr Asp Met Leu His Thr 7130 7135 7140 Ser Pro Glu Leu Val Thr Ser Ser Pro Pro Lys Leu Ser His Thr 7145 7150 7155 Ser Asp Glu Arg Leu Thr Thr Gly Lys Asp Thr Thr Asn Thr Glu 7160 7165 7170 Ala Val His Pro Ser Thr Asn Thr Ala Ala Ser Asn Val Glu Ile 7175 7180 7185 Pro Ser Ser Gly His Glu Ser Pro Ser Ser Ala Leu Ala Asp Ser 7190 7195 7200 Glu Thr Ser Lys Ala Thr Ser Pro Met Phe Ile Thr Ser Thr Gln 7205 7210 7215 Glu Asp Thr Thr Val Ala Ile Ser Thr Pro His Phe Leu Glu Thr 7220 7225 7230 Ser Arg Ile Gln Lys Glu Ser Ile Ser Ser Leu Ser Pro Lys Leu 7235 7240 7245 Arg Glu Thr Gly Ser Ser Val Glu Thr Ser Ser Ala Ile Glu Thr 7250 7255 7260 Ser Ala Val Leu Ser Glu Val Ser Ile Gly Ala Thr Thr Glu Ile 7265 7270 7275 Ser Arg Thr Glu Val Thr Ser Ser Ser Arg Thr Ser Ile Ser Gly 7280 7285 7290 Ser Ala Glu Ser Thr Met Leu Pro Glu Ile Ser Thr Thr Arg Lys 7295 7300 7305 Ile Ile Lys Phe Pro Thr Ser Pro Ile Leu Ala Glu Ser Ser Glu 7310 7315 7320 Met Thr Ile Lys Thr Gln Thr Ser Pro Pro Gly Ser Thr Ser Glu 7325 7330 7335 Ser Thr Phe Thr Leu Asp Thr Ser Thr Thr Pro Ser Leu Val Ile 7340 7345 7350 Thr His Ser Thr Met Thr Gln Arg Leu Pro His Ser Glu Ile Thr 7355 7360 7365 Thr Leu Val Ser Arg Gly Ala Gly Asp Val Pro Arg Pro Ser Ser 7370 7375 7380 Leu Pro Val Glu Glu Thr Ser Pro Pro Ser Ser Gln Leu Ser Leu 7385 7390 7395 Ser Ala Met Ile Ser Pro Ser Pro Val Ser Ser Thr Leu Pro Ala 7400 7405 7410 Ser Ser His Ser Ser Ser Ala Ser Val Thr Ser Leu Leu Thr Pro 7415 7420 7425 Gly Gln Val Lys Thr Thr Glu Val Leu Asp Ala Ser Ala Glu Pro 7430 7435 7440 Glu Thr Ser Ser Pro Pro Ser Leu Ser Ser Thr Ser Val Glu Ile 7445 7450 7455 Leu Ala Thr Ser Glu Val Thr Thr Asp Thr Glu Lys Ile His Pro 7460 7465 7470 Phe Ser Asn Thr Ala Val Thr Lys Val Gly Thr Ser Ser Ser Gly 7475 7480 7485 His Glu Ser Pro Ser Ser Val Leu Pro Asp Ser Glu Thr Thr Lys 7490 7495 7500 Ala Thr Ser Ala Met Gly Thr Ile Ser Ile Met Gly Asp Thr Ser 7505 7510 7515 Val Ser Thr Leu Thr Pro Ala Leu Ser Asn Thr Arg Lys Ile Gln 7520 7525 7530 Ser Glu Pro Ala Ser Ser Leu Thr Thr Arg Leu Arg Glu Thr Ser 7535 7540 7545 Thr Ser Glu Glu Thr Ser Leu Ala Thr Glu Ala Asn Thr Val Leu 7550 7555 7560 Ser Lys Val Ser Thr Gly Ala Thr Thr Glu Val Ser Arg Thr Glu 7565 7570 7575 Ala Ile Ser Phe Ser Arg Thr Ser Met Ser Gly Pro Glu Gln Ser 7580

7585 7590 Thr Met Ser Gln Asp Ile Ser Ile Gly Thr Ile Pro Arg Ile Ser 7595 7600 7605 Ala Ser Ser Val Leu Thr Glu Ser Ala Lys Met Thr Ile Thr Thr 7610 7615 7620 Gln Thr Gly Pro Ser Glu Ser Thr Leu Glu Ser Thr Leu Asn Leu 7625 7630 7635 Asn Thr Ala Thr Thr Pro Ser Trp Val Glu Thr His Ser Ile Val 7640 7645 7650 Ile Gln Gly Phe Pro His Pro Glu Met Thr Thr Ser Met Gly Arg 7655 7660 7665 Gly Pro Gly Gly Val Ser Trp Pro Ser Pro Pro Phe Val Lys Glu 7670 7675 7680 Thr Ser Pro Pro Ser Ser Pro Leu Ser Leu Pro Ala Val Thr Ser 7685 7690 7695 Pro His Pro Val Ser Thr Thr Phe Leu Ala His Ile Pro Pro Ser 7700 7705 7710 Pro Leu Pro Val Thr Ser Leu Leu Thr Ser Gly Pro Ala Thr Thr 7715 7720 7725 Thr Asp Ile Leu Gly Thr Ser Thr Glu Pro Gly Thr Ser Ser Ser 7730 7735 7740 Ser Ser Leu Ser Thr Thr Ser His Glu Arg Leu Thr Thr Tyr Lys 7745 7750 7755 Asp Thr Ala His Thr Glu Ala Val His Pro Ser Thr Asn Thr Gly 7760 7765 7770 Gly Thr Asn Val Ala Thr Thr Ser Ser Gly Tyr Lys Ser Gln Ser 7775 7780 7785 Ser Val Leu Ala Asp Ser Ser Pro Met Cys Thr Thr Ser Thr Met 7790 7795 7800 Gly Asp Thr Ser Val Leu Thr Ser Thr Pro Ala Phe Leu Glu Thr 7805 7810 7815 Arg Arg Ile Gln Thr Glu Leu Ala Ser Ser Leu Thr Pro Gly Leu 7820 7825 7830 Arg Glu Ser Ser Gly Ser Glu Gly Thr Ser Ser Gly Thr Lys Met 7835 7840 7845 Ser Thr Val Leu Ser Lys Val Pro Thr Gly Ala Thr Thr Glu Ile 7850 7855 7860 Ser Lys Glu Asp Val Thr Ser Ile Pro Gly Pro Ala Gln Ser Thr 7865 7870 7875 Ile Ser Pro Asp Ile Ser Thr Arg Thr Val Ser Trp Phe Ser Thr 7880 7885 7890 Ser Pro Val Met Thr Glu Ser Ala Glu Ile Thr Met Asn Thr His 7895 7900 7905 Thr Ser Pro Leu Gly Ala Thr Thr Gln Gly Thr Ser Thr Leu Asp 7910 7915 7920 Thr Ser Ser Thr Thr Ser Leu Thr Met Thr His Ser Thr Ile Ser 7925 7930 7935 Gln Gly Phe Ser His Ser Gln Met Ser Thr Leu Met Arg Arg Gly 7940 7945 7950 Pro Glu Asp Val Ser Trp Met Ser Pro Pro Leu Leu Glu Lys Thr 7955 7960 7965 Arg Pro Ser Phe Ser Leu Met Ser Ser Pro Ala Thr Thr Ser Pro 7970 7975 7980 Ser Pro Val Ser Ser Thr Leu Pro Glu Ser Ile Ser Ser Ser Pro 7985 7990 7995 Leu Pro Val Thr Ser Leu Leu Thr Ser Gly Leu Ala Lys Thr Thr 8000 8005 8010 Asp Met Leu His Lys Ser Ser Glu Pro Val Thr Asn Ser Pro Ala 8015 8020 8025 Asn Leu Ser Ser Thr Ser Val Glu Ile Leu Ala Thr Ser Glu Val 8030 8035 8040 Thr Thr Asp Thr Glu Lys Thr His Pro Ser Ser Asn Arg Thr Val 8045 8050 8055 Thr Asp Val Gly Thr Ser Ser Ser Gly His Glu Ser Thr Ser Phe 8060 8065 8070 Val Leu Ala Asp Ser Gln Thr Ser Lys Val Thr Ser Pro Met Val 8075 8080 8085 Ile Thr Ser Thr Met Glu Asp Thr Ser Val Ser Thr Ser Thr Pro 8090 8095 8100 Gly Phe Phe Glu Thr Ser Arg Ile Gln Thr Glu Pro Thr Ser Ser 8105 8110 8115 Leu Thr Leu Gly Leu Arg Lys Thr Ser Ser Ser Glu Gly Thr Ser 8120 8125 8130 Leu Ala Thr Glu Met Ser Thr Val Leu Ser Gly Val Pro Thr Gly 8135 8140 8145 Ala Thr Ala Glu Val Ser Arg Thr Glu Val Thr Ser Ser Ser Arg 8150 8155 8160 Thr Ser Ile Ser Gly Phe Ala Gln Leu Thr Val Ser Pro Glu Thr 8165 8170 8175 Ser Thr Glu Thr Ile Thr Arg Leu Pro Thr Ser Ser Ile Met Thr 8180 8185 8190 Glu Ser Ala Glu Met Met Ile Lys Thr Gln Thr Asp Pro Pro Gly 8195 8200 8205 Ser Thr Pro Glu Ser Thr His Thr Val Asp Ile Ser Thr Thr Pro 8210 8215 8220 Asn Trp Val Glu Thr His Ser Thr Val Thr Gln Arg Phe Ser His 8225 8230 8235 Ser Glu Met Thr Thr Leu Val Ser Arg Ser Pro Gly Asp Met Leu 8240 8245 8250 Trp Pro Ser Gln Ser Ser Val Glu Glu Thr Ser Ser Ala Ser Ser 8255 8260 8265 Leu Leu Ser Leu Pro Ala Thr Thr Ser Pro Ser Pro Val Ser Ser 8270 8275 8280 Thr Leu Val Glu Asp Phe Pro Ser Ala Ser Leu Pro Val Thr Ser 8285 8290 8295 Leu Leu Asn Pro Gly Leu Val Ile Thr Thr Asp Arg Met Gly Ile 8300 8305 8310 Ser Arg Glu Pro Gly Thr Ser Ser Thr Ser Asn Leu Ser Ser Thr 8315 8320 8325 Ser His Glu Arg Leu Thr Thr Leu Glu Asp Thr Val Asp Thr Glu 8330 8335 8340 Asp Met Gln Pro Ser Thr His Thr Ala Val Thr Asn Val Arg Thr 8345 8350 8355 Ser Ile Ser Gly His Glu Ser Gln Ser Ser Val Leu Ser Asp Ser 8360 8365 8370 Glu Thr Pro Lys Ala Thr Ser Pro Met Gly Thr Thr Tyr Thr Met 8375 8380 8385 Gly Glu Thr Ser Val Ser Ile Ser Thr Ser Asp Phe Phe Glu Thr 8390 8395 8400 Ser Arg Ile Gln Ile Glu Pro Thr Ser Ser Leu Thr Ser Gly Leu 8405 8410 8415 Arg Glu Thr Ser Ser Ser Glu Arg Ile Ser Ser Ala Thr Glu Gly 8420 8425 8430 Ser Thr Val Leu Ser Glu Val Pro Ser Gly Ala Thr Thr Glu Val 8435 8440 8445 Ser Arg Thr Glu Val Ile Ser Ser Arg Gly Thr Ser Met Ser Gly 8450 8455 8460 Pro Asp Gln Phe Thr Ile Ser Pro Asp Ile Ser Thr Glu Ala Ile 8465 8470 8475 Thr Arg Leu Ser Thr Ser Pro Ile Met Thr Glu Ser Ala Glu Ser 8480 8485 8490 Ala Ile Thr Ile Glu Thr Gly Ser Pro Gly Ala Thr Ser Glu Gly 8495 8500 8505 Thr Leu Thr Leu Asp Thr Ser Thr Thr Thr Phe Trp Ser Gly Thr 8510 8515 8520 His Ser Thr Ala Ser Pro Gly Phe Ser His Ser Glu Met Thr Thr 8525 8530 8535 Leu Met Ser Arg Thr Pro Gly Asp Val Pro Trp Pro Ser Leu Pro 8540 8545 8550 Ser Val Glu Glu Ala Ser Ser Val Ser Ser Ser Leu Ser Ser Pro 8555 8560 8565 Ala Met Thr Ser Thr Ser Phe Phe Ser Thr Leu Pro Glu Ser Ile 8570 8575 8580 Ser Ser Ser Pro His Pro Val Thr Ala Leu Leu Thr Leu Gly Pro 8585 8590 8595 Val Lys Thr Thr Asp Met Leu Arg Thr Ser Ser Glu Pro Glu Thr 8600 8605 8610 Ser Ser Pro Pro Asn Leu Ser Ser Thr Ser Ala Glu Ile Leu Ala 8615 8620 8625 Thr Ser Glu Val Thr Lys Asp Arg Glu Lys Ile His Pro Ser Ser 8630 8635 8640 Asn Thr Pro Val Val Asn Val Gly Thr Val Ile Tyr Lys His Leu 8645 8650 8655 Ser Pro Ser Ser Val Leu Ala Asp Leu Val Thr Thr Lys Pro Thr 8660 8665 8670 Ser Pro Met Ala Thr Thr Ser Thr Leu Gly Asn Thr Ser Val Ser 8675 8680 8685 Thr Ser Thr Pro Ala Phe Pro Glu Thr Met Met Thr Gln Pro Thr 8690 8695 8700 Ser Ser Leu Thr Ser Gly Leu Arg Glu Ile Ser Thr Ser Gln Glu 8705 8710 8715 Thr Ser Ser Ala Thr Glu Arg Ser Ala Ser Leu Ser Gly Met Pro 8720 8725 8730 Thr Gly Ala Thr Thr Lys Val Ser Arg Thr Glu Ala Leu Ser Leu 8735 8740 8745 Gly Arg Thr Ser Thr Pro Gly Pro Ala Gln Ser Thr Ile Ser Pro 8750 8755 8760 Glu Ile Ser Thr Glu Thr Ile Thr Arg Ile Ser Thr Pro Leu Thr 8765 8770 8775 Thr Thr Gly Ser Ala Glu Met Thr Ile Thr Pro Lys Thr Gly His 8780 8785 8790 Ser Gly Ala Ser Ser Gln Gly Thr Phe Thr Leu Asp Thr Ser Ser 8795 8800 8805 Arg Ala Ser Trp Pro Gly Thr His Ser Ala Ala Thr His Arg Ser 8810 8815 8820 Pro His Ser Gly Met Thr Thr Pro Met Ser Arg Gly Pro Glu Asp 8825 8830 8835 Val Ser Trp Pro Ser Arg Pro Ser Val Glu Lys Thr Ser Pro Pro 8840 8845 8850 Ser Ser Leu Val Ser Leu Ser Ala Val Thr Ser Pro Ser Pro Leu 8855 8860 8865 Tyr Ser Thr Pro Ser Glu Ser Ser His Ser Ser Pro Leu Arg Val 8870 8875 8880 Thr Ser Leu Phe Thr Pro Val Met Met Lys Thr Thr Asp Met Leu 8885 8890 8895 Asp Thr Ser Leu Glu Pro Val Thr Thr Ser Pro Pro Ser Met Asn 8900 8905 8910 Ile Thr Ser Asp Glu Ser Leu Ala Thr Ser Lys Ala Thr Met Glu 8915 8920 8925 Thr Glu Ala Ile Gln Leu Ser Glu Asn Thr Ala Val Thr Gln Met 8930 8935 8940 Gly Thr Ile Ser Ala Arg Gln Glu Phe Tyr Ser Ser Tyr Pro Gly 8945 8950 8955 Leu Pro Glu Pro Ser Lys Val Thr Ser Pro Val Val Thr Ser Ser 8960 8965 8970 Thr Ile Lys Asp Ile Val Ser Thr Thr Ile Pro Ala Ser Ser Glu 8975 8980 8985 Ile Thr Arg Ile Glu Met Glu Ser Thr Ser Thr Leu Thr Pro Thr 8990 8995 9000 Pro Arg Glu Thr Ser Thr Ser Gln Glu Ile His Ser Ala Thr Lys 9005 9010 9015 Pro Ser Thr Val Pro Tyr Lys Ala Leu Thr Ser Ala Thr Ile Glu 9020 9025 9030 Asp Ser Met Thr Gln Val Met Ser Ser Ser Arg Gly Pro Ser Pro 9035 9040 9045 Asp Gln Ser Thr Met Ser Gln Asp Ile Ser Thr Glu Val Ile Thr 9050 9055 9060 Arg Leu Ser Thr Ser Pro Ile Lys Thr Glu Ser Thr Glu Met Thr 9065 9070 9075 Ile Thr Thr Gln Thr Gly Ser Pro Gly Ala Thr Ser Arg Gly Thr 9080 9085 9090 Leu Thr Leu Asp Thr Ser Thr Thr Phe Met Ser Gly Thr His Ser 9095 9100 9105 Thr Ala Ser Gln Gly Phe Ser His Ser Gln Met Thr Ala Leu Met 9110 9115 9120 Ser Arg Thr Pro Gly Asp Val Pro Trp Leu Ser His Pro Ser Val 9125 9130 9135 Glu Glu Ala Ser Ser Ala Ser Phe Ser Leu Ser Ser Pro Val Met 9140 9145 9150 Thr Ser Ser Ser Pro Val Ser Ser Thr Leu Pro Asp Ser Ile His 9155 9160 9165 Ser Ser Ser Leu Pro Val Thr Ser Leu Leu Thr Ser Gly Leu Val 9170 9175 9180 Lys Thr Thr Glu Leu Leu Gly Thr Ser Ser Glu Pro Glu Thr Ser 9185 9190 9195 Ser Pro Pro Asn Leu Ser Ser Thr Ser Ala Glu Ile Leu Ala Ile 9200 9205 9210 Thr Glu Val Thr Thr Asp Thr Glu Lys Leu Glu Met Thr Asn Val 9215 9220 9225 Val Thr Ser Gly Tyr Thr His Glu Ser Pro Ser Ser Val Leu Ala 9230 9235 9240 Asp Ser Val Thr Thr Lys Ala Thr Ser Ser Met Gly Ile Thr Tyr 9245 9250 9255 Pro Thr Gly Asp Thr Asn Val Leu Thr Ser Thr Pro Ala Phe Ser 9260 9265 9270 Asp Thr Ser Arg Ile Gln Thr Lys Ser Lys Leu Ser Leu Thr Pro 9275 9280 9285 Gly Leu Met Glu Thr Ser Ile Ser Glu Glu Thr Ser Ser Ala Thr 9290 9295 9300 Glu Lys Ser Thr Val Leu Ser Ser Val Pro Thr Gly Ala Thr Thr 9305 9310 9315 Glu Val Ser Arg Thr Glu Ala Ile Ser Ser Ser Arg Thr Ser Ile 9320 9325 9330 Pro Gly Pro Ala Gln Ser Thr Met Ser Ser Asp Thr Ser Met Glu 9335 9340 9345 Thr Ile Thr Arg Ile Ser Thr Pro Leu Thr Arg Lys Glu Ser Thr 9350 9355 9360 Asp Met Ala Ile Thr Pro Lys Thr Gly Pro Ser Gly Ala Thr Ser 9365 9370 9375 Gln Gly Thr Phe Thr Leu Asp Ser Ser Ser Thr Ala Ser Trp Pro 9380 9385 9390 Gly Thr His Ser Ala Thr Thr Gln Arg Phe Pro Gln Ser Val Val 9395 9400 9405 Thr Thr Pro Met Ser Arg Gly Pro Glu Asp Val Ser Trp Pro Ser 9410 9415 9420 Pro Leu Ser Val Glu Lys Asn Ser Pro Pro Ser Ser Leu Val Ser 9425 9430 9435 Ser Ser Ser Val Thr Ser Pro Ser Pro Leu Tyr Ser Thr Pro Ser 9440 9445 9450 Gly Ser Ser His Ser Ser Pro Val Pro Val Thr Ser Leu Phe Thr 9455 9460 9465 Ser Ile Met Met Lys Ala Thr Asp Met Leu Asp Ala Ser Leu Glu 9470 9475 9480 Pro Glu Thr Thr Ser Ala Pro Asn Met Asn Ile Thr Ser Asp Glu 9485 9490 9495 Ser Leu Ala Ala Ser Lys Ala Thr Thr Glu Thr Glu Ala Ile His 9500 9505 9510 Val Phe Glu Asn Thr Ala Ala Ser His Val Glu Thr Thr Ser Ala 9515 9520 9525 Thr Glu Glu Leu Tyr Ser Ser Ser Pro Gly Phe Ser Glu Pro Thr 9530 9535 9540 Lys Val Ile Ser Pro Val Val Thr Ser Ser Ser Ile Arg Asp Asn 9545 9550 9555 Met Val Ser Thr Thr Met Pro Gly Ser Ser Gly Ile Thr Arg Ile 9560 9565 9570 Glu Ile Glu Ser Met Ser Ser Leu Thr Pro Gly Leu Arg Glu Thr 9575 9580 9585 Arg Thr Ser Gln Asp Ile Thr Ser Ser Thr Glu Thr Ser Thr Val 9590 9595 9600 Leu Tyr Lys Met Pro Ser Gly Ala Thr Pro Glu Val Ser Arg Thr 9605 9610 9615 Glu Val Met Pro Ser Ser Arg Thr Ser Ile Pro Gly Pro Ala Gln 9620 9625 9630 Ser Thr Met Ser Leu Asp Ile Ser Asp Glu Val Val Thr Arg Leu 9635 9640 9645 Ser Thr Ser Pro Ile Met Thr Glu Ser Ala Glu Ile Thr Ile Thr 9650 9655 9660 Thr Gln Thr Gly Tyr Ser Leu Ala Thr Ser Gln Val Thr Leu Pro 9665 9670 9675 Leu Gly Thr Ser Met Thr Phe Leu Ser Gly Thr His Ser Thr Met 9680 9685 9690 Ser Gln Gly Leu Ser His Ser Glu Met Thr Asn Leu Met Ser Arg 9695 9700 9705 Gly Pro Glu Ser Leu Ser Trp Thr Ser Pro Arg Phe Val Glu Thr 9710 9715 9720 Thr Arg Ser Ser Ser Ser Leu Thr Ser Leu Pro Leu Thr Thr Ser 9725 9730 9735 Leu Ser Pro Val Ser Ser Thr Leu Leu Asp Ser Ser Pro Ser Ser 9740 9745 9750 Pro Leu Pro Val Thr Ser Leu Ile Leu Pro Gly Leu Val Lys Thr 9755 9760 9765 Thr Glu Val Leu Asp Thr Ser Ser Glu Pro Lys Thr Ser Ser Ser 9770 9775 9780 Pro Asn

Leu Ser Ser Thr Ser Val Glu Ile Pro Ala Thr Ser Glu 9785 9790 9795 Ile Met Thr Asp Thr Glu Lys Ile His Pro Ser Ser Asn Thr Ala 9800 9805 9810 Val Ala Lys Val Arg Thr Ser Ser Ser Val His Glu Ser His Ser 9815 9820 9825 Ser Val Leu Ala Asp Ser Glu Thr Thr Ile Thr Ile Pro Ser Met 9830 9835 9840 Gly Ile Thr Ser Ala Val Asp Asp Thr Thr Val Phe Thr Ser Asn 9845 9850 9855 Pro Ala Phe Ser Glu Thr Arg Arg Ile Pro Thr Glu Pro Thr Phe 9860 9865 9870 Ser Leu Thr Pro Gly Phe Arg Glu Thr Ser Thr Ser Glu Glu Thr 9875 9880 9885 Thr Ser Ile Thr Glu Thr Ser Ala Val Leu Tyr Gly Val Pro Thr 9890 9895 9900 Ser Ala Thr Thr Glu Val Ser Met Thr Glu Ile Met Ser Ser Asn 9905 9910 9915 Arg Ile His Ile Pro Asp Ser Asp Gln Ser Thr Met Ser Pro Asp 9920 9925 9930 Ile Ile Thr Glu Val Ile Thr Arg Leu Ser Ser Ser Ser Met Met 9935 9940 9945 Ser Glu Ser Thr Gln Met Thr Ile Thr Thr Gln Lys Ser Ser Pro 9950 9955 9960 Gly Ala Thr Ala Gln Ser Thr Leu Thr Leu Ala Thr Thr Thr Ala 9965 9970 9975 Pro Leu Ala Arg Thr His Ser Thr Val Pro Pro Arg Phe Leu His 9980 9985 9990 Ser Glu Met Thr Thr Leu Met Ser Arg Ser Pro Glu Asn Pro Ser 9995 10000 10005 Trp Lys Ser Ser Leu Phe Val Glu Lys Thr Ser Ser Ser Ser Ser 10010 10015 10020 Leu Leu Ser Leu Pro Val Thr Thr Ser Pro Ser Val Ser Ser Thr 10025 10030 10035 Leu Pro Gln Ser Ile Pro Ser Ser Ser Phe Ser Val Thr Ser Leu 10040 10045 10050 Leu Thr Pro Gly Met Val Lys Thr Thr Asp Thr Ser Thr Glu Pro 10055 10060 10065 Gly Thr Ser Leu Ser Pro Asn Leu Ser Gly Thr Ser Val Glu Ile 10070 10075 10080 Leu Ala Ala Ser Glu Val Thr Thr Asp Thr Glu Lys Ile His Pro 10085 10090 10095 Ser Ser Ser Met Ala Val Thr Asn Val Gly Thr Thr Ser Ser Gly 10100 10105 10110 His Glu Leu Tyr Ser Ser Val Ser Ile His Ser Glu Pro Ser Lys 10115 10120 10125 Ala Thr Tyr Pro Val Gly Thr Pro Ser Ser Met Ala Glu Thr Ser 10130 10135 10140 Ile Ser Thr Ser Met Pro Ala Asn Phe Glu Thr Thr Gly Phe Glu 10145 10150 10155 Ala Glu Pro Phe Ser His Leu Thr Ser Gly Phe Arg Lys Thr Asn 10160 10165 10170 Met Ser Leu Asp Thr Ser Ser Val Thr Pro Thr Asn Thr Pro Ser 10175 10180 10185 Ser Pro Gly Ser Thr His Leu Leu Gln Ser Ser Lys Thr Asp Phe 10190 10195 10200 Thr Ser Ser Ala Lys Thr Ser Ser Pro Asp Trp Pro Pro Ala Ser 10205 10210 10215 Gln Tyr Thr Glu Ile Pro Val Asp Ile Ile Thr Pro Phe Asn Ala 10220 10225 10230 Ser Pro Ser Ile Thr Glu Ser Thr Gly Ile Thr Ser Phe Pro Glu 10235 10240 10245 Ser Arg Phe Thr Met Ser Val Thr Glu Ser Thr His His Leu Ser 10250 10255 10260 Thr Asp Leu Leu Pro Ser Ala Glu Thr Ile Ser Thr Gly Thr Val 10265 10270 10275 Met Pro Ser Leu Ser Glu Ala Met Thr Ser Phe Ala Thr Thr Gly 10280 10285 10290 Val Pro Arg Ala Ile Ser Gly Ser Gly Ser Pro Phe Ser Arg Thr 10295 10300 10305 Glu Ser Gly Pro Gly Asp Ala Thr Leu Ser Thr Ile Ala Glu Ser 10310 10315 10320 Leu Pro Ser Ser Thr Pro Val Pro Phe Ser Ser Ser Thr Phe Thr 10325 10330 10335 Thr Thr Asp Ser Ser Thr Ile Pro Ala Leu His Glu Ile Thr Ser 10340 10345 10350 Ser Ser Ala Thr Pro Tyr Arg Val Asp Thr Ser Leu Gly Thr Glu 10355 10360 10365 Ser Ser Thr Thr Glu Gly Arg Leu Val Met Val Ser Thr Leu Asp 10370 10375 10380 Thr Ser Ser Gln Pro Gly Arg Thr Ser Ser Ser Pro Ile Leu Asp 10385 10390 10395 Thr Arg Met Thr Glu Ser Val Glu Leu Gly Thr Val Thr Ser Ala 10400 10405 10410 Tyr Gln Val Pro Ser Leu Ser Thr Arg Leu Thr Arg Thr Asp Gly 10415 10420 10425 Ile Met Glu His Ile Thr Lys Ile Pro Asn Glu Ala Ala His Arg 10430 10435 10440 Gly Thr Ile Arg Pro Val Lys Gly Pro Gln Thr Ser Thr Ser Pro 10445 10450 10455 Ala Ser Pro Lys Gly Leu His Thr Gly Gly Thr Lys Arg Met Glu 10460 10465 10470 Thr Thr Thr Thr Ala Leu Lys Thr Thr Thr Thr Ala Leu Lys Thr 10475 10480 10485 Thr Ser Arg Ala Thr Leu Thr Thr Ser Val Tyr Thr Pro Thr Leu 10490 10495 10500 Gly Thr Leu Thr Pro Leu Asn Ala Ser Met Gln Met Ala Ser Thr 10505 10510 10515 Ile Pro Thr Glu Met Met Ile Thr Thr Pro Tyr Val Phe Pro Asp 10520 10525 10530 Val Pro Glu Thr Thr Ser Ser Leu Ala Thr Ser Leu Gly Ala Glu 10535 10540 10545 Thr Ser Thr Ala Leu Pro Arg Thr Thr Pro Ser Val Phe Asn Arg 10550 10555 10560 Glu Ser Glu Thr Thr Ala Ser Leu Val Ser Arg Ser Gly Ala Glu 10565 10570 10575 Arg Ser Pro Val Ile Gln Thr Leu Asp Val Ser Ser Ser Glu Pro 10580 10585 10590 Asp Thr Thr Ala Ser Trp Val Ile His Pro Ala Glu Thr Ile Pro 10595 10600 10605 Thr Val Ser Lys Thr Thr Pro Asn Phe Phe His Ser Glu Leu Asp 10610 10615 10620 Thr Val Ser Ser Thr Ala Thr Ser His Gly Ala Asp Val Ser Ser 10625 10630 10635 Ala Ile Pro Thr Asn Ile Ser Pro Ser Glu Leu Asp Ala Leu Thr 10640 10645 10650 Pro Leu Val Thr Ile Ser Gly Thr Asp Thr Ser Thr Thr Phe Pro 10655 10660 10665 Thr Leu Thr Lys Ser Pro His Glu Thr Glu Thr Arg Thr Thr Trp 10670 10675 10680 Leu Thr His Pro Ala Glu Thr Ser Ser Thr Ile Pro Arg Thr Ile 10685 10690 10695 Pro Asn Phe Ser His His Glu Ser Asp Ala Thr Pro Ser Ile Ala 10700 10705 10710 Thr Ser Pro Gly Ala Glu Thr Ser Ser Ala Ile Pro Ile Met Thr 10715 10720 10725 Val Ser Pro Gly Ala Glu Asp Leu Val Thr Ser Gln Val Thr Ser 10730 10735 10740 Ser Gly Thr Asp Arg Asn Met Thr Ile Pro Thr Leu Thr Leu Ser 10745 10750 10755 Pro Gly Glu Pro Lys Thr Ile Ala Ser Leu Val Thr His Pro Glu 10760 10765 10770 Ala Gln Thr Ser Ser Ala Ile Pro Thr Ser Thr Ile Ser Pro Ala 10775 10780 10785 Val Ser Arg Leu Val Thr Ser Met Val Thr Ser Leu Ala Ala Lys 10790 10795 10800 Thr Ser Thr Thr Asn Arg Ala Leu Thr Asn Ser Pro Gly Glu Pro 10805 10810 10815 Ala Thr Thr Val Ser Leu Val Thr His Pro Ala Gln Thr Ser Pro 10820 10825 10830 Thr Val Pro Trp Thr Thr Ser Ile Phe Phe His Ser Lys Ser Asp 10835 10840 10845 Thr Thr Pro Ser Met Thr Thr Ser His Gly Ala Glu Ser Ser Ser 10850 10855 10860 Ala Val Pro Thr Pro Thr Val Ser Thr Glu Val Pro Gly Val Val 10865 10870 10875 Thr Pro Leu Val Thr Ser Ser Arg Ala Val Ile Ser Thr Thr Ile 10880 10885 10890 Pro Ile Leu Thr Leu Ser Pro Gly Glu Pro Glu Thr Thr Pro Ser 10895 10900 10905 Met Ala Thr Ser His Gly Glu Glu Ala Ser Ser Ala Ile Pro Thr 10910 10915 10920 Pro Thr Val Ser Pro Gly Val Pro Gly Val Val Thr Ser Leu Val 10925 10930 10935 Thr Ser Ser Arg Ala Val Thr Ser Thr Thr Ile Pro Ile Leu Thr 10940 10945 10950 Phe Ser Leu Gly Glu Pro Glu Thr Thr Pro Ser Met Ala Thr Ser 10955 10960 10965 His Gly Thr Glu Ala Gly Ser Ala Val Pro Thr Val Leu Pro Glu 10970 10975 10980 Val Pro Gly Met Val Thr Ser Leu Val Ala Ser Ser Arg Ala Val 10985 10990 10995 Thr Ser Thr Thr Leu Pro Thr Leu Thr Leu Ser Pro Gly Glu Pro 11000 11005 11010 Glu Thr Thr Pro Ser Met Ala Thr Ser His Gly Ala Glu Ala Ser 11015 11020 11025 Ser Thr Val Pro Thr Val Ser Pro Glu Val Pro Gly Val Val Thr 11030 11035 11040 Ser Leu Val Thr Ser Ser Ser Gly Val Asn Ser Thr Ser Ile Pro 11045 11050 11055 Thr Leu Ile Leu Ser Pro Gly Glu Leu Glu Thr Thr Pro Ser Met 11060 11065 11070 Ala Thr Ser His Gly Ala Glu Ala Ser Ser Ala Val Pro Thr Pro 11075 11080 11085 Thr Val Ser Pro Gly Val Ser Gly Val Val Thr Pro Leu Val Thr 11090 11095 11100 Ser Ser Arg Ala Val Thr Ser Thr Thr Ile Pro Ile Leu Thr Leu 11105 11110 11115 Ser Ser Ser Glu Pro Glu Thr Thr Pro Ser Met Ala Thr Ser His 11120 11125 11130 Gly Val Glu Ala Ser Ser Ala Val Leu Thr Val Ser Pro Glu Val 11135 11140 11145 Pro Gly Met Val Thr Ser Leu Val Thr Ser Ser Arg Ala Val Thr 11150 11155 11160 Ser Thr Thr Ile Pro Thr Leu Thr Ile Ser Ser Asp Glu Pro Glu 11165 11170 11175 Thr Thr Thr Ser Leu Val Thr His Ser Glu Ala Lys Met Ile Ser 11180 11185 11190 Ala Ile Pro Thr Leu Ala Val Ser Pro Thr Val Gln Gly Leu Val 11195 11200 11205 Thr Ser Leu Val Thr Ser Ser Gly Ser Glu Thr Ser Ala Phe Ser 11210 11215 11220 Asn Leu Thr Val Ala Ser Ser Gln Pro Glu Thr Ile Asp Ser Trp 11225 11230 11235 Val Ala His Pro Gly Thr Glu Ala Ser Ser Val Val Pro Thr Leu 11240 11245 11250 Thr Val Ser Thr Gly Glu Pro Phe Thr Asn Ile Ser Leu Val Thr 11255 11260 11265 His Pro Ala Glu Ser Ser Ser Thr Leu Pro Arg Thr Thr Ser Arg 11270 11275 11280 Phe Ser His Ser Glu Leu Asp Thr Met Pro Ser Thr Val Thr Ser 11285 11290 11295 Pro Glu Ala Glu Ser Ser Ser Ala Ile Ser Thr Thr Ile Ser Pro 11300 11305 11310 Gly Ile Pro Gly Val Leu Thr Ser Leu Val Thr Ser Ser Gly Arg 11315 11320 11325 Asp Ile Ser Ala Thr Phe Pro Thr Val Pro Glu Ser Pro His Glu 11330 11335 11340 Ser Glu Ala Thr Ala Ser Trp Val Thr His Pro Ala Val Thr Ser 11345 11350 11355 Thr Thr Val Pro Arg Thr Thr Pro Asn Tyr Ser His Ser Glu Pro 11360 11365 11370 Asp Thr Thr Pro Ser Ile Ala Thr Ser Pro Gly Ala Glu Ala Thr 11375 11380 11385 Ser Asp Phe Pro Thr Ile Thr Val Ser Pro Asp Val Pro Asp Met 11390 11395 11400 Val Thr Ser Gln Val Thr Ser Ser Gly Thr Asp Thr Ser Ile Thr 11405 11410 11415 Ile Pro Thr Leu Thr Leu Ser Ser Gly Glu Pro Glu Thr Thr Thr 11420 11425 11430 Ser Phe Ile Thr Tyr Ser Glu Thr His Thr Ser Ser Ala Ile Pro 11435 11440 11445 Thr Leu Pro Val Ser Pro Gly Ala Ser Lys Met Leu Thr Ser Leu 11450 11455 11460 Val Ile Ser Ser Gly Thr Asp Ser Thr Thr Thr Phe Pro Thr Leu 11465 11470 11475 Thr Glu Thr Pro Tyr Glu Pro Glu Thr Thr Ala Ile Gln Leu Ile 11480 11485 11490 His Pro Ala Glu Thr Asn Thr Met Val Pro Arg Thr Thr Pro Lys 11495 11500 11505 Phe Ser His Ser Lys Ser Asp Thr Thr Leu Pro Val Ala Ile Thr 11510 11515 11520 Ser Pro Gly Pro Glu Ala Ser Ser Ala Val Ser Thr Thr Thr Ile 11525 11530 11535 Ser Pro Asp Met Ser Asp Leu Val Thr Ser Leu Val Pro Ser Ser 11540 11545 11550 Gly Thr Asp Thr Ser Thr Thr Phe Pro Thr Leu Ser Glu Thr Pro 11555 11560 11565 Tyr Glu Pro Glu Thr Thr Ala Thr Trp Leu Thr His Pro Ala Glu 11570 11575 11580 Thr Ser Thr Thr Val Ser Gly Thr Ile Pro Asn Phe Ser His Arg 11585 11590 11595 Gly Ser Asp Thr Ala Pro Ser Met Val Thr Ser Pro Gly Val Asp 11600 11605 11610 Thr Arg Ser Gly Val Pro Thr Thr Thr Ile Pro Pro Ser Ile Pro 11615 11620 11625 Gly Val Val Thr Ser Gln Val Thr Ser Ser Ala Thr Asp Thr Ser 11630 11635 11640 Thr Ala Ile Pro Thr Leu Thr Pro Ser Pro Gly Glu Pro Glu Thr 11645 11650 11655 Thr Ala Ser Ser Ala Thr His Pro Gly Thr Gln Thr Gly Phe Thr 11660 11665 11670 Val Pro Ile Arg Thr Val Pro Ser Ser Glu Pro Asp Thr Met Ala 11675 11680 11685 Ser Trp Val Thr His Pro Pro Gln Thr Ser Thr Pro Val Ser Arg 11690 11695 11700 Thr Thr Ser Ser Phe Ser His Ser Ser Pro Asp Ala Thr Pro Val 11705 11710 11715 Met Ala Thr Ser Pro Arg Thr Glu Ala Ser Ser Ala Val Leu Thr 11720 11725 11730 Thr Ile Ser Pro Gly Ala Pro Glu Met Val Thr Ser Gln Ile Thr 11735 11740 11745 Ser Ser Gly Ala Ala Thr Ser Thr Thr Val Pro Thr Leu Thr His 11750 11755 11760 Ser Pro Gly Met Pro Glu Thr Thr Ala Leu Leu Ser Thr His Pro 11765 11770 11775 Arg Thr Glu Thr Ser Lys Thr Phe Pro Ala Ser Thr Val Phe Pro 11780 11785 11790 Gln Val Ser Glu Thr Thr Ala Ser Leu Thr Ile Arg Pro Gly Ala 11795 11800 11805 Glu Thr Ser Thr Ala Leu Pro Thr Gln Thr Thr Ser Ser Leu Phe 11810 11815 11820 Thr Leu Leu Val Thr Gly Thr Ser Arg Val Asp Leu Ser Pro Thr 11825 11830 11835 Ala Ser Pro Gly Val Ser Ala Lys Thr Ala Pro Leu Ser Thr His 11840 11845 11850 Pro Gly Thr Glu Thr Ser Thr Met Ile Pro Thr Ser Thr Leu Ser 11855 11860 11865 Leu Gly Leu Leu Glu Thr Thr Gly Leu Leu Ala Thr Ser Ser Ser 11870 11875 11880 Ala Glu Thr Ser Thr Ser

Thr Leu Thr Leu Thr Val Ser Pro Ala 11885 11890 11895 Val Ser Gly Leu Ser Ser Ala Ser Ile Thr Thr Asp Lys Pro Gln 11900 11905 11910 Thr Val Thr Ser Trp Asn Thr Glu Thr Ser Pro Ser Val Thr Ser 11915 11920 11925 Val Gly Pro Pro Glu Phe Ser Arg Thr Val Thr Gly Thr Thr Met 11930 11935 11940 Thr Leu Ile Pro Ser Glu Met Pro Thr Pro Pro Lys Thr Ser His 11945 11950 11955 Gly Glu Gly Val Ser Pro Thr Thr Ile Leu Arg Thr Thr Met Val 11960 11965 11970 Glu Ala Thr Asn Leu Ala Thr Thr Gly Ser Ser Pro Thr Val Ala 11975 11980 11985 Lys Thr Thr Thr Thr Phe Asn Thr Leu Ala Gly Ser Leu Phe Thr 11990 11995 12000 Pro Leu Thr Thr Pro Gly Met Ser Thr Leu Ala Ser Glu Ser Val 12005 12010 12015 Thr Ser Arg Thr Ser Tyr Asn His Arg Ser Trp Ile Ser Thr Thr 12020 12025 12030 Ser Ser Tyr Asn Arg Arg Tyr Trp Thr Pro Ala Thr Ser Thr Pro 12035 12040 12045 Val Thr Ser Thr Phe Ser Pro Gly Ile Ser Thr Ser Ser Ile Pro 12050 12055 12060 Ser Ser Thr Ala Ala Thr Val Pro Phe Met Val Pro Phe Thr Leu 12065 12070 12075 Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met Arg His 12080 12085 12090 Pro Gly Ser Arg Lys Phe Asn Ala Thr Glu Arg Glu Leu Gln Gly 12095 12100 12105 Leu Leu Lys Pro Leu Phe Arg Asn Ser Ser Leu Glu Tyr Leu Tyr 12110 12115 12120 Ser Gly Cys Arg Leu Ala Ser Leu Arg Pro Glu Lys Asp Ser Ser 12125 12130 12135 Ala Thr Ala Val Asp Ala Ile Cys Thr His Arg Pro Asp Pro Glu 12140 12145 12150 Asp Leu Gly Leu Asp Arg Glu Arg Leu Tyr Trp Glu Leu Ser Asn 12155 12160 12165 Leu Thr Asn Gly Ile Gln Glu Leu Gly Pro Tyr Thr Leu Asp Arg 12170 12175 12180 Asn Ser Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser Met Pro 12185 12190 12195 Thr Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Val Gly Thr Ser 12200 12205 12210 Gly Thr Pro Ser Ser Ser Pro Ser Pro Thr Thr Ala Gly Pro Leu 12215 12220 12225 Leu Met Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr 12230 12235 12240 Glu Glu Asp Met Arg Arg Thr Gly Ser Arg Lys Phe Asn Thr Met 12245 12250 12255 Glu Ser Val Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys Asn Thr 12260 12265 12270 Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg 12275 12280 12285 Pro Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr 12290 12295 12300 His Arg Leu Asp Pro Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu 12305 12310 12315 Tyr Trp Glu Leu Ser Lys Leu Thr Asn Asp Ile Glu Glu Leu Gly 12320 12325 12330 Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr 12335 12340 12345 His Gln Ser Ser Val Ser Thr Thr Ser Thr Pro Gly Thr Ser Thr 12350 12355 12360 Val Asp Leu Arg Thr Ser Gly Thr Pro Ser Ser Leu Ser Ser Pro 12365 12370 12375 Thr Ile Met Ala Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn 12380 12385 12390 Phe Thr Ile Thr Asn Leu Gln Tyr Gly Glu Asp Met Gly His Pro 12395 12400 12405 Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu 12410 12415 12420 Leu Gly Pro Ile Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser 12425 12430 12435 Gly Cys Arg Leu Thr Ser Leu Arg Ser Glu Lys Asp Gly Ala Ala 12440 12445 12450 Thr Gly Val Asp Ala Ile Cys Ile His His Leu Asp Pro Lys Ser 12455 12460 12465 Pro Gly Leu Asn Arg Glu Arg Leu Tyr Trp Glu Leu Ser Gln Leu 12470 12475 12480 Thr Asn Gly Ile Lys Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asn 12485 12490 12495 Ser Leu Tyr Val Asn Gly Phe Thr His Arg Thr Ser Val Pro Thr 12500 12505 12510 Ser Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly Thr Ser Gly 12515 12520 12525 Thr Pro Phe Ser Leu Pro Ser Pro Ala Thr Ala Gly Pro Leu Leu 12530 12535 12540 Val Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Lys Tyr Glu 12545 12550 12555 Glu Asp Met His Arg Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu 12560 12565 12570 Arg Val Leu Gln Thr Leu Leu Gly Pro Met Phe Lys Asn Thr Ser 12575 12580 12585 Val Gly Leu Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Ser 12590 12595 12600 Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr His 12605 12610 12615 Arg Leu Asp Pro Lys Ser Pro Gly Val Asp Arg Glu Gln Leu Tyr 12620 12625 12630 Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile Lys Glu Leu Gly Pro 12635 12640 12645 Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr His 12650 12655 12660 Trp Ile Pro Val Pro Thr Ser Ser Thr Pro Gly Thr Ser Thr Val 12665 12670 12675 Asp Leu Gly Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Thr 12680 12685 12690 Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr 12695 12700 12705 Asn Leu Lys Tyr Glu Glu Asp Met His Cys Pro Gly Ser Arg Lys 12710 12715 12720 Phe Asn Thr Thr Glu Arg Val Leu Gln Ser Leu Leu Gly Pro Met 12725 12730 12735 Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu 12740 12745 12750 Thr Leu Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp 12755 12760 12765 Ala Ile Cys Thr His Arg Leu Asp Pro Lys Ser Pro Gly Val Asp 12770 12775 12780 Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile 12785 12790 12795 Lys Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val 12800 12805 12810 Asn Gly Phe Thr His Gln Thr Ser Ala Pro Asn Thr Ser Thr Pro 12815 12820 12825 Gly Thr Ser Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Ser Ser 12830 12835 12840 Leu Pro Ser Pro Thr Ser Ala Gly Pro Leu Leu Val Pro Phe Thr 12845 12850 12855 Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met His 12860 12865 12870 His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln 12875 12880 12885 Gly Leu Leu Gly Pro Met Phe Lys Asn Thr Ser Val Gly Leu Leu 12890 12895 12900 Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Asn Gly 12905 12910 12915 Ala Ala Thr Gly Met Asp Ala Ile Cys Ser His Arg Leu Asp Pro 12920 12925 12930 Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu Tyr Trp Glu Leu Ser 12935 12940 12945 Gln Leu Thr His Gly Ile Lys Glu Leu Gly Pro Tyr Thr Leu Asp 12950 12955 12960 Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser Val 12965 12970 12975 Ala Pro Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly Thr 12980 12985 12990 Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Thr Ala Val Pro 12995 13000 13005 Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln 13010 13015 13020 Tyr Gly Glu Asp Met Arg His Pro Gly Ser Arg Lys Phe Asn Thr 13025 13030 13035 Thr Glu Arg Val Leu Gln Gly Leu Leu Gly Pro Leu Phe Lys Asn 13040 13045 13050 Ser Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Ile Ser Leu 13055 13060 13065 Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys 13070 13075 13080 Thr His His Leu Asn Pro Gln Ser Pro Gly Leu Asp Arg Glu Gln 13085 13090 13095 Leu Tyr Trp Gln Leu Ser Gln Met Thr Asn Gly Ile Lys Glu Leu 13100 13105 13110 Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe 13115 13120 13125 Thr His Arg Ser Ser Gly Leu Thr Thr Ser Thr Pro Trp Thr Ser 13130 13135 13140 Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Ser Pro Val Pro Ser 13145 13150 13155 Pro Thr Thr Thr Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe 13160 13165 13170 Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asn Met Gly His Pro Gly 13175 13180 13185 Ser Arg Lys Phe Asn Ile Thr Glu Ser Val Leu Gln Gly Leu Leu 13190 13195 13200 Lys Pro Leu Phe Lys Ser Thr Ser Val Gly Pro Leu Tyr Ser Gly 13205 13210 13215 Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly Val Ala Thr 13220 13225 13230 Arg Val Asp Ala Ile Cys Thr His Arg Pro Asp Pro Lys Ile Pro 13235 13240 13245 Gly Leu Asp Arg Gln Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr 13250 13255 13260 His Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser 13265 13270 13275 Leu Tyr Val Asn Gly Phe Thr Gln Arg Ser Ser Val Pro Thr Thr 13280 13285 13290 Ser Thr Pro Gly Thr Phe Thr Val Gln Pro Glu Thr Ser Glu Thr 13295 13300 13305 Pro Ser Ser Leu Pro Gly Pro Thr Ala Thr Gly Pro Val Leu Leu 13310 13315 13320 Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu 13325 13330 13335 Asp Met Arg Arg Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg 13340 13345 13350 Val Leu Gln Gly Leu Leu Met Pro Leu Phe Lys Asn Thr Ser Val 13355 13360 13365 Ser Ser Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu 13370 13375 13380 Lys Asp Gly Ala Ala Thr Arg Val Asp Ala Val Cys Thr His Arg 13385 13390 13395 Pro Asp Pro Lys Ser Pro Gly Leu Asp Arg Glu Arg Leu Tyr Trp 13400 13405 13410 Lys Leu Ser Gln Leu Thr His Gly Ile Thr Glu Leu Gly Pro Tyr 13415 13420 13425 Thr Leu Asp Arg His Ser Leu Tyr Val Asn Gly Phe Thr His Gln 13430 13435 13440 Ser Ser Met Thr Thr Thr Arg Thr Pro Asp Thr Ser Thr Met His 13445 13450 13455 Leu Ala Thr Ser Arg Thr Pro Ala Ser Leu Ser Gly Pro Met Thr 13460 13465 13470 Ala Ser Pro Leu Leu Val Leu Phe Thr Ile Asn Phe Thr Ile Thr 13475 13480 13485 Asn Leu Arg Tyr Glu Glu Asn Met His His Pro Gly Ser Arg Lys 13490 13495 13500 Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Val 13505 13510 13515 Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu 13520 13525 13530 Thr Leu Leu Arg Pro Lys Lys Asp Gly Ala Ala Thr Lys Val Asp 13535 13540 13545 Ala Ile Cys Thr Tyr Arg Pro Asp Pro Lys Ser Pro Gly Leu Asp 13550 13555 13560 Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Ser Ile 13565 13570 13575 Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val 13580 13585 13590 Asn Gly Phe Thr Gln Arg Ser Ser Val Pro Thr Thr Ser Ile Pro 13595 13600 13605 Gly Thr Pro Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Val Ser 13610 13615 13620 Lys Pro Gly Pro Ser Ala Ala Ser Pro Leu Leu Val Leu Phe Thr 13625 13630 13635 Leu Asn Phe Thr Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met Gln 13640 13645 13650 His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln 13655 13660 13665 Gly Leu Leu Arg Ser Leu Phe Lys Ser Thr Ser Val Gly Pro Leu 13670 13675 13680 Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly 13685 13690 13695 Thr Ala Thr Gly Val Asp Ala Ile Cys Thr His His Pro Asp Pro 13700 13705 13710 Lys Ser Pro Arg Leu Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser 13715 13720 13725 Gln Leu Thr His Asn Ile Thr Glu Leu Gly Pro Tyr Ala Leu Asp 13730 13735 13740 Asn Asp Ser Leu Phe Val Asn Gly Phe Thr His Arg Ser Ser Val 13745 13750 13755 Ser Thr Thr Ser Thr Pro Gly Thr Pro Thr Val Tyr Leu Gly Ala 13760 13765 13770 Ser Lys Thr Pro Ala Ser Ile Phe Gly Pro Ser Ala Ala Ser His 13775 13780 13785 Leu Leu Ile Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Arg 13790 13795 13800 Tyr Glu Glu Asn Met Trp Pro Gly Ser Arg Lys Phe Asn Thr Thr 13805 13810 13815 Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Leu Phe Lys Asn Thr 13820 13825 13830 Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg 13835 13840 13845 Pro Glu Lys Asp Gly Glu Ala Thr Gly Val Asp Ala Ile Cys Thr 13850 13855 13860 His Arg Pro Asp Pro Thr Gly Pro Gly Leu Asp Arg Glu Gln Leu 13865 13870 13875 Tyr Leu Glu Leu Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly 13880 13885 13890 Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr 13895 13900 13905 His Arg Ser Ser Val Pro Thr Thr Ser Thr Gly Val Val Ser Glu 13910 13915 13920 Glu Pro Phe Thr Leu Asn Phe Thr Ile Asn Asn Leu Arg Tyr Met 13925 13930 13935 Ala Asp Met Gly Gln Pro Gly Ser Leu Lys Phe Asn Ile Thr Asp 13940 13945 13950 Asn Val Met Gln His Leu Leu Ser Pro Leu Phe Gln Arg Ser Ser 13955 13960 13965 Leu Gly Ala Arg Tyr Thr Gly Cys Arg Val Ile Ala Leu Arg Ser 13970

13975 13980 Val Lys Asn Gly Ala Glu Thr Arg Val Asp Leu Leu Cys Thr Tyr 13985 13990 13995 Leu Gln Pro Leu Ser Gly Pro Gly Leu Pro Ile Lys Gln Val Phe 14000 14005 14010 His Glu Leu Ser Gln Gln Thr His Gly Ile Thr Arg Leu Gly Pro 14015 14020 14025 Tyr Ser Leu Asp Lys Asp Ser Leu Tyr Leu Asn Gly Tyr Asn Glu 14030 14035 14040 Pro Gly Pro Asp Glu Pro Pro Thr Thr Pro Lys Pro Ala Thr Thr 14045 14050 14055 Phe Leu Pro Pro Leu Ser Glu Ala Thr Thr Ala Met Gly Tyr His 14060 14065 14070 Leu Lys Thr Leu Thr Leu Asn Phe Thr Ile Ser Asn Leu Gln Tyr 14075 14080 14085 Ser Pro Asp Met Gly Lys Gly Ser Ala Thr Phe Asn Ser Thr Glu 14090 14095 14100 Gly Val Leu Gln His Leu Leu Arg Pro Leu Phe Gln Lys Ser Ser 14105 14110 14115 Met Gly Pro Phe Tyr Leu Gly Cys Gln Leu Ile Ser Leu Arg Pro 14120 14125 14130 Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Thr Thr Cys Thr Tyr 14135 14140 14145 His Pro Asp Pro Val Gly Pro Gly Leu Asp Ile Gln Gln Leu Tyr 14150 14155 14160 Trp Glu Leu Ser Gln Leu Thr His Gly Val Thr Gln Leu Gly Phe 14165 14170 14175 Tyr Val Leu Asp Arg Asp Ser Leu Phe Ile Asn Gly Tyr Ala Pro 14180 14185 14190 Gln Asn Leu Ser Ile Arg Gly Glu Tyr Gln Ile Asn Phe His Ile 14195 14200 14205 Val Asn Trp Asn Leu Ser Asn Pro Asp Pro Thr Ser Ser Glu Tyr 14210 14215 14220 Ile Thr Leu Leu Arg Asp Ile Gln Asp Lys Val Thr Thr Leu Tyr 14225 14230 14235 Lys Gly Ser Gln Leu His Asp Thr Phe Arg Phe Cys Leu Val Thr 14240 14245 14250 Asn Leu Thr Met Asp Ser Val Leu Val Thr Val Lys Ala Leu Phe 14255 14260 14265 Ser Ser Asn Leu Asp Pro Ser Leu Val Glu Gln Val Phe Leu Asp 14270 14275 14280 Lys Thr Leu Asn Ala Ser Phe His Trp Leu Gly Ser Thr Tyr Gln 14285 14290 14295 Leu Val Asp Ile His Val Thr Glu Met Glu Ser Ser Val Tyr Gln 14300 14305 14310 Pro Thr Ser Ser Ser Ser Thr Gln His Phe Tyr Leu Asn Phe Thr 14315 14320 14325 Ile Thr Asn Leu Pro Tyr Ser Gln Asp Lys Ala Gln Pro Gly Thr 14330 14335 14340 Thr Asn Tyr Gln Arg Asn Lys Arg Asn Ile Glu Asp Ala Leu Asn 14345 14350 14355 Gln Leu Phe Arg Asn Ser Ser Ile Lys Ser Tyr Phe Ser Asp Cys 14360 14365 14370 Gln Val Ser Thr Phe Arg Ser Val Pro Asn Arg His His Thr Gly 14375 14380 14385 Val Asp Ser Leu Cys Asn Phe Ser Pro Leu Ala Arg Arg Val Asp 14390 14395 14400 Arg Val Ala Ile Tyr Glu Glu Phe Leu Arg Met Thr Arg Asn Gly 14405 14410 14415 Thr Gln Leu Gln Asn Phe Thr Leu Asp Arg Ser Ser Val Leu Val 14420 14425 14430 Asp Gly Tyr Ser Pro Asn Arg Asn Glu Pro Leu Thr Gly Asn Ser 14435 14440 14445 Asp Leu Pro Phe Trp Ala Val Ile Leu Ile Gly Leu Ala Gly Leu 14450 14455 14460 Leu Gly Val Ile Thr Cys Leu Ile Cys Gly Val Leu Val Thr Thr 14465 14470 14475 Arg Arg Arg Lys Lys Glu Gly Glu Tyr Asn Val Gln Gln Gln Cys 14480 14485 14490 Pro Gly Tyr Tyr Gln Ser His Leu Asp Leu Glu Asp Leu Gln 14495 14500 14505 17425PRTArtificial Sequencesynthetic 174Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 17517PRTArtificial Sequencesynthetic 175Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Gly 17638PRTArtificial Sequencesynthetic 176Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Leu Tyr Tyr Cys 35 17711PRTArtificial Sequencesynthetic 177Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 1788PRTArtificial SequencesyntheticVARIANT(8)..(8)Xaa = Ala or Ser 178Gly Phe Thr Phe Asp Asp Tyr Xaa 1 5 1798PRTArtificial SequencesyntheticVARIANT(8)..(8)Xaa = Lys or Ile 179Ile Ser Trp Asn Ser Gly Ser Xaa 1 5 18017PRTArtificial SequencesyntheticVARIANT(3)..(3)Xaa = Tyr or AspVARIANT(12)..(12)Xaa = Tyr or HisVARIANT(15)..(15)Xaa = Met or LeuVARIANT(15)..(15)Xaa = Met or Leu 180Ala Lys Xaa Gly Ser Gly Tyr Gly Lys Phe Tyr Xaa Tyr Gly Xaa Asp 1 5 10 15 Val 181125PRTArtificial Sequencesynthetic 181Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Tyr Ser Gly Tyr Gly Tyr Phe Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 182330PRTArtificial Sequencesynthetic 182Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 183330PRTArtificial Sequencesynthetic 183Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 184326PRTArtificial Sequencesynthetic 184Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 185326PRTArtificial Sequencesynthetic 185Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 186327PRTArtificial Sequencesynthetic 186Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 187327PRTArtificial Sequencesynthetic 187Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 188329PRTArtificial Sequencesynthetic 188Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135 140 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 145 150 155 160 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 225 230 235 240 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250 255 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 260 265 270 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 305 310 315 320 Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 189329PRTArtificial Sequencesynthetic 189Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 115 120 125 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 130 135 140 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 145 150 155 160 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 165 170 175 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 180 185 190 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 195 200 205 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 210 215 220 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 225 230 235 240 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 245 250 255 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 260 265 270 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 275 280 285 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 290 295 300 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 305 310 315 320 Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 190326PRTArtificial Sequencesynthetic 190Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Leu Gly Lys 325 191326PRTArtificial Sequencesynthetic 191Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Leu Gly Lys 325 192232PRTArtificial Sequencesynthetic 192Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 193232PRTArtificial Sequencesynthetic 193Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu

Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 194228PRTArtificial Sequencesynthetic 194Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys 225 195228PRTArtificial Sequencesynthetic 195Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 100 105 110 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Pro Gly Lys 225 196229PRTArtificial Sequencesynthetic 196Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 197229PRTArtificial Sequencesynthetic 197Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 198231PRTArtificial Sequencesynthetic 198Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 65 70 75 80 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 100 105 110 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 130 135 140 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230 199231PRTArtificial Sequencesynthetic 199Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 65 70 75 80 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 100 105 110 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 130 135 140 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230 200228PRTArtificial Sequencesynthetic 200Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Leu Gly Lys 225 201228PRTArtificial Sequencesynthetic 201Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val 1 5 10 15 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Leu Gly Lys 225

Claims

1. A cytotoxic composition comprising a bispecific antigen-binding molecule that (i) specifically binds an effector cell with an EC.sub.50 of greater than about 100 nM and ii) specifically binds a target tumor cell, wherein the EC.sub.50 is a binding affinity value measured in an in vitro FACS binding assay.

2. The composition of claim 1, wherein the antigen-binding molecule specifically binds human CD3 with an EC.sub.50 of greater than 500 nM or greater than about 1 .mu.M.

3. The composition of claim 1, wherein the bispecific antigen-binding molecule comprises a first antigen-binding fragment (Fab1), derived from a first antibody, that is not capable of specifically binding to an effector cell, as measured in an in vitro FACS binding assay or an in vitro surface plasmon resonance binding assay.

4. The composition of claim 1, wherein the bispecific antigen-binding molecule exhibits no detectable binding to an effector cell or to CD3 antigen.

5. The composition of any one of claims 1-4, wherein the bispecific antigen-binding molecule comprises a second antigen-binding fragment (Fab2) derived from a second antibody that specifically binds the target tumor cell with an EC.sub.50 value of less than about 50 nM.

6. The composition of any one of claims 1-5, wherein the bispecific antigen-binding molecule specifically binds each of human CD3 and cynomolgus CD3 with an EC.sub.50 value of greater than about 500 nM, or greater than about 1 .mu.M.

7. The composition of any one of claims 1-6, wherein the target tumor cell is a human tumor cell.

8. The composition of any one of claims 1-7, wherein the bispecific antigen-binding molecule induces T cell-mediated tumor cell killing with an EC.sub.50 value of less than about 1.3 nM, as measured in an in vitro T cell-mediated tumor cell killing assay.

9. The composition of any one of claims 1-8, wherein the target tumor cell expresses a tumor-associated antigen selected from the group consisting of AFP, ALK, BAGE proteins, BIRC5 (survivin), BIRC7, .beta.-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-.beta., TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

10. The composition of any one of claims 1-9, wherein the tumor-associated antigen is CD20, EGFRvIII, PSMA (FOLH1), STEAP2, or MUC16.

11. The composition of any one of claims 1-10, wherein the bispecific antigen-binding molecule comprises a first heavy chain comprising: a CDR1 comprising amino acid residues 1-7 of SEQ ID NO:178, a CDR2 comprising amino acid residues 1-7 of SEQ ID NO:179, and/or a CDR3 comprising amino acid residues 4-11 of SEQ ID NO:180.

12. The composition of claim 11, wherein the first heavy chain comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12 or 20.

13. The composition of any one of claim 11 or 12, wherein the first heavy chain comprises a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 or 54.

14. The composition of any one of claims 11 to 13, wherein the first heavy chain comprises a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, or SEQ ID NO: 152.

15. The composition of any one of claims 11 to 14, wherein the first heavy chain comprises variable domain framework regions having an amino acid sequence selected from FR1 (SEQ ID NO: 174), FR2 (SEQ ID NO: 175), FR3 (SEQ ID NO: 176), and FR4 (SEQ ID NO: 177).

16. The composition of any one of claims 1-11, wherein the bispecific antigen-binding molecule comprises a HCVR and LCVR amino acid sequence pair (HCVR/LCVR) selected from the group consisting of: SEQ ID NOs: 2/162; 10/162; 18/162; 26/162; 34/162; 42/162; 50/162; 58/162; 66/162; 74/162; 82/162; 90/162; 98/162; 106/162; 114/162; 122/162; 130/162; 138/162; 146/162.

17. The composition of any one of claims 1-11, wherein the bispecific antigen binding molecule comprises a first heavy chain comprising a HCVR comprising HCDR1-HCDR2-HCDR3 domains having the amino acid sequences of SEQ ID NOs: 178-179-180, respectively.

18. A method of making the composition of any one of claims 1-17, the method comprising: a. identifying the amino acid sequence of a first heavy chain derived from a first antibody that specifically binds CD3 with an EC.sub.50 value of less than about 40 nM, b. modifying selected amino acid residues in the heavy chain variable region of the first antibody to produce a modified antibody, c. pairing the modified antibody with a second heavy chain derived from a second antibody that specifically binds a target tumor antigen to produce a bispecific antibody, d. testing the bispecific antibody in a binding affinity assay, and if the binding affinity to CD3 has an EC.sub.50 value of greater than about 500 nM, then e. preparing a composition comprising the bispecific antibody and a pharmaceutically acceptable carrier or diluent.

19. The method of claim 18, wherein the first antibody heavy chain comprises a first C.sub.H3 domain and the second antibody comprises a second C.sub.H3 domain suitable for bispecific pairing and isolation.

20. The method of claim 19, wherein the first and second C.sub.H3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference.

21. A pharmaceutical composition comprising the composition of any one of claims 1-17 or made by the method of any one of claims 18-20, comprising a pharmaceutically acceptable carrier or diluent.

22. A method for treating cancer in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 21.

23. The method of claim 22, wherein the cancer is selected from the group consisting of: pancreatic cancer, melanoma, glioblastoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanomaglioma, breast cancer, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, renal oncocytoma, renal transitional cell carcinoma, urothelial carcinoma, adenocarcinoma, or small cell carcinoma.

24. The method of claim 22 or 23, wherein the subject is afflicted with a tumor that is resistant to, or incompletely responsive to monospecific therapy alone.

25. A method for mediating tumor cell lysis, or suppressing tumor growth in a subject, the method comprising administering to the subject a therapeutic amount of the composition of claim 21.

26. The method of claim 25, wherein the amount is sufficient to reduce tumor burden, produce tumor regression, inhibit tumor growth or reduce tumor development in the subject.

27. The method of claim 26, wherein subject is afflicted with a tumor expressing a tumor-associated antigen selected from the group consisting of AFP, ALK, BAGE proteins, BIRC5 (survivin), BIRC7, .beta.-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-.beta., TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

28. Use of a cytotoxic bispecific antibody having reduced clearance for suppression of tumor growth in a subject, wherein the bispecific antibody comprises no detectable binding affinity to an effector cell or weak binding affinity to an effector cell at least greater than 200 EC.sub.50 or K.sub.D, and specifically binds with high affinity to a target tumor cell, wherein binding affinity is measured in an in vitro FACS binding assay or in vitro surface plasmon resonance assay.

29. A method of producing a cytotoxic bispecific antibody, comprising: a. identifying a first human antibody or antigen-binding fragment thereof that interacts with an effector cell antigen from multiple species; b. identifying the germline amino acid residues of the heavy chain variable region (HCVR) of the first human antibody; c. comparing the amino acid sequence of the HCVR of the first human antibody to the amino acid sequence of the corresponding germline HCVR; d. identifying amino acids within a modified region of the HCVR of the first human antibody; whereby a modified region in the first human antibody displays at least one amino acid modification by substitution, deletion or addition of a single amino acid residue compared to the same region in the germline HCVR; e. producing a plurality of modified antibodies each comprising at least one modified region of the HCVR; f. screening each of the plurality of modified antibodies for monovalent affinity to the effector cell antigen; g. selecting those modified antibodies that exhibit weaker binding affinity for the effector cell antigen compared to the first human antibody; and h. pairing a selected antibody with a second antibody that interacts with a tumor-associated antigen to produce a cytotoxic bispecific antibody.